PASK INHIBITOR

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional

Application No. 62/398,596, which was filed on September 23, 2016. The content of this earlier filed application is hereby incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant number

R01DK71962 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted on September 21, 2017 as a text named

"21101_0339Pl_Sequence_Listing.txt," created on September 14, 2017 and having a size of 16,384 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

PAS domain containing protein kinase (PASK) is an evolutionarily conserved protein kinase implicated in energy homeostasis and metabolic regulation across eukaryotic species. The identification of a role for PASK in differentiation in different cell types would be useful as a cell therapy for the treatment of various diseases and disorders.

SUMMARY

The present disclosure provides a mechanism for PASK in promoting the differentiation of progenitor and stem cells. As described herein, inhibition of PASK can be useful in preventing cell differentiation. Described herein are methods of preventing differentiation of stem cells, the methods comprising contacting stem cells with a PAS domain containing protein kinase (PASK) inhibitor.

Described herein are methods of reprogramming mouse embryonic fibroblast cells, the methods comprising contacting mouse embryonic fibroblast cells with a PAS domain containing protein kinase (PASK) inhibitor.

Described herein are methods of inhibiting differentiation of pluripotent embryonic stem cells into neurons, the methods comprising contacting pluripotent embryonic stem cells with a PAS domain containing protein kinase (PASK) inhibitor.

Described herein are methods of inhibiting differentiation of C3H10T1/2 mesenchymal stem cells into adipocytes, the methods comprising contacting C3H10T1/2 mesenchymal stem cells with a PAS domain containing protein kinase (PASK) inhibitor.

Described herein are methods of inhibiting differentiation of mouse C2C12 muscle progenitor cells into muscle fibers, the methods comprising contacting mouse C2C12 muscle progenitor cells with a PAS domain containing protein kinase (PASK) inhibitor.

Described herein are methods of increasing the number of preadipocytes present in sample, the methods comprising administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having preadipocytes and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress preadipocytes cell differentiation into adipocytes; and suppressing PASK activity in the sample; thereby increasing the number of preadipocytes present in the sample.

Described herein are methods of increasing the number of neural stem cells present in a sample, the method comprising administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having neural stem cells and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress neural stem cells differentiation into neurons; and suppressing PASK activity in the sample; thereby increasing the number of neural stem cells present in the sample.

Described herein are methods of increasing the number of myoblasts present in a sample, the method comprising administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having myoblasts and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress myoblasts cell differentiation into myoctyes; and suppressing PASK activity in the sample; thereby increasing the number of myoblasts present in the sample.

Described herein are methods of producing a culture of undifferentiated cells, the method comprising providing a population of preadipocytes having with a PAS domain containing protein kinase (PASK); and contacting the population of cells in a) with a PASK inhibitor, wherein the PASK inhibitor suppresses or prevents differentiation of the said cells; thereby producing a culture of undifferentiated cells.

Described herein are methods of producing a culture of undifferentiated cells, the method comprising providing a population of myoblasts having a PAS domain containing protein kinase (PASK); and contacting the population of cells in a) with PASK inhibitor, wherein the PASK inhibitor suppresses or prevents differentiation of the said cells;

thereby producing a culture of undifferentiated cells.

Described herein are methods of inhibiting expression of a PAS domain containing protein kinase (PASK), the method comprising contacting a cell expressing PASK with a short-interfering ribonucleic acid (siRNA) molecule complementary to the nucleic acid sequence capable of encoding the PAS domain containing protein kinase (PASK), thereby inhibiting the expression of PASK.

Described herein are methods of inhibiting expression of a PAS domain containing protein kinase (PASK) in a subject, the method comprising administering to a subject an effective amount of a pharmaceutical composition comprising a short- interfering ribonucleic acid (siRNA) molecule complementary to the nucleic acid sequence capable of encoding the PAS domain containing protein kinase (PASK), thereby inhibiting the expression of PASK in the subject.

Described herein are methods of inhibiting expression of a gene encoding a PAS domain containing protein kinase (PASK), the method comprising introducing into a stem cell containing and expressing the gene encoding the PASK, an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein and one or more guide RNAs that target the gene encoding the PASK, whereby the one or more guide RNAs target the genomic loci of the gene encoding the PASK and the Cas protein cleaves the genomic loci of the gene encoding the PASK, whereby expression of the gene encoding the PASK is inhibited; and, wherein the Cas protein and the guide RNA do not naturally occur together.

BRIEF DESCRIPTION OF THE DRAWINGS FIGs. 1 A-K shows that PASK is required for skeletal muscle regeneration after acute muscle injury. FIG. 1A shows the differentiation of C2C12 myoblasts that were assessed after PASK was either knocked down using pooled siRNA or inhibited using 25μΜ BioE-1197. Scale bar = 40μΜ. FIG. IB shows the comparison of fusion index between control and siRNA (Si_PASK) or inhibitor (BioE-1197)-treated C2C12 cells from FIG. 1A. n=3 independent experiments each with 100 cells counted. Error bars ± S.D.; * P<0.05. FIG. 1C shows that GFP, Flag-WT or Flag-KD (K1028R) human PASK were expressed using retrovirus in PASK-silenced C2C12 cells. Differentiation was initiated 24 h after transgene introduction and was analyzed using anti-MHC staining on day 3 as in FIG. 1A. Expression of PASK was visualized using anti-Flag staining. Scale bar = 20μηι. FIG. ID shows the Fusion index (as in FIG. IB) of cells from FIG. 1C that were individually scored as with (+) or without (-) GFP, WT PASK or KD PASK expression. Cells expressing WT PASK show restoration of fusion index. n=3 independent experiments each with 100 cells counted. Error bars ± S.D. ** P<0.005. FIG. IE shows the results of qRT-PCR analysis of C2C12 cells showing abundance of Mylpf and Actal mRNAs in PASK-silenced cells expressing GFP, WT or KD PASK. 18S rRNA was used as normalizer. Error bars ± S.D. *P<0.05, #P<0.05 WT vs KD PASK in control samples, # P<0.05 WT vs KD hPASK in Si PASK samples. FIG. IF shows myogenesis of primary myoblasts derived from WT and Pask-/- skeletal muscle that were assessed using anti-MHC staining after 4 days of differentiation. Nuclei are stained with DAPI. Scale bar = ΙΟΟμΜ. FIG. 1G shows the quantification of fusion index from FIG. IF at Day 4. *** PO.0005. FIG. 1H shows qRT-PCR analysis of fold change in the expression of Pask and Myh3 mRNA following BaC12 induced muscle injury to TA muscle relative to uninjured (DPI 0). 18S rRNA was used as normalizer. FIG. II is a Western blot analysis of isolated TA muscle following BaC12 induced muscle injury from WT and Pask-/- mice. FIG. 1J is a representative cross-section of TA muscle 5d post injury showing levels of Myh3 (green) expression between WT (Pask+/+) and Pask-/- animals. Nuclei are stained with DAPI (blue). Scale bars = ΙΟΟμΜ. n=5. FIG. IK shows the quantification of the Myh3 positive area/total as determined by measuring Myh3 staining intensities across 3 representative sections from each independent animal, n=5. Error bars ± S.D. ***P<0.0005.

FIGs. 2A-L shows that Pask is required for transcriptional activation of MyoG in response to differentiation cues. FIG. 2A is a schematic of myogenesis from satellite cells that depicts the progression of transcription factors during myogenesis. FIG. 2B shows qRT-PCR analysis of WT and Pask ^'1' satellite cells prior to (day 0) or at the indicated time after initiation of differentiation with ΙΟΟηΜ insulin in serum free DMEM. 18S rRNA was used as normalizer. Transcript levels of WT cells at Day 0 were set at 1 to calculate fold changes during differentiation. Error bars ± S.D. *P<0.05, **P<0.005,

***P<0.001. FIG. 2C is a Western blot analysis of the indicated proteins at 5 days post- injury from isolated TA muscles of WT and Pask ^~ mice. FIG. 2D shows the

immunofluorescence microscopic examination of Pax7 expression in control, Pask- siRNA or 25 μΜ BioE-1197 treated samples on Day 0 of differentiation. FIG. 2E shows the quantification of Pax7+ cell numbers from experiment in FIG. 2D along the differentiation time course. n=3 independent experiments each with 100 cells counted. Error bars= S.D. *p<0.05. FIG. 2F is an immunofluorescence microscopy showing MyoG and Pask in control, Pask-siRNA or 25μΜ BioE-119- treated cells at Day 1 of differentiation. FIG. 2G shows the percent MyoG ⁺ cells in Control, Pas^-siRNA or 25μΜ BioE-1197-treated cells during differentiation as in FIG. 2F. n=3 independent experiments each with 100 cells counted. Error bars ± S.D. ** P<0.005, * P<0.05. FIG. 2H shows the percent co-expression of Pask ⁺ and MyoG ⁺ cells at Day 1 of differentiation as in FIG. 2F. n=3 independent experiments each with 100 cells counted. Error bars ± S.D. **P<0.005. FIG. 21 shows representative images fromPas^-silenced C2C12 cells expressing GFP control or Flag-human Pask at 24 h after initiation of differentiation to detect endogenous Pax7 (left) or MyoG (right) together with GFP or Flag-Pask. Scale bar = 20μΜ. FIG. 2J shows the quantification of Pax7 ⁺ and MyoG ⁺ cells counting those cells that are GFP ⁺ or Pask ⁺ from FIG. 21. n=3 independent experiments each with 100 cells counted. Error bars ± S.D. *P<0.05 Control vs. Pask silenced GFP ⁺ cells, #P<0.05 Pask silenced GFP ⁺ vs. Pask silenced hPask ⁺ cells. FIG. 2K shows that an empty GFP vector or GFP vector containing hPask were retrovirally introduced to proliferating C2C12 cells at a sub-confluent density in growth media. FIG. 2L shows qRT-PCR analysis of GFP or hPask-expressing C2C12 myoblasts after puromycin selection from FIG. 21. Cells were collected from growth media (GM) or 24 h after addition of differentiation media (DM). n=3, Error bars ± S.D. *P<0.05, **P<0.005, ***P<0.0005.

FIGs. 3A-F show that Pask is required for myogenic conversion of C3H10T1/2 cells by MyoD. FIG. 3A is a schematic depiction of the mechanism by which MyoD- induces myogenic conversion of adipogenic C3H10T1/2 cells. FIG. 3B shows qRT-PCR analysis of the indicated mRNAs in C3H10T1/2 cells expressing MyoD in the presence of DMSO or 25μΜ BioE-1197 during differentiation. n=3, Error bars ± S.D ***P<0.0005, P<0.005. FIG. 3C shows MyoD-expressing C3H10T1/2 cells were allowed to differentiate in the presence of DMSO or 25μΜ BioE-1197 and processed for immunofluorescence microscopy using anti-MyoG antibody. Scale bar = 20μηι. FIG. 3D shows the quantification of MyoD ⁺ and MyoG ⁺ cells in the presence of DMSO or BioE- 1197 from FIG. 3C. n=3 independent experiments each with 100 cells counted. Error bars ± S.D.**P<0.005. FIG. 3E shows the fusion index on Day 1 and 2 of differentiation for C3H10T1/2 cells expressing MyoD in the presence of DMSO or 25μΜ BioE-1197. Inset shows representative MHC staining on Day 2 of differentiation. Scale bar = 20μιη. FIG. 3F shows the qRT-PCR analysis of endogenous mouse Mylpf and Myog and rat Myog upon GFP or rat MyoG expression in MyoD-expressing C3H10T1/2 cells treated with either DMSO or 25μΜ BioE-1197. n=3, Error bars ± S.D. ***P<0.0005,

**P<0.005.

FIGs. 4A-I shows that Pask directly interacts with and phosphorylates Wdr5 at Ser49. FIG. 4A shows that endogenous Pask was immunoprecipitated from C2C12 cells either before (day -1, 0) or after induction of differentiation (Day 1). FIG. 4B shows V5-tagged LacZ, WT Pask or KD (K1028R) Pask was co-expressed with Flag-YFP or Flag-Wdr5 in 293T cells. FIG. 4C shows V5-hPask was expressed in HEK293T cells with control or Flag-Wdr5 vector. FIG. 4D shows in vitro phosphorylation of purified His-Wdr5 was performed using WT or KD Pask and analyzed by autoradiogram of the reaction mixture after western blotting, with total protein visualized by Ponceau S staining. pPask indicates autophosphorylation of WT-Pask during kinase reaction. FIG. 4E shows that the Pask-Wdr5 complex was immunoprecipitated from cells incubated with 2P in the presence of DMSO or 25μΜ BioE-1197. FIG. 4F shows endogenous Pask was immunoprecipitated from C2C12 cells growing in growth media or 12 h after replacement with differentiation media containing ΙΟηΜ Insulin and was incubated with purified Flag- Wdr5 and [γ- ² Ρ] ATP. Autoradiogram shows incorporation of ² P into Pask (p-Pask) and Wdr5 (p-Wdr5). FIG. 4G is a schematic showing Ser49 and upstream sequence in Wdr5, compared to the site of Pask phosphorylation in Ugpl, a bona fide substrate of S.

cerevisiae Pask. FIG. 4H shows GST-tagged WT, S49A or S49E Wdr5 was incubated with Pask and [γ- ² Ρ] ATP and phosphorylation was detected by autoradiography after SDS-PAGE. [I] WT or S49A Wdr5 was co-immunoprecipitated with Pask from cells incubated with ² P-phosphate and analyzed as in FIG. 4E.

FIGs. 5A-H show that the phospho-mimetic S49E Wdr5 mutant rescues myogenesis in Pask-silenced cells. FIG. 5 A shows that GFP or WT, S49A or S49E Wdr5 were retrovirally expressed in Pask-siRNA C2C12 cells. n=3. Error bars ± S.D * P<0.05. FIG. 5B shows Flag-tagged WT, S49A or S49E Wdr5 was expressed in Pask-siRNA C2C12 cells. Arrows show Wdr5S49E-expressing cells and the corresponding cell autonomous decrease in Pax7 expression. Scale bar = 20μΜ. FIG. 5C shows the quantification of percent Pax7+ cells from FIG. 5B as a function of the presence (+) or absence (-) of GFP or Wdr5. n=3 independent experiments each with 100 cells counted. Error bars ± S.D. *P<0.05. Similar to the results shown in FIG. 5B, FIG. 5D shows the same except cells were stained for MyoG on Day 1 of differentiation. FIG. 5E shows quantification of percent MyoG+ cells from FIG. 5D as a function of the presence (+) or absence (-) of GFP or Wdr5. n=3 independent experiments each with 100 cells counted. Error bars ± S.D. *P<0.05. FIG. 5F shows the same as in FIG. 5B, except cells were stained for MHC on Day 3 of differentiation. FIG. 5G shows the quantification of fusion index from FIG. 5F as a function of the presence (+) or absence (-) of GFP or Wdr5. n=3 independent experiments each with 100 cells counted. Error bars ± S.D. *P<0.05. FIG. 5H shows C2C12 myoblasts were infected with retrovirus expressing GFP, Flag tagged WT or KD Pask or WT, S49A or S49E Wdr5 and infected cells were selected with puromycin in growth media for 48 hrs.

FIGs. 6A-G shows that Pask is required for recruitment of Wdr5 and MyoD to the Myog promoter during differentiation. FIG. 6A is a depiction of the Myog genomic locus depicting MyoD and RNAPolII occupancy as well as H3K4me2 and H3K4me3 abundance at 60 h of differentiation from the ENCODE dataset for the C2C12 cell line. TSS: Transcription Start Site. Colored horizontal bars indicate the positions of ChIP amplicons a, b or c. FIG. 6B shows fold H3K4me3 enrichment in control, Pask-siRNA or BioE-1197-treated C2C12 cells that were assessed by ChlP-qPCR of the indicated amplicon followed by normalization against H3K4me3-deficient negative control region of the actb gene. n=3. Error bars ± S.D. *P<0.05, ** P<0.005. FIG. 6C shows that H3K4me3 ChIP was performed from control or Pask silenced C2C12 cells expressing GFP, WT or KD Pask or WT, S49A or S49E Wdr5 at Day 1 of differentiation. n=3. Error bars ± S.D. * P<0.05. FIG. 6D shows that Flag ChIP was performed from Pask silenced C2C12 cells expressing GFP, WT or KD Pask or WT, S49A or S49E Wdr5 at Day 1 of differentiation. n=3. Error bars ± S.D. * PO.05. FIG. 6E shows that endogenous Wdr5 ChIP was performed from control or Pask-siRNA C2C12 cells at Day 0 or Day 1 of differentiation and fold enrichment on the Myog promoter was determined by qRT-PCR. Error bars ± S.D. * P<0.05. FIG. 6F shows that MyoD ChIP was performed from control or Pask-siRNA C2C12 cells at Day 0, Day 1 or Day 2 of differentiation and fold enrichment of MyoD occupancy on the Myog promoter was determined by qRT-PCR. n=3. Error bars ± S.D. ** PO.05, ***P<0.0005. FIG. 6G shows MyoD ChIP was performed from proliferating (Day 0) or differentiating (Day 1) C2C12 cells expressing GFP, Wdr5WT, Wdr5S49A or Wdr5S49E. n=3. Error bars ± S.D. #, ***P<0.0005, Wdr5 S49E vs GFP, Wdr5WT or Wdr5S49A at Day 0 and Day 1 respectively.

FIGs. 7A-F shows that differentiation induced H3K4mel to H3K4me3 conversion is dependent upon Pask phosphorylation of Wdr5. H3K4mel (FIG. 7A) and total H3 ChIP (FIG. 7B) were performed from control or Pask-siRNA C2C12 cells at the indicated day of differentiation and fold enrichment on the Myog promoter was determined by qRT-PCR using primer set b. Error bars ± S.D. * P<0.05, **P<0.005. FIG. 7C shows that the differentiation potential of C2C12 myoblasts subjected to Pask or M113 siRNA treatments was assessed by qRT-PCR using primers specific for Myog, Mylpf, M113 and Pask. H3K4mel (FIG. 7D) or total H3 ChIP (FIG. 7E) was performed from proliferating (Day 0) or differentiating (Day 1) C2C12 cells expressing GFP, Wdr5WT, Wdr5S49A or Wdr5S49E. n=3. Error bars ± S.D. *, # PO.05, Wdr5 S49E vs GFP, Wdr5WT or Wdr5S49A at Day 0 and Day 1 respectively. FIG. 7F is a model depicting the role of Pask and Wdr5 phosphorylation in regulating MyoD recruitment to the Myog promoter during differentiation. FIGs. 8A-E shows that Pask is enriched in stem cells and is required for terminal differentiation of neuronal, adipogenic and myogenic cell types. FIG. 8A shows a comparison of Pask mRNA expression profile from GeneAtlas MOE430 dataset obtained from BioGPS. FIG. 8B shows induction of Pask mRNA expression during iPSC formation from indicated differentiated cell types plotted as percentile rank across all genes. FIG. 8C shows Pask mRNA expression during differentiation of hESCs into cardiomyocytes and in comparison with adult heart expressed as a percentile rank across all genes. FIG. 8D is a depiction of the mouse Pask locus showing positions of Oct4 and Nanog transcription factor binding and corresponding chromatin marks indicative of active transcription in ESCs. ChlP-Seq data was obtained from ENCODE browser, see Examples for citation. FIG. 8E shows the relative mRNA after shRNA treatments.

FIGs. 9A-C shows that Pask is required for differentiation of ES cells but not for iPS reprograming. FIG. 9A shows mouse embryonic fibroblasts (MEFs) containing an IRES-GFP cassette expressed from the Oct4 locus were induced for reprogramming by mSTEMCCA factors as described in material and methods in the presence of DMSO or 50μΜ BioE-1197. Representative images of iPS colonies at day 8 post-initiation of reprogramming showing expression of GFP and its corresponding brightfield image are shown. FIG. 9B shows the quantification of GFP+ colonies 8 days after initiation of reprograming from experiment in FIG. 9A. FIG. 9C shows mouse ES cells were treated with either DMSO or 25μΜ BioE-1197 24 h prior to induction of neuronal

differentiation. n=3 Error bars ± S.D.

FIGs. 10A-D shows that Pask is required for adipogenesis of mesenchymal stem cell. FIG. 10A shows C3H10T1/2 cells that were treated with either DMSO or 25μΜ BioE-1197 48 h prior to induction of adipocyte differentiation. FIG. 10B shows the quantification of percent of cells with lipid droplets after 8 days of differentiation from FIG. 1 OA. n=3. Error bars ± S.D. ***p<0.0001. FIG. IOC shows qRT-PCR analysis of markers of adipocyte differentiation in DMSO or BioE-1197 treated samples from FIG. 10A. n=3. Error bars ± S.D. **p<0.005. FIG. 10D shows the proliferation rate of C3H10T1/2 cells that were measured by seeding cells at 10,000 cells/well and counting cells using a hemocytometer over the ensuing three days.

FIGs. 11-F shows that genetic and pharmacological inhibition of Pask suppresses myogenesis of mouse and human myoblasts. FIG. 11A shows MHC staining of C2C12 cells with Cas9 guided deletion of Pask during differentiation. FIG. 11B shows the fusion index calculation from FIG. 11A. FIG. 11C is a Western blot analysis of MHC expression during differentiation of C2C12 cells with Cas9 guided Pask deletion. FIG. 11D shows human primary myoblasts that were subjected to differentiation by either 2% horse serum or ΙΟΟηΜ insulin in presence of DMSO or BioE-1197. FIG. HE shows mRNA expression levels of human MYLPF and ACTA1 that were determined from human primary myoblasts subjected to insulin stimulated differentiation at indicated time point. FIG. 1 IF shows the proliferation rate of mouse primary myoblasts derived from WT or Pask-/- mice treated with DMSO or BioE-1197.

FIGs. 12A-C show that Pask is required for transcriptional activation of MyoG in response to differentiation cues. FIG. 12A is a Western blot analysis of the abundance of the indicated proteins during myoblast differentiation in control or Pask-silenced C2C12 cells using 2% Horse Serum media. FIG. 12B shows qRT-PCR analysis of DMSO or 25 μΜ BioE-1197 treated C2C12 cells prior to (day 0) or 3 days after ΙΟηΜ insulin- stimulated differentiation. 18S rRNA was used as normalizer. Error bars=S.D. *p<0.05, ***p<0.0005. FIG. 12C shows that WT or KD (K1028R) Pask or GFP were expressed in Pask-silenced C2C12 cells via retroviral infection 48 h prior to differentiation. Error bars = S.D. *p<0.05.

FIGs. 13A-B shows that Pask does not regulate MyoD+ cell population. FIG. 13A is an immunofluorescence microscopy showing MyoD expression and localization in control, Pask-siRNA or 25 μΜ BioE-1197 treated cells on Day 0 of differentiation. FIG. 13B shows the quantification of MyoD+ cell numbers from experiment in FIG. 13A along the differentiation time course. n=3 independent experiments each with 100 cells counted.

FIGs. 14A-C shows a Pask promoter occupied by MyoG and MyoD during differentiation. FIG. 14A shows ChlP-Seq data from the ENCODE dataset for H3K4me3 levels, MyoG, MyoD and POL II binding at the indicated time-points at the Pask promoter during C2C12 differentiation. Vertical turquois line represents the peak of MyoG/MyoD binding at the predicted E-Box motif (sequence in brown). The transcription start site is indicated by an arrow. The green and red horizontal bars represent Pask a and Pask _b ChIP primer sets used. FIG. 14B shows MyoG and MyoD ChIP was performed from proliferating (Day 0) or differentiating (Day 1 and Day 2) C2C12 cells and analyzed by Pask a and Pask b primer sets. Myh3 primers spanning - lOObp from TSS were used as a positive control for MyoG binding. n=3, Error bars ± S.D. ***p<0.0005. FIG. 14C shows a region containing ~300bp upstream of the predicted Pask transcriptional start site, which contains a putative E-box element, was cloned upstream of firefly luciferase. RLU: Relative Light Units.

FIGs. 15A-B show that Pask is required for myogenic conversion of mesenchymal stem cells by MyoD. FIG. 15A shows the immunofluorescence of MyoD expression in DMSO or BioE-1197 treated samples at Day 1 and Day 2. FIG. 15B shows the immunofluorescence of MyoG expression in DMSO or BioE-1197 treated samples at Day l and Day 2.

FIGs. 16A-F show that Pask directly interacts with and phosphorylates Wdr5 at Ser49. FIG. 16A shows flag-tagged YFP or indicated members of various protein complexes of which Wdr5 is a member were expressed in 293T cells. FIG. 16B shows bacterially purified GST-Wdr5 or GST control was attached to glutathione sepharose and purified His-tagged Pask was passed over and the beads were washed extensively. FIG. 16C shows the domain truncation of Pask to determine Wdr5 binding region. FIG. 16D shows the in vitro phosphorylation of Wdr5 by Pask in the presence of DMSO or 25μΜ 1197. FIG. 16E shows that bacterially purified GST-(Flag)-WT, S49A or S49E Wdr5 were used for GST pull-down assays of Pask as in FIG. 16B. FIG. 16F shows HEK293T cells expressing Flag tagged YFP or the indicated Wdr5 variants were lysed and Flag- tagged proteins were imunoprecipitated.

FIG. 17 shows Wdr5 ^S49E expression rescues genetic loss of Pask.

FIGs. 18A-D shows Pask and phosphomimetic Wdr5 promote H3K4mel to H3K4me3 conversion and MyoD recruitment to the Myog promoter. FIG. 18A is a depiction of the Myog locus showing abundance of position of enhancer and promoter region as well as H3K4me3 levels and MyoD occupancy 60hrs after initiation of differentiation from ENCODE datasets deposited by Barbara Wold lab. FIG. 18B shows ChlP-qPCR of differentiation time course of Control vs Pask silenced C2C12 cells using MyoD, H3K4mel, H3K27ac and H3K4me3 antibodies. Actb negative region was used as normalizer. *p<0.05, **p<0.005, ***p<0.001. FIG. 18C shows H3K4mel ChIP of control and Pask silenced C2C12 cells using primers spanning the indicated gene and position relative to the TSS. actb negative control region was used as the normalizer. *P<0.05, **P<0.005. FIG. 18D shows H3K4me3 ChIP of control and Pask silenced C2C12 cells using primers spanning the indicated gene and position relative to the TSS. actb negative control region was used as the normalizer. *P<0.05, **P<0.005,

***P<0.001.

FIG. 19 is a table providing cell lines, antibodies and primer sequences used herein.

FIG. 20 shows that Pax7+ cell number increases upon PASK inhibition.

FIG. 21 depicts the effects of WT, S49A or S49E mutant Wdr5 expression on Pax7 expression in C2C12 myoblasts.

FIGS. 22A-C shows the restoration of Pax7 expression and myogenesis program differentiation defective myoblasts after BioE-1197 pretreatment. FIG. 22 A is a schematic representation of the method used to isolate differentiation defective myoblasts and the treatment used in this experiment. Pax7 levels were detected (FIG. 22B) at passage # as indicated in (FIG. 22B). FIG. 22C shows the differentiation capacity of differentiation defective myoblasts.

DETAILED DESCRIPTION

The present disclosure can be understood more readily by reference to the following detailed description of the invention, the figures and the examples included herein.

Before the present methods and compositions are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

Moreover, it is to be understood that unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, and the number or type of aspects described in the specification.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

DEFINITIONS

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

The word "or" as used herein means any one member of a particular list and also includes any combination of members of that list.

Ranges can be expressed herein as from "about" or "approximately" one particular value, and/or to "about" or "approximately" another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," or "approximately," it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value " 10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11 , 12, 13, and 14 are also disclosed.

As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. As used herein, the term "sample" is meant a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), which is assayed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.

As used herein, the term "subject" refers to the target of administration, e.g., a human. Thus the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In one aspect, a subject is a mammal. In another aspect, a subject is a human. The term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

As used herein, the term "patient" refers to a subject afflicted with a disease or disorder. The term "patient" includes human and veterinary subjects. In some aspects of the disclosed methods, the "patient" has been diagnosed with a need for treatment for cancer, such as, for example, prior to the administering step.

As used herein, the term "comprising" can include the aspects "consisting of and

"consisting essentially of. " "Comprising can also mean "including but not limited to. "

"Inhibit," "inhibiting" and "inhibition" as used herein, mean to diminish or decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% inhibition or reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, in an aspect, the inhibition or reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. In an aspect, the inhibition or reduction is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90- 100% as compared to native or control levels. In an aspect, the inhibition or reduction is 0-25, 25-50, 50-75, or 75-100% as compared to native or control levels. "Modulate", "modulating" and "modulation" as used herein mean a change in activity or function or number. The change may be an increase or a decrease, an enhancement or an inhibition of the activity, function or number.

"Promote," "promotion," and "promoting" refer to an increase in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the initiation of the activity, response, condition, or disease. This may also include, for example, a 10% increase in the activity, response, condition, or disease as compared to the native or control level. Thus, in an aspect, the increase or promotion can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or more, or any amount of promotion in between compared to native or control levels. In an aspect, the increase or promotion is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared to native or control levels. In an aspect, the increase or promotion is 0-25, 25-50, 50-75, or 75-100%, or more, such as 200, 300, 500, or 1000% more as compared to native or control levels. In an aspect, the increase or promotion can be greater than 100 percent as compared to native or control levels, such as 100, 150, 200, 250, 300, 350, 400, 450, 500% or more as compared to the native or control levels.

As used herein, the term "determining" can refer to measuring or ascertaining a quantity or an amount or a change in activity. For example, determining the amount of a disclosed polypeptide in a sample as used herein can refer to the steps that the skilled person would take to measure or ascertain some quantifiable value of the polypeptide in the sample. The art is familiar with the ways to measure an amount of the disclosed polypeptides and disclosed nucleotides in a sample.

As used herein, the terms "disease" or "disorder" or "condition" are used interchangeably referring to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease or disorder or condition can also related to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be practiced within the scope of the appended claims.

INTRODUCTION

Pask (PAS domain containing protein Kinase) is an evolutionarily conserved protein kinase that has been implicated in signaling to coordinate nutrient sensing with metabolic control across eukaryotic phylogeny (Hao and Rutter, 2008). The principal modality by which mammalian Pask appears to exert metabolic control is the regulation of gene expression. For example, Pask was shown to regulate the synthesis of fatty acids and triglycerides in the liver via activation of Sterol Regulatory Element Binding Protein- 1 (SREBP-1) transcriptional activity in response to feeding and insulin (Wu et al., 2014). Probably related to this function, pharmacologic inhibition or genetic ablation of Pask resulted in decreased liver fat content and improved insulin sensitivity in rodent models of diabetes and obesity (Hao et al, 2007; Wu et al, 2014). In pancreatic β-cells, Pask responds to extracellular glucose and stimulates the transcription of the gene encoding insulin via regulation of the PDX-1 transcription factor (An et al., 2006; Semache et al, 2013). In spite of these observations, it has remained unclear how Pask coordinates these seemingly diverse transcriptional responses in different cell types.

Pask is expressed at a low level in most adult tissues (Katschinski et al., 2003). Interestingly, elevated Pask mRNA abundance in stem or progenitor cell types in several transcriptome datasets was observed. As described herein, genetic and pharmacologic methods of modulating Pask activity were used to uncover a function of Pask in regulating the differentiation of stem and progenitor cells into, for example, neuronal, adipocytes or myocytes lineages. As described herein, the mechanism underlying the role of Pask in regulating stem and progenitor cell differentiation can depend upon, for instance, direct phosphorylation of Wdr5. Wdr5 is a component of several chromatin modifying complexes, including mixed lineage leukemia (Mil) histone H3 Lysine 4 (H3K4) methyltransferase complexes (Ruthenburg et al, 2007; Wysocka et al, 2005). Wdr5 is a histone H3 binding protein (Wysocka et al, 2005) that is postulated to present the H3 N-terminal tail to the Mil or Setl enzymes for methylation at lysine 4 (Ruthenburg et al, 2006; Schuetz et al, 2006).

Lysine 4 of Histone H3 is sequentially methylated to the mono- (H3K4mel), di- (H3K4me2) and tri-methyl (H3K4me3) forms by methyltransferases (Shilatifard, 2012). H3K4mel is typically found at enhancers, which are binding sites for regulatory DNA- binding transcription factors (Rada-Iglesias et al, 2011 ; Shlyueva et al, 2014). A recent study demonstrated, however, that H3K4mel functions as a transcriptional repressive mark at the promoters of lineage specifying genes (Cheng et al, 2014). In contrast, H3K4me3 marks are usually associated with transcriptionally active promoters, or with poised promoters when found together with repressive H3K27me3 marks (Bernstein et al., 2006). These histone modifications collaborate with pioneering transcription factors to elicit programs of gene expression that drive differentiation of stem and progenitor cells (Zaret and Carroll, 201 1). As described herein, myogenic progenitor cells were used as a model of inducible differentiation to show that phosphorylation of a single Wdr5 serine by Pask is needed and sufficient for the conversion of repressive H3K4mel marks to activating H3K4me3 marks at the lineage-specifying myogenin {Myog) promoter. This concomitantly enhances MyoD recruitment and chromatin remodeling of the Myog promoter and stimulates transcription of Myog to initiate terminal differentiation. Taken together, the results described herein establish Wdr5 phosphorylation by Pask as an important node in the signaling and transcriptional network that initiates and executes differentiation.

Taken together, the results described herein have identified Pask and Wdr5 phosphorylation as necessary for differentiation of myocytes in vitro. Because the myocyte differentiation epigenetic program is similar to those found in other

differentiation paradigms, including those shown to be Pask dependent, Pask may function through similar mechanisms in other systems. Specifically, the data described herein shows that Pask phosphorylates Wdr5 to promote H3K4mel to H3K4me3 conversion on the promoters of lineage-specifying genes to facilitate chromatin remodeling, gene expression and differentiation. METHODS

Described herein are methods of preventing or inhibiting differentiation of a stem cell. The prevention or inhibition can be permanent or temporary. In an aspect, the method can comprise contacting a stem cell with a PAS domain containing protein kinase (PASK) inhibitor. In an aspect, the PASK inhibitor is BioE- 1197. In an aspect, the contact between the stem cell and a PASK inhibitor can be conducted or otherwise carried out in vitro. In an aspect, the stem cell can be a pluripotent stem cell, a mouse embryonic fibroblast cell, a progenitor cell, a C3H10T1/2 mesenchymal stem cell, a mouse C2C12 muscle progenitor cell, or a primary myoblast cell. In an aspect, the stem cell can comprise a PAS domain.

Described herein are methods of reprogramming mouse embryonic fibroblast cells. The method can comprise contacting mouse embryonic fibroblast cells with a PAS domain containing protein kinase (PASK) inhibitor. In an aspect, the PASK inhibitor can be BioE-1197. In an aspect, the contact between the mouse embryonic fibroblast cells and a PASK inhibitor can be conducted or otherwise carried out in vitro. In an aspect, the mouse embryonic fibroblast cells can comprise a PAS domain. In an aspect, the mouse embryonic fibroblast cells can be reprogrammed such that they are less likely to or do not differentiate.

Disclosed herein are methods of inhibiting differentiation of a pluripotent embryonic stem cell into a neuron. In an aspect, the method can comprise contacting a pluripotent embryonic stem cell with a PAS domain containing protein kinase (PASK) inhibitor. In an aspect, the PASK inhibitor can be BioE-1197. In an aspect, the contact between the pluripotent embryonic stem cell and a PASK inhibitor can be conducted or otherwise carried out in vitro.

Disclosed herein are methods of inhibiting differentiation of a C3H10T1/2 mesenchymal stem cell into an adipocyte. In an aspect, the method can comprise contacting a C3H10T1/2 mesenchymal stem cell with a PAS domain containing protein kinase (PASK) inhibitor. In an aspect, the PASK inhibitor can be BioE-1197. In an aspect, the contact between the C3H10T1/2 mesenchymal stem cell and a PASK inhibitor can be conducted or otherwise carried out in vitro.

Disclosed herein are methods of inhibiting differentiation of a mouse C2C12 muscle progenitor cell into a muscle fiber. In an aspect, the method can comprise contacting a mouse C2C12 muscle progenitor cell with a PAS domain containing protein kinase (PASK) inhibitor. In an aspect, the PASK inhibitor can be BioE-1197. In an aspect, the contact between the mouse C2C12 muscle progenitor cell and a PASK inhibitor can be conducted or otherwise carried out in vitro.

Disclosed herein are methods of increasing the number of preadipocytes present in a sample. In an aspect, the method can comprise: a) administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having preadipocytes and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress preadipocytes cell differentiation into adipocytes; and b) suppressing PASK activity in the sample; thereby increasing the number of preadipocytes present in the sample. In an aspect, the PASK inhibitor can be BioE-1197. In an aspect, the sample is a human sample. In an aspect, the increase in the number of preadipocytes can occur in vitro prior to

transplantation of the preadipocytes into a patient in need of cell therapy. In an aspect, the patient in need of cell therapy can be at risk for developing or has sarcopenia or disease-associated sarcopenia or a stem cell loss disorder.

Disclosed herein are methods of increasing the number of neural stem cells present in a sample. In an aspect, method can comprise a) administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having neural stem cells and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress neural stem cells differentiation into neurons; and b) suppressing PASK activity in the sample;

thereby increasing the number of neural stem cells present in the sample. In an aspect, the PASK inhibitor can be BioE-1197. In an aspect, the sample is a human sample. In an aspect, the increase in the number of neural stem cells can occur in vitro prior to transplantation of the neural stem cells into a patient in need of cell therapy. In an aspect, the patient in need of cell therapy can be at risk for developing or has sarcopenia or disease-associated sarcopenia or a stem cell loss disorder.

Disclosed herein are methods of increasing the number of myoblasts present in a sample. In an aspect, the method can comprise a) administering a composition to suppress PAS domain containing protein kinase (PASK) activity to a sample having myoblasts and PASK, wherein the composition is administered in an amount and for a length of time sufficient to suppress the ability of the PASK to suppress myoblasts cell differentiation into myoctyes; and b) suppressing PASK activity in the sample; thereby increasing the number of myoblasts present in the sample. In an aspect, the PASK inhibitor can be BioE-1197. In an aspect, the sample can be a human sample. In an aspect, the increase in the number of myoblasts can occur in vitro prior to transplantation of the myoblasts into a patient in need of cell therapy. In an aspect, the patient in need of cell therapy can be at risk for developing or has sarcopenia or disease-associated sarcopenia or a stem cell loss disorder.

Disclosed herein are methods of producing a culture of undifferentiated cells. In an aspect, the method can comprise: a) providing a population of preadipocytes having with a PAS domain containing protein kinase (PASK); and b) contacting the population of cells in a) with a PASK inhibitor, wherein the PASK inhibitor suppresses or prevents differentiation of the said cells; thereby producing a culture of undifferentiated cells. In an aspect, the undifferentiated cells can be mammalian cells. In an aspect, the PASK inhibitor can be BioE- 1197.

Disclosed herein are methods of producing a culture of undifferentiated cells. In an aspect, the method can comprise: a) providing a population of neural stem cells having a PAS domain containing protein kinase (PASK); and b) contacting the population of cells in a) with a PASK inhibitor, wherein the PASK inhibitor suppresses or prevents differentiation of the said cells; thereby producing a culture of undifferentiated cells. In an aspect, the undifferentiated cells can be mammalian cells. In an aspect, the PASK inhibitor can be BioE- 1197.

Disclosed herein are methods of producing a culture of undifferentiated cells. In an aspect, the method can comprise: a) providing a population of myoblasts having a PAS domain containing protein kinase (PASK); and b) contacting the population of cells in a) with PASK inhibitor, wherein the PASK inhibitor suppresses or prevents differentiation of the said cells; thereby producing a culture of undifferentiated cells. In an aspect, the undifferentiated cells can be mammalian cells. In an aspect, the PASK inhibitor can be BioE- 1197.

Disclosed herein are methods of inhibiting the expression of a PAS domain containing protein kinase (PASK). In an aspect, the method can comprise contacting a cell expressing a PASK with a short-interfering ribonucleic acid (siRNA) molecule complementary to the nucleic acid sequence capable of encoding the PAS domain containing protein kinase (PASK), thereby inhibiting the expression of PAS K. The cell can be an undifferentiated stem cell. In an aspect, the cell can be a stem cell. The stem cell can a pluripotent stem cell, a mouse embryonic fibroblast cell, a progenitor cell, a C3H10T1/2 mesenchymal stem cell, a mouse C2C 12 muscle progenitor cell, or a primary myoblast cell. In an aspect, the stem cell can comprise a PAS domain. In an aspect, the cell can be a fibroblast, a preadipocyte, neural stem cell or a myoblast.

Disclosed herein are methods of inhibiting expression of a PAS domain containing protein kinase (PASK) in a subject. In an aspect, the method can comprise administering to a subject an effective amount of a pharmaceutical composition comprising a short-interfering ribonucleic acid (siRNA) molecule complementary to the nucleic acid sequence capable of encoding the PAS domain containing protein kinase (PASK), thereby inhibiting the expression of PASK in the subject. The subject can be a human. In an aspect, the subject can be a patient in need of therapy or treatment. In an aspect, the subject can be at risk for developing or has sarcopenia or disease-associated sarcopenia or a stem cell loss disorder.

The disclosed siRNAs, RNA that comprises or forms a double-stranded structure containing a first strand comprising a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second strand comprising a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene, wherein the first and the second ribonucleotide sequences can be separate complementary sequences that hybridize to each other to form said double-stranded structure, can be delivered to cells in a variety of known mechanisms.

In some aspects, siRNA can be delivered using an expression construct that encodes the siRNA. Thus, cells can be transfected with the expression construct to get the siRNA inside the cell.

In some aspects, contacting the cell or providing the cell with an RNA comprises introducing the RNA comprising a double-stranded structure into the cell using a nanoparticle carrier. Other known delivery vehicles can be used. In some aspects, the naked RNA is delivered. For in vivo delivery, the vehicle (RNA and associated delivery agent) can be small. For example, the vehicle can be, but is not limited to, less than 100 nm in diameter, less than 50 nm, less that 20 nm, or less than 10 nm.

Disclosed herein are methods of inhibiting expression of a gene encoding a PAS domain containing protein kinase (PASK). Genome editing can be carried out to inhibit or prevent expression of a gene. Genome editing techniques are well-known in the art including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR associated system (CAS) technology.

In an aspect, the method can comprise introducing into a stem cell containing and expressing the gene encoding the PASK, an engineered, non-naturally occurring

CRISPR-Cas system comprising a Cas protein and one or more guide RNAs that target the gene encoding the PASK, whereby the one or more guide RNAs target the genomic loci of the gene encoding the PASK and the Cas protein cleaves the genomic loci of the gene encoding the PASK, whereby expression of the gene encoding the PASK is inhibited; and, wherein the Cas protein and the guide RNA do not naturally occur together. In an aspect, the stem cell is a mammalian cell. The stem cell can a pluripotent stem cell, a mouse embryonic fibroblast cell, a progenitor cell, a C3H10T1/2

mesenchymal stem cell, a mouse C2C12 muscle progenitor cell, or a primary myoblast cell. In an aspect, the cell can be a fibroblast, a preadipocyte, neural stem cell or a myoblast. In an aspect, the Cas protein is a Cas9 protein. In an aspect, the Cas protein is codon optimized for expression in a eukaryotic cell.

As used herein, "CRISPR system" and "CRISPR-Cas system" refers to transcripts and other elements involved in the expression of or directing the activity of CRISPR- associated ("Cas") genes, including sequences encoding a Cas gene, a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In some aspects, one or more elements of a CRISPR system can be derived from a type I, type II, or type III CRISPR system. In some aspects, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. Generally, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a proto spacer in the context of an endogenous CRISPR system). In an aspect, the guide RNA can comprise a guide sequence fused to a tracr sequence. In an aspect, the cell can be a eukaryotic cell. In an aspect, the cell can be a mammalian cell. In an aspect, the cell can be a mammalian cell. In an aspect, the cell can be a human cell. In an aspect, the Cas protein can comprise one or more nuclear localization signals (NLS). In some aspects, the Cas protein can be a type II CRISPR system enzyme. In some aspects, the Cas protein can be a Cas9 protein. In some aspects, the Cas9 protein can S. pneumoniae, S. pyogenes, or S. thermophilus Cas9. In some aspects, the Cas protein can be a mutated Cas9 derived from any of these organisms. The Cas protein can also be a Cas9 homolog or ortholog. In some aspects, the Cas protein is codon optimized for expression in a eukaryotic cell. In some aspects, the Cas protein directs cleavage of one or two strands at the location of the target sequence.

As used herein, the term "target sequence" is used to refer to a sequence to which a guide sequence can be designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient

complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence can comprise any polynucleotide, such as DNA or RNA polynucleotides. In some aspects, a target sequence can be located in the nucleus or cytoplasm of a cell. In some aspects, the target sequence can be within an organelle of a eukaryotic cell (e.g., mitochondrion). A sequence or template that can be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template" or "editing polynucleotide" or "editing sequence." The target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA). It is believed that the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized the CRISPR complex. The precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). A skilled person will be able to identify further PAM sequences for use with a given CRISPR enzyme. In an aspect, the PAM comprises NGG (where N is any nucleotide, (G)uanine, (G)uanine). In an aspect, the target sequence corresponds to PASK. In an aspect, the expression of the gene product can decreased. In an aspect, CRISPR system can be introduced into a cell by a delivery system including but not limited to viral particles, liposomes, electroporation, microinjection or conjugation.

Disclosed herein are methods of modulating expression of a gene in a cell. In an aspect, the gene can be a gene that can encode a PAS domain containing protein kinase (PASK). The method of modulating expression can be any of the methods described herein (e.g., CRISPR, siRNA).

In some aspects, silencing one or more gene comprise techniques well-known in the art. For example, silencing can be performed by silencing transcription or by silencing translation, both of which result in a suppression of the expression of the gene. Several known techniques can be used for silencing, such as, but not limited to, RNAi, CRISPR, or siRNA. Silencing can comprise administering a silencing agent.

The methods disclosed herein can use a variety of cells. Examples of cells include but are not limited to stem cells, such as embryonic stem cells. METHODS OF TREATING

Disclosed herein are methods of treating a subject or patient. In an aspect, the subject or patient is a human. The method can comprise administering a therapeutically effective amount of the in vitro produced and cells. The in vitro produced cells can be produced by any of the methods disclosed herein. In an aspect, the cells are

undifferentiated stem cells. In an aspect, the cells can be pluripotent stem cells, a progenitor cells, mesenchymal stem cells, embryonic stem cells, primary myoblast cells, fibroblasts, preadipocytes, neural stem cells or a myoblasts.

Disclosed herein are methods of treating a patient in need of cell therapy. In an aspect, the subject can be in need of cell therapy or a cellular transplantation or transfusion. The method can comprise administering a therapeutically effective amount of any of the cells produced and/or disclosed herein to the subject or patient. The method can comprise identifying a patient in need of treatment. The method can comprise administering to the patient a therapeutically effective amount of undifferentiated stem cells. These cells can comprise a PASK that is inhibited or with lower or inhibited expression that were produced or generated via any of the methods disclosed herein.

Therapeutic administration encompasses prophylactic applications. Based on genetic testing and other prognostic methods, a physician in consultation with their patient can choose a prophylactic administration where the patient has a clinically determined predisposition or increased susceptibility (in some cases, a greatly increased susceptibility) to a type of condition disorder or disease.

In some aspects, the patient can be at risk for developing sarcopenia, disease- associated sarcopenia or a stem cell loss disorder or has sarcopenia or disease-associated sarcopenia or a stem cell loss disorder. In some aspects, the subject can be identified using standard clinical tests known to those skilled in the art. Diseases associated or related to sarcopenia include but are not limited to rheumatologic conditions (e.g., rheumatoid arthritis), obesity, Type 2 diabetes and insulin resistance. Stem cells can be used to treat diseases and conditions of the blood and immune systems. Stem cell loss disorder includes but is not limited to leukemias (acute and chronic), lymphomas, myelodyspalstic syndromes, myeloproliferative disorders (e.g., anemias),

lymphoproliferative disorders, phagocyte disorders, metabolic disorders (including inherited metabolic disorders), histiocytic disorders, inherited erythrocyte abnormalities, immune system disorders (including inherited immune system disorders), plasma cell disorders, malignancies, disorders associated with the nervous system (e.g., multiple sclerosis), and disorders associated with the muscular system.

The cells described herein can be administered to the subject (e.g., a human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of clinical disease. Accordingly, in some aspects, the patient is a human patient. In therapeutic applications, compositions are administered to a subject (e.g., a human patient) already with or diagnosed with a condition, disorder or disease in an amount sufficient to at least partially improve a sign or symptom or to inhibit the progression of (and preferably arrest) the symptoms of the condition, its complications, and

consequences. An amount adequate to accomplish this is defined as a "therapeutically effective amount. " A therapeutically effective amount of the cells described herein can be an amount that achieves a cure, but that outcome is only one among several that can be achieved. One or more of the symptoms can be less severe. Recovery can be accelerated in an individual who has been treated.

The therapeutically effective amount of the cells described herein and used in the methods as disclosed herein applied to mammals (e.g., humans) can be determined by one of ordinary skill in the art with consideration of individual differences in age, weight, and other general conditions (as mentioned above).

The cells including undifferentiated cells (e.g., stem cells) as described herein can be prepared for parenteral administration. Cells prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, intravaginal, or rectal), or transdermal (e.g., topical) administration. Aerosol inhalation can also be used to deliver the cells.

PHARMACEUTICAL COMPOSITIONS

As disclosed herein, are pharmaceutical compositions, comprising protein kinase

(PASK) inhibitor, siRNA molecules or CRISPR-cas systems. In some aspects, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants that can be used as media for a pharmaceutically acceptable substance. The pharmaceutically acceptable carriers can be lipid-based or a polymer-based colloid. Examples of colloids include liposomes, hydrogels, microparticles, nanoparticles and micelles. The compositions can be formulated for administration by any of a variety of routes of administration and can include one or more physiologically acceptable excipients, which can vary depending on the route of administration.

As used herein, the term "excipient" means any compound or substance, including those that can also be referred to as "carriers" or "diluents." Preparing pharmaceutical and physiologically acceptable compositions is considered routine in the art, and thus, one of ordinary skill in the art can consult numerous authorities for guidance if needed. The compositions can also include additional agents (e.g., preservatives).

The pharmaceutical compositions as disclosed herein can be prepared for, for example, parenteral administration. Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, intervertebral subcutaneous, or intraperitoneal. Paternal administration can be in the form of a single bolus dose, or may be, for example, by a continuous pump. Topical administration includes ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery. Aerosol inhalation can also be used to deliver any of the compositions described herein. Pulmonary administration includes inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal. In some aspects, the compositions can be prepared for parenteral administration that includes dissolving or suspending the CRISPR-Cas systems, si-RNA molecules, nucleic acids, polypeptide sequences or vectors in an acceptable carrier, including but not limited to an aqueous carrier, such as water, buffered water, saline, buffered saline (e.g., PBS), and the like. One or more of the excipients included can help approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like. Where the compositions include a solid component (as they may for oral administration), one or more of the excipients can act as a binder or filler (e.g., for the formulation of a tablet, a capsule, and the like). Where the compositions are formulated for application to the skin or to a mucosal surface, one or more of the excipients can be a solvent or emulsifier for the formulation of a cream, an ointment, and the like.

The pharmaceutical compositions can be sterile and sterilized by conventional sterilization techniques or sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation, which is encompassed by the present disclosure, can be combined with a sterile aqueous carrier prior to administration. The pH of the pharmaceutical compositions typically will be between 3 and 1 1 (e.g., between about 5 and 9) or between 6 and 8 (e.g., between about 7 and 8). The resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment. The compositions can also be formulated as powders, elixirs, suspensions, emulsions, solutions, syrups, aerosols, lotions, creams, ointments, gels, suppositories, sterile injectable solutions and sterile packaged powders. The active ingredient can be nucleic acids or vectors described herein in combination with one or more

pharmaceutically acceptable carriers. As used herein "pharmaceutically acceptable" means molecules and compositions that do not produce or lead to an untoward reaction (i.e., adverse, negative or allergic reaction) when administered to a subject as intended (i.e., as appropriate).

EXAMPLES

Example 1: Pask is required for terminal differentiation in multiple cell lineages in vitro and muscle regeneration in vivo.

To study the regulation and function of Pask, Pask mRNA abundance was examined in several publicly available gene expression datasets. Elevated Pask mRNA was observed across diverse stem and progenitor cell types compared to differentiated cells and tissues (FIG. 8 A). For example, Pask was more abundant in mouse embryonic stem (ES) cells and progenitor cell types such as C2C12 myoblasts, C3H10T1/2 mesenchymal stem cells, Neuro2a neuroblastoma cells and immune progenitor cells compared to mouse embryonic fibroblasts, other somatic cell types and adult tissues (FIG. 8A) (BioGPS:Pask, GeneAtlas MOE430). Furthermore, an increase in Pask expression during reprogramming of hepatocytes, fibroblasts and melanocytes to induced pluripotent stem cells (iPSCs) was also observed. The increased Pask expression in iPSCs was comparable to the abundance observed in undifferentiated ES cells (FIG. 8B) (Ohi et al., 2011). Conversely, terminal differentiation of human ESCs into cardiac muscle resulted in a progressive decline in Pask expression before ultimately reaching the low abundance found in the adult heart (FIG. 8C) (Cao et al., 2008) showing a positive correlation between Pask expression and sternness.

In examining potential drivers of Pask expression in transcription factor ChlP-Seq databases from mouse ESCs, it was observed that the Pask promoter was occupied by the Oct4 and Nanog pluripotency transcription factors (FIG. 8D) (Marson et al, 2008). The Oct4 and Nanog binding region of the Pask promoter is evolutionarily conserved and is decorated in ESCs with transcriptionally favorable histone H3 lysine 27 acetylation (H3K27ac) and H3 Lysine 4 trimethylation (H3K4me3) modifications and RNA

Polymerase II recruitment, suggestive of transcriptional activation (FIG. 8D) (Karlic et al., 2010; Moorefield, 2013). Silencing of Oct4 or Nanog in ES cells resulted in modest, but statistically significant, suppression of Pask expression (FIG. 8E) (Loh et al, 2006). Thus, gene expression and promoter analysis show robust stem and progenitor cell- specific expression of Pask, possibly driven by the Oct4 and Nanog transcription factors, in embryonic stem cells.

To determine if this high Pask expression in stem cells, including in iPSCs, is indicative of Pask being functionally important for either iPSC generation or

differentiation, the effect of Pask inhibition on iPSC reprogramming from mouse embryonic fibroblasts (MEFs) was assessed. Extensive studies using in vitro, cell and animal models have established the Pask selectivity and lack of toxicity of the BioE-1197 Pask inhibitor, making it a reliable and acute means of suppressing Pask activity in cells (Wu et al, 2014). In the presence or absence of BioE-1197, reprogramming of mouse embryonic fibroblasts (MEFs) expressing IRES-driven Green Fluorescent Protein (GFP) from the endogenous Pou5fl (Oct4) locus (Lengner et al., 2007) was induced, enabling robust GFP expression upon successful reprogramming. As shown in FIG. 9A-B, Pask inhibition had no effect on GFP ⁺ colony formation, indicating that Pask is dispensable for iPSC reprogramming.

Next, the role of Pask for differentiation of pluripotent ES cells into neurons using retinoic acid (Kim et al, 2009) in the presence or absence of BioE-1197 was examined. Differentiation in control cells caused Oct4 expression to be lost and increased expression of the neuron-specific Glycine Receptor Alpha 1 (Glral) (FIG. 9C). Neuronal differentiated was initiated by addition of l ^g/ml of Retinoic Acid (RA) in the presence of DMSO or 25μΜ BioE-1197. Differentiation was determined by qRT-PCR analysis of the indicated genes 8 days after initiation of differentiation. In contrast, each of these signs of terminal differentiation were lost or blunted by treatment with BioE-1197, showing a role of Pask for neuronal differentiation of ES cells.

Progenitor cells such as C3H10T1/2 mesenchymal stem cells and C2C12 myoblasts also robustly express Pask (FIG. 10A). Adipocyte differentiation was induced as described herein. Cells were processed for Oil Red O staining 8 days after

differentiation.C3H10Tl/2 cells differentiate into adipocytes in response to appropriate signaling cues (Pinney and Emerson, 1989; Reznikoff et al, 1973; Zhou et al, 2013). To determine if Pask is required for differentiation of these progenitor cells similar to neuronal differentiation of ES cells, C3H10T1/2 cells were treated with adipogenic stimuli in the presence or absence of BioE-1197. Vehicle-treated control cells efficiently differentiated into mature adipocytes, as evidenced by accumulation of lipid droplets in more than 80% of cells (FIG. 10A, quantified in FIG. 10B). In contrast, Pask inhibition greatly impaired adipocyte differentiation, as about 10% of cells contained observable lipid droplets. Impaired expression of the Adipoq and Fabp4 adipocyte markers (Zhou et al., 2013) (FIG. IOC) in BioE-1197 treated samples was also observed compared with DMSO control. BioE-1197 had no effect on cell doubling time in proliferative

C3H10T1/2 cells (FIG. 10D).

The role for Pask in the differentiation of mouse C2C12 muscle progenitor cells, which differentiate into multi-nucleated muscle fibers in response to horse serum or insulin (FIG. 1A, control) (Yaffe and Saxel, 1977) was evaluated. Control cells were transfected with pooled non-targeting siRNA. DMSO vehicle control (v/v) was used for BioE-1197 samples and it was indistinguishable from siRNA control. 48 h after siRNA or BioE-1197 treatment, differentiation was stimulated using ΙΟηΜ insulin and myotube formation was visualized using anti-MHC (MF20-Red) antibody 3 days later. Pooled siRNA knockdown or inhibition of Pask strongly impaired the formation of multi- nucleated myotubes (FIG. 1A, quantified in FIG. IB). Similarly, CRISPR/Cas9-mediated Pask mutation also resulted in suppression of insulin-induced differentiation as indicated by the absence of multi -nucleated myotubes (FIG. 11 A, quantified in 1 IB) and prevented the induction of MHC protein expression (FIG. 11C). In addition, BioE-1197 treatment also blunted the induction of myosin heavy chain in human primary myoblasts in response to both horse serum and insulin treatment (FIG. 1 ID). Protein extracts were prepared 3 days of differentiation and probed for MHC or β-tubulin. Similar to mouse myoblasts, induction of human MYLPF (myosin light chain) wAACTAl (skeletal muscle actin) mRNAs was also blunted or abrogated by Pask inhibition during differentiation of human myoblasts (FIG. HE).

The defect in myotube formation caused by knockdown of endogenous mouse

Pask was reversed by the expression of siRNA-resistant human wild type (WT) Pask, but not a K1028R kinase-dead (KD) Pask mutant (FIG. 1C, quantified in FIG. ID) (Kikani et al, 2010), indicating the importance of Pask catalytic activity for myoblast fusion.

Importantly, this effect appears to be cell autonomous as a fraction of cells in the population that express WT Pask (WT ⁺ ) show rescue of the fusion defect, whereas cells not expressing hPask within the same culture (WT ^" ) remained mono-nucleated and fusion defective. Additionally, Mylpf (myosin light chain) and Actal mRNAs, which are both markers of myoblast differentiation, failed to be induced in Pask knockdown cells.

Expression of both genes was rescued by expression of WT but not KD human PASK (FIG. IE).

The proliferation and differentiation of primary myoblasts isolated from hindlimb muscles of WT and Pask ^~ mice in the presence or absence of BioE- 1197 were examined. Under proliferative culture conditions, neither genetic deletion nor inhibition of Pask had any effect on proliferation rate (FIG. 1 IF). In response to differentiation cues, myoblasts derived from WT mice efficiently formed multi-nucleated myotubes (FIG. IF, quantified in FIG. 1G). In contrast, genetic elimination of Pask (i.e., myoblasts derived from germline Pask ^~ mice) caused a -60% decrease in differentiation efficiency.

Interestingly, the effect of Pask inhibition in WT cells was much more pronounced, almost completely eliminating myotube formation. BioE- 1197 had no effect, however, on Pask ^'1' myoblasts (FIG. IF, quantified in FIG. 1G). This result demonstrates that the effects of the BioE- 1197 Pask inhibitor are dependent on the presence of Pask, a clear indication that these effects are mediated by Pask inhibition. Moreover, these results demonstrate that myoblasts derived from germline Pask ^~A mice exhibit a Pask- independent compensation that appears, at least in vitro, to partially rescue myotube formation.

The myoblast differentiation process is activated in vivo to repair and replenish the myotome after muscle injury. During muscle regeneration, satellite cells become activated, proliferate and eventually fuse in response to differentiating signals to restore mature myotubes. It was observed that as early as 3 days post injury, Pask mRNA (FIG. 1H) and protein (FIG. II) expression was robustly upregulated. This is consistent with high Pask expression in stem cells compared with differentiated cells (FIG. 8A), since muscle stem cells begin to proliferate upon muscle injury before eventually fusing to replenish the injured myotome. To test the functional relevance of increased Pask expression during muscle regeneration, the kinetics of muscle regeneration in WT and Pask ^'1' mice was compared. As shown in FIG. II, whereas Myh3, a marker of regenerative myogenesis, was robustly induced as early as Day 5 in WT animals, Pask ^'1' animals showed little induction of Myh3 expression. Similarly, WT animals show robust induction of Myh3 immunohistochemical staining, but this was blunted in Pask ^'1' muscle (FIG. 1 J, quantified in FIG. IK). Taken together, these data show that mammalian Pask plays an important role in the differentiation of stem cells across many lineages in vitro and during muscle regeneration in vivo.

These data show that Pask expression is high in stem and progenitor cell types (FIG. 8A-C). In ES cells, the Pask promoter is occupied by the pluripotency promoting transcription factors, Oct4 and Nanog (FIG. 8D). This transcription factor occupancy coincided with Pask transcriptional activation as indicated by the occupancy of RNA PolII as well as the activating H3K4me3 and H3K27ac chromatin modifications. During muscle regeneration in vivo, Pask expression is dramatically induced as early as 3 days after injury (FIG. 1H), coincident with expansion of the satellite cell population and stimulation of My oG expression (Braun and Gautel, 2011 ).

Cell lines and differentiation paradigm. HEK 293T, C2C12 myoblasts and C3H10T1/2 mesenchymal stem cells were obtained from American Type Culture Collection (ATCC). Human skeletal muscle primary myoblasts (Cat# SKB-F) were obtained from Zenbio labs Inc. These cell lines were verified for authenticity by ATCC and Zenbio labs and all cell lines were routinely tested to be free of mycoplasma using PlasmoTest (Invivogen Inc.). HEK293T, C2C12 and C3H10T1/2 cells were maintained in DMEM with 10% FBS (fetal bovine serum) and 1% PS (penicillin and streptomycin) (growth media, GM) at <60% confluency. Human skeletal muscle myoblasts were maintained in proprietary media obtained from Zenbio labs. Mouse embryonic cell culture conditions and differentiation were as described previously (Shaky a et al, 2015). C3H10T1/2 differentiation was as described previously (Villanueva et al., 2011) except that BioE-1197 or DMSO was added 48 h prior to induction of the differentiation regime. C2C12 differentiation was initiated at 95% confluency by switching from GM to DMEM with 2% horse serum and 1%PS (DM) or DMEM + ΙΟηΜ Insulin + 1% PS (Insulin differentiation media). Day 0 time-point samples were collected at 95% confluency before switching to DM. During differentiation, fresh DM was applied every 24 h until the end of the experiment.

iPSC generation. mSTEMCCA construct containing mouse Oct4, Sox2, Klf4 and c-Myc was obtained from R. Mostoslavsky. Lentivirus was produced by co-transfecting 293T cells with l.7μg each of packaging plasmids (pMDLg/pRRE,pRSV-Rev) and 1.7 μg envelope plasmid (pVSVG) and 5μg mSTEMCCA. 1X105 Oct4-GFP MEFs generated from Pou5fltm2Jae/J (Jackson Lab) were plated on the feeders in 6-well plates and were infected with lentivirus for 48 hours in the presence of 4μg/ml polybrene (Sigma). The ESC medium with or without 50μΜ BioE-1197 was changed every day. From day 8, GFP colonies were counted and images were captured with an Olympus 1X51 inverted microscope.

Plasmids and retroviral infection. Human WT and K1028R (KD) (Kikani et al,

2010) versions of Pask were cloned into the pQCXIP vector with N-terminal Flag tags. pCDNA FlagWdr5 (Addgene Cat#15552) and pGEX-5Xl-Flag-Wdr5 (Addgene

Cat#15553), pCDNA3 Flag Rbbp5 (Addgene Cat# 15550) and pCDNA3-Flag-PTIP (Addgene Cat # 15557) were deposited by Dr. Kai Ge (Cho et al, 2007). S49A and S49E mutations in pCDNA3 Flag-Wdr5 were made using sewing PCR-based mutagenesis in these vectors. For retroviral expression, WT, S49A and S49E Wdr5 were subcloned into pQCXIP vector. pQCXIP/GFP was described previously (Chen et al, 2014). pCL-Flag- PCAF (KAT2B, Addgene Cat#8941) was deposited by Dr. Yoshihiro Nakatani (Yang et al., 1996). pCDNA-Set9 (Addgene Cat#24084) was deposited by Dr. Danny Reinberg. pCDNA-Flag-Menin (Addgene Cat# 32079) was deposited by Dr. Matthew Meyerson. pCL-Babe-MyoD (Addgene Cat# 20917) was deposited by Dr. Stephen Tapscott (Yang et al., 2009). Rat MyoG was cloned into the pQCXIP vector after PCR from a rat cDNA library. Retroviral production for infection was as described (Kikani et al, 2012).

Satellite cell isolation. Satellite cells were isolated from 10-12 weeks old WT and Pask-/- littermates according to published protocol (Danoviz and Yablonka-Reuveni,

2012). Briefly TA muscles from hind limbs of WT or Pask-/- mice were isolated, minced in DMEM and enzymatically digested with 0.1% Pronase for lhr. After repeated tituration, the cell suspension was filtered through 40uM filter. Cells were plated on matrigel precoated plates and allowed to grow for four days. The differentiation of these satellite cells derived myoblasts was stimulated by addition of ΙΟΟηΜ insulin in serum free DMEM.

Pask siRNA knockdown and inhibition. siRNA-mediated gene knockdown was performed by transfecting 50nM of pooled siRNA against control (Cat #ID-001810-10- 05), mouse Pask (L-065533-00-0005) or mouse Myodl (L-041113-00-0005) purchased from Dharmacon using Lipofectamine RNAimax (Life Technologies). Knockdown was carried out with the cells at 40% confluence in suspension. Cells were allowed to attach and grow for 48 h when they reached 95% confluence, which marked the Day 0 point. Cells were either harvested at this point or differentiation was initiated by switching from GM to DM. For Pask inhibition by BioE-1197, C2C12 or human primary skeletal muscle myoblasts were seeded at 40% density in the presence of 25 μΜ BioE-1 197 or equal volume of DMSO. Cells were allowed to grow for 48 h at which point they reached 95% confluence and differentiation was initiated by switching from GM to DM in presence of 25μΜ BioE-1197 or DMSO.

Quantitative reverse transcription PCR. Total RNA was prepared from C2C12 or human myoblasts subjected to differentiation conditions as described. qRT-PCR was performed from cDNA prepared from RNA using mRNA target-specific primers. Three independent experiments, done in triplicate with identical experimental parameters, were used for statistical analysis of mRNA transcript abundance. Student t-test was used for statistical significance with significance value set to P<0.05. Sequences for qRT-PCR primers used in this study are listed in FIG. 19.

Protein extracts, co-immunoprecipitation and western blot analysis . For quantitation of protein abundance during differentiation, cells were lysed in RIP A buffer, cellular debris was eliminated by centrifugation, and lysates were separated by SDS- PAGE. For co-immunoprecipitation, cells were lysed in native lysis buffer described previously (Kikani et al, 2010). Immunoprecipitation was performed using the designated antibodies bound to Protein G beads (Pierce Cat# 22852). Protein complexes were washed with lysis buffer 5 times, denatured and separated by SDS-PAGE. FIG. 19 provides a list of antibodies used and their resource ID.

Muscle injury. Injury and subsequent analysis of skeletal muscle regeneration was performed using previously established methods (Murphy et al., 2011).

Chromatin Immunoprecipitation. Chromatin Immunoprecipitation (ChIP) from C2C 12 cells was performed according to (Hollenhorst et al, 2007) with the following modifications. 1X107 C2C12 cells/15cm plate were treated for differentiation according to procedures described above. At the appropriate time-point, cells were washed twice with PBS and cross-linked with 1% formaldehyde in PBS for 10 min at room

temperature. The cross-linking was quenched by the addition of glycine to a 125mM final concentration. Nuclear pellets were sonicated using a Branson 450 Sonifier to prepare sheared chromatin extracts. Appropriate ChIP grade antibodies, as indicated in resource ID, were incubated with Dynabeads (Life technologies Cat # 1 1202D - anti- Mouse or 11204 - anti -Rabbit), to which sheared chromatin was added for

immunoprecipitation. Crosslinks were reversed and DNA was purified using the Qiagen PCR purification kit. A sample with no immunoprecipitation step was processed in parallel as the input sample. qRT-PCR analysis of ChIP DNA was performed according to (Hollenhorst et al, 2007). Fold enrichment is calculated as the ratio of signal of the target region over the signal of a negative control genomic region (~20,000bp upstream of the actb gene).

Statistical analysis. Data are presented as mean ± standard deviation (SD).

Student's t test with -tailed equal variance with paired type analysis was used for calculating statistical significance between control and test sample. P<0.05 is accepted as significant difference between control vs test sample. Experiments were performed in independent sets of triplicates.

Example 2: Pask and Myogenin establish a positive transcriptional feedback loop that enforces myocyte differentiation.

Following injury, satellite cells execute a transcriptional program that culminates in the formation of syncytial myocytes that constitute a healthy muscle fiber (FIG. 2A) (Bentzinger et al, 2012). Quiescent Pax7+ satellite cells are induced to proliferate and initiate expression of MyoD and/or Myf5. In MyoD+ myoblasts, MyoD drives the expression of MyoG in response to differentiation cues. MyoG then executes the terminal differentiation program by down-regulating Pax7 expression (Olguin and Olwin, 2004) and activating the genes necessary for myoblast fusion and muscle function, including myosin heavy chain (My Iff) and muscle specific actin (Actal).

To identify the underlying mechanism for the defect in myoblast differentiation caused by loss of Pask activity, the expression of these key transcription factors and their targets during the differentiation of WT and Pask-/- satellite cells were compared. As expected, the abundance of the Pax7 and Myf5 mRNAs declined at day 1 of

differentiation in WT cells (FIG. 2B) (Bentzinger et al, 2012). In conjunction, Myog mRNA abundance increased at Day 1, as did expression of its target genes Mylpf and Actal (FIG. 2B). Pask-/- cells exhibited, even at Day 0, a more satellite cell-like gene expression profile, with Pax7 and Myf5 being elevated relative to WT and Myog and its targets Mylpf and Actal having decreased expression. At Day 1 of differentiation, these patterns were maintained, with Pax7 and Myf5 mRNAs remaining elevated and Myog, Mylpf and Actal remaining decreased (FIG. 2B). The expression of Myod, on the other hand, was not affected by the loss of Pask. the protein levels of Pask, Pax7 and MyoG were also compared during in vivo muscle regeneration between WT and Pask-/- animals (FIG. 2C). Consistent with the mRNA expression data from satellite cells, Pax7 protein was aberrantly elevated in Pask-/- animals compared with WT animals at Day 5 post- injury. Pask-/- animals also showed a profound defect in the induction of MyoG and its target Myh3 (FIG. 2C).

To determine if these effects of genetic loss of Pask can be recapitulated by acute means of controlling Pask expression or activity in C2C12 myoblasts, mRNA or protein levels of Pax7, MyoG and MHC after Pask knockdown or inhibition were examined (FIG. 12A-B). At Day 1 of differentiation, when Pask silencing was most effective, Pax7 abundance remained elevated and the induction of early differentiation marker MyoG and its target MHC was delayed in Pask-silenced C2C12 cells (FIG. 12A). Pask inhibition by BioE-1197 recapitulated these effects at the mRNA level during insulin-induced C2C12 differentiation (FIG. 12B). These transcriptional defects in myogenesis caused by Pask knockdown were rescued by ectopic expression of human WT but not kinase dead (KD) Pask (FIG. 12C). Three days after induction of differentiation, mRNA abundance of Pax7, Myog and Actal was assessed by qRT-PCR. Taken together, these findings show that Pask is required to initiate the commitment phase of myoblast differentiation and maintain the myogenic transcriptional program.

To properly place Pask in the myogenic transcription factor cascade during differentiation (FIG. 2A), co-immunostaining experiments were carried out to profile the expression of endogenous Pask with Pax7, MyoD or MyoG in proliferating (Day 0) and differentiating (Day 1 and 3) myoblasts in control, BioE-1197 treated or Pask knockdown conditions. At Day 0, -40% of control cells were Pax7+ and this fraction declined over the course of differentiation (FIG. 2D, quantified in 2E). When Pask was inhibited or knocked down, the number of Pax7+ cells was elevated at Day 0 and during

differentiation, matching the mRNA data (Figure 2B, FIG. 12A-B). In contrast, Pask knockdown or inhibition had no effect on number or subcellular localization of MyoD at any point during differentiation, consistent with FIG. 2B (FIG. 13 A, quantified in FIG. 13B). The effects of loss of Pask activity were most evident in the appearance of MyoG+ cells during differentiation. In control cells, the fraction of MyoG+ cells dramatically increased from Day 0 (-7%) to Day 1 (-60%) and Day 2 (-80%) (FIG. 2F, quantified in 2G). MyoG+ cell numbers were substantially reduced by either Pask inhibition or knockdown. A striking correlation of Pask and MyoG expression was also observed. At Day 1, a strong co-expression between Pask and MyoG was noted, wherein -50% of

Pask+ cells were MyoG+ whereas -15% of Pask- cells were MyoG+ (FIG. 2F, quantified in 2H). As expected, inhibition of Pask function with BioE-1197 eliminated this correlation, with both populations losing MyoG staining. Importantly, in the Pask knockdown samples, MyoG expression was present almost exclusively in those cells in the population that escaped Pask silencing (FIG. 2F, quantified in 2H).

Both the increase in Pax7+ cells and the decrease in MyoG+ cells observed upon Pask knockdown were completely rescued by expression of WT hPask (FIG. 21, quantified in 2J). Surprisingly, transient over-expression of human Pask, even in the absence of extrinsic differentiation cues, was sufficient to induce formation of multi- nucleated myocytes (FIG. 2K) and to induce the entire differentiation transcriptional program in growth medium (GM) (FIG. 2L). Cells were allowed to grow in growth media for 72 h and imaged for GFP. This included suppression of Pax7 as well as induction of Myod, Myog and Mylpf. Addition of differentiation medium (DM) further enhanced myoblast differentiation in hPask-expressing cells, as evidenced by the elevated expression of Myog and Mylpf (FIG. 2L). Together, these data establish Pask as necessary for the timely execution of the terminal differentiation program in myoblasts and as sufficient to initiate that program even in the absence of extrinsic differentiation signals.

The robust cell autonomous correlation between Pask and MyoG expression is probably a reflection of the necessity of Pask for MyoG expression, but it also prompted the exploration of the possibility that MyoG might also regulate Pask expression during differentiation. Interestingly, previous ChlP-Seq data had suggested that MyoG, and to a lesser extent MyoD, occupies the Pask promoter during C2C12 differentiation coincident with H3K4me3 modification and RNA-PolII recruitment (FIG. 14A) (Yue et al, 2014). It was independently validated herein that MyoG was indeed recruited to the proximal Pask promoter upon initiation of differentiation (FIG. 14B) and that MyoG expression stimulates expression from a Pask promoter-driven reporter gene (FIG. 14C). This construct was co-expressed with GFP, MyoG or MyoD in HEK293T cells. Luciferase activity, indicative of activation of the Pask promoter, was measured using a luminometer and normalized to CMV-driven Renilla. These data show the possibility that Pask expression in differentiating myoblasts might be driven by MyoG to further support commitment to differentiate. Thus, taken together our data shows that Pask and MyoG engage in a positive, self-reinforcing feedback loop to enforce the terminal differentiation program once it has been signaled to initiate.

These data show that MyoG and MyoD bind the Pask promoter (FIG. 14A-B), which is marked by broad domains of H3K4me3 occupancy similar to those found in other lineage-specifying genes (Benayoun et al, 2014). Terminal differentiation of ES cells and C2C12 myoblasts result in a decline in Pask expression, which ultimately reached a low steady-state abundance when differentiation is complete. This pattern is consistent with the expression of other key regulators of terminal differentiation like MyoD and hints at the importance of Pask in lineage commitment process.

Methods and materials used are described herein and above.

Example 3: Pask collaborates with MyoD to drive MyoG expression and myogenesis.

The gene expression data presented above show the possibility that either failure to suppress Pax7 expression or failure to induce MyoG expression underlies the differentiation defect caused by the absence of active Pask. Because Pask protein expression correlated most strongly with that of MyoG and MyoG is known to repress Pax7 expression (Olguin et al, 2007), it was hypothesized that Pask collaborates with MyoD to induce MyoG expression. MyoD is a pioneering transcription factor that is sufficient to induce a myogenic cell fate even in non-muscle progenitor cells.

C3H10T1/2 mesenchymal stem cells normally express the PPARy2 adipogenic transcription factor and efficiently differentiate into adipocytes in response to adipogenic differentiation cues (FIG. 3 A) (Zhao et al, 2013). The myogenic transcriptional program is epigenetically silenced in C3H10T1/2 cells, but it can be activated in response to MyoD expression (Penn et al., 2004; Tapscott et al., 1988). MyoD stimulates MyoG expression, which then collaborates with MyoD to establish myogenic commitment and repress Ppary2 expression (FIG. 3A). Using this experimental paradigm, it was evaluated as to whether Pask is necessary for MyoD-dependent MyoG expression and myogenesis in C3H10T1/2 cells. As shown in FIG. 3B, MyoD-induced expression of Myog and Mylpf was impaired by Pask inhibition while MyoD expression was unaffected. Moreover, Ppary2 expression, which was lost in control cells due to myogenic lineage conversion, was maintained in cells treated with BioE-1197 (FIG. 3B). In addition, as assessed by immunofluorescence microscopy, the fraction of MyoG+ cells at either Day 1 or Day 2 of differentiation was markedly decreased by Pask inhibition, while MyoD positivity was unaffected (FIG. 3C, quantified in 3D; FIG. 15A-B). In addition to the loss of MyoG expression, cells treated with BioE-1197 exhibited a profound failure in the formation of multi -nucleated myotubes (FIG. 3E). Based on these results, it was hypothesized that the differentiation failure of Pask-inhibited cells was not due to failed inactivation of Pax7, which is not expressed in C3H10T1/2 cells, but instead is due to an inability of MyoD to induce MyoG expression in the absence of Pask. Indeed, it was found that ectopic expression of rat MyoG was sufficient to bypass the Pask requirement for Mylpf expression even though endogenous mouse Myog remained suppressed in BioE-1197 treated cells (FIG. 3F). These data place Pask within the myogenic transcriptional cascade, with Pask being necessary to act in concert with MyoD to induce MyoG expression and myogenic differentiation.

Immunofluorescence microscopy. C2C12 cells growing on coverslips were fixed with 4% Paraformaldehyde and permeabilized with 0.2% Triton-X 100. Following lh of blocking with 10% normal goat serum, the indicated primary antibodies were added for overnight incubation at 4 °C. Following three washes with ice cold PBS, cells were incubated with anti-mouse Alexa fluor 568 (for Pax7, MyoD, MyoG and MHC) or anti- rabbit Alexa fluor 488 (Flag or Pask) secondary antibodies for 1 hour in dark at room temperature. The coverslips were mounted using Prolong- Anti-fade mounting media containing DAPI. Fusion index was used as a measure of differentiation and was calculated as the percent of nuclei in MHC+ cells in relation with total nuclei. For quantification of microscopic images, at least 100 cells were counted from three separate experiments in a sample-blinded manner. Statistical significance was calculated using Student's t-test with P<0.05 set as the significance level.

Methods and materials used are described herein and above. Example 4: Pask phosphorylates Wdr5.

Catalytically inactive Pask was unable to rescue the differentiation defect caused by silencing of endogenous Pask (see FIG. 1C-E) and a catalytic inhibitor mimicked genetic inactivation of Pask (FIGs. 1A, 2F-G). It was concluded, therefore, that Pask- dependent phosphorylation of one or more specific substrates must be required for myogenic gene expression and differentiation. Since none of the known Pask substrates are likely to serve this role, experiments were carried out to identify new substrates that might mediate the effects of Pask on differentiation. A strategy was employed that combines experimental and bioinformatics approaches. Specifically, all proteins that had been described to physically interact with Pask in high-throughput protein-protein interactome datasets (BIOGRID: Pask) and were identified in unpublished Pask interactome studies were identified. Next, this dataset was filtered for proteins that contain the consensus phosphorylation motif for Pask ([HKR]-X-[KR]-X-X-[ST]) that was previously identified (Kikani et al, 2010). Wdr5 was the single candidate substrate protein that emerged, based on its interaction with Pask in a dataset and in a global affinity capture screen (Ewing et al, 2007).

First, validation of the physical interaction of Pask and Wdr5 was sought and experiments were carried out in C2C12 cells across a differentiation time-course. As shown in FIG. 4A, Wdr5 associated with Pask during the proliferative phase (-1 and 0 days). However, that interaction was significantly enhanced following the onset of differentiation (Day 1) (FIG. 4A). It was also found that co-expressed epitope-tagged forms of Pask and Wdr5 co-immunoprecipitated and that this did not require Pask catalytic activity as the K1028R Pask mutant (KD) precipitated Wdr5 equivalently to WT Pask (FIG. 4B). Immunoprecipitates were analyzed by western blot for Pask and Wdr5, indicating an enrichment of co-immunoprecipitation at Day 1 of differentiation. V5 or Flag-tagged proteins were immunoprecipitated and examined by western blot using anti- Flag or V5 antibody.

Wdr5 is a member of several protein complexes that catalyze histone methylation or acetylation (Migliori et al, 2012; Shilatifard, 2012; Trievel and Shilatifard, 2009). To properly place the Pask-Wdr5 interaction within these complexes and to determine if Pask associates with any intact Wdr5 -containing complexes, exclusive members of each of the major Wdr5- containing complexes were expressed. This included Menin (Mlll/2 complex), PTIP (M113/4 complex), GCN5 (Kat2A Histone Acetyltransferase (HAT) complex) and Set9 (Setd7a - H3K4me3 complex) as well as Rbbp5, which is a member of the core Wdr5 sub-complex that is common to all Mil and Set complexes. Among these, Wdr5 was able to co-purify endogenous Pask (FIG. 16A), showing that Pask is not a stable member of intact Wdr5 -containing complexes, but might associate selectively with free Wdr5. Supporting this notion, the interaction between Pask and Wdr5 appears to be direct as GST-tagged Wdr5, purified from bacteria, bound to Pask purified from insect cells (FIG. 16B). Flag tagged proteins were immunoprecipitated using anti-Flag antibody. Co-immunoprecipitation of Pask was detected via western blotting of anti-Flag immunoprecipitates (FIG. 16A). Bound protein was detected by Coomassie staining or anti-Pask immunoblot (FIG. 16B). Furthermore, while overexpressed Pask was cytoplasmic (typically perinuclear) in the majority of cells (Rutter et al., 2001), with -10% of cells exhibiting nuclear Pask in absence of Wdr5 co-expression, Wdr5 co- expression with Pask significantly enhanced the nuclear accumulation of Pask (FIG. 4C). V5 and Flag were stained using Alexa Flour 568 or Alexa Flour 488, respectively. The fraction of cells with nuclear Pask localization was scored as a function of the presence (+) or absence (-) of Wdr5. To define the minimal region of Pask that is responsible for Wdr5 interaction, a series of N- and C-terminal Pask truncation mutations were generated and assessed for their ability to co-purify Wdr5. It was found that the N-terminal 914 residues of Pask were dispensable for Wdr5 binding (FIG. 16C). V5-tagged versions of various Pask truncation mutants were co-expressed with Flag-Wdr5 in HEK293T cells. Wdr5 was detected using anti-Flag antibody after V5 immunoprecipitation. The interaction was lost, however, upon deletion of residues 915-948, showing that Wdr5 likely interacts with Pask just upstream of the canonical protein kinase domain.

Wdr5 was phosphorylated efficiently by WT Pask, but not KD Pask, in in vitro kinase reactions (FIG. 4D) and this was abrogated by the BioE-1197 Pask inhibitor (FIG. 16D). BioE-1197 also robustly blunted the in situ phosphorylation of Pask and Pask- bound Wdr5 using in-cell 32P labeling (FIG. 4E). Immunoprecipitates were analyzed by SDS-PAGE and autoradiography or western blot. As Pask association with Wdr5 was enhanced at the onset of differentiation, the next set of experiments were carried out to determine if Pask activity towards Wdr5 is stimulated at this time. Consistent with a previous report (Kikani et al., 2010), Pask exhibits modest activity in the absence of stimulation in C2C12 myoblasts (FIG. 4F). At 12 h post-addition of insulin containing differentiation media, however, Pask was activated as assessed by increased

autophosphorylation and Wdr5 phosphorylation, thus showing that Pask kinase activity is stimulated during differentiation.

Wdr5 was selected based on it containing a sequence that matched the Pask consensus substrate motif (Kikani et al., 2010). That site, with Serine 49 as the putative phospho-acceptor residue, is strikingly similar to that of the best-characterized substrate of yeast Pask, Ugpl (FIG. 4G), which is also robustly phosphorylated by human Pask (Kikani et al., 2010; Rutter et al, 2002). In particular, the -5His and -3Lys residues, which were shown to be the most important for determining Pask phosphorylation (Kikani et al., 2010), are present in this putative Wdr5 phosphorylation motif. As expected, it was found that Ser49 is required for phosphorylation by Pask since mutation to either glutamate (Glu) or alanine (Ala) nearly abolished phosphorylation in vitro (FIG. 4H). Pask-associated WT Wdr5 was phosphorylated in cells as shown above, but the

S49A mutant showed a marked reduction in 32P incorporation (FIG. 41). Together, these results show that Pask interacts with Wdr5 and phosphorylates it at Ser49 both in vitro and in cells.

Finally, the phosphorylation status was determined to assess whether Ser49 affects Wdr5 association with either Pask or members of its histone modifying complexes using unphosphorylatable S49A or phospho-mimetic S49E variants of Wdr5. Pask purified from insect cells bound to bacterial-expressed Wdr5S49E more weakly than WT, whereas Wdr5S49A bound Pask more avidly (FIG. 16E). A very similar pattern was observed in co-immunoprecipitation of endogenous Pask with Wdr5WT, WDRS49A and Wdr5S49E from cells (FIG. 16F). Endogenous interaction of various members of protein complexes with WT or mutant Wdr5 were determined by Western blotting using the indicated antibodies. It was found that the association with endogenous Pask is weakened with Wdr5S49E and strengthened with Wdr5S49A compared with Wdr5WT. These data are consistent with the common observation that an unphosphorylated substrate (mimicked by the Ala mutant) engages with its kinase with higher affinity compared to its phosphorylated forms (mimicked by the Glu mutant). When examining components of the Mil 1-4, SET1 and KAT2a complexes, it was found that Wdr5WT, WDRS49A and Wdr5S49E all bound to each member of these complexes equivalently, showing that Wdr5 phosphorylation by Pask does not alter recruitment of Wdr5 into its many chromatin-modifying complexes.

Pask and Wdr5 phosphorylation promote terminal differentiation. Pharmacologic inhibition of Pask activity caused a loss of terminal differentiation in three differentiation paradigms: ES cells to a neuronal fate, C3H10T1/2 mesenchymal stem cells to adipocytes and C2C12 myoblasts to myotubes. Utilizing the myoblast differentiation system, Wdr5 was identified and described herein as a Pask substrate that mediates these differentiation effects. Wdr5 has been previously implicated in controlling stem cell maintenance. For example, Oct4 associates with Wdr5 and recruits H3K4me3 complexes to its target promoters to induce transcriptional activation and maintenance of pluripotency (Ang et al., 2011). In C2C12 myoblasts, Wdr5 was shown to interact with Pax7 to enforce myogenic specification (Kawabe et al, 2012; McKinnell et al, 2008; Rudnicki et al, 2008). These results provide a new regulatory role for Wdr5 in promoting differentiation via the expression of key target genes. More importantly, these results define a novel regulatory paradigm wherein the function of Wdr5 is modulated by Pask-dependent phosphorylation. While it is likely, it, however, remains to be established whether Wdr5 phosphorylation is a conserved and required mechanism whereby Pask promotes the differentiation of other stem and progenitor cells.

Pask as a signaling intermediate in cell differentiation. Differentiation cues engage and activate the signaling pathways and transcriptional networks that combine to drive terminal differentiation (Basson, 2012). While a pioneering transcription factor like MyoD can occupy its target promoters in a sequence-specific manner, the full activation of the MyoD transcriptional response depends upon signaling from differentiation cues (Berkes and Tapscott, 2005). These cues, including those used in vitro treatment with insulin, are thought to enhance MyoD binding to its target promoters and recruit histone modifying proteins to initiate the expression of differentiation genes (Berkes and Tapscott, 2005; Blum and Dynlacht, 2013; Braun and Gautel, 2011 ; Dilworth and Blais, 2011). Ectopic expression of Pask or phospho-mimetic Wdr5 was sufficient to induce all of these effects in the absence of differentiation cues (FIG. 2K-J, FIG. 5H). These data raise the possibility that Pask might be an important node in the signaling network connecting differentiation cues with myogenic gene expression. Consistent with that hypothesis, these data show that Pask activity was stimulated by differentiation cues as was the Pask-Wdr5 interaction (FIG. 4F). Thus, these results show that differentiation cues act, at least in part, to drive the myogenic transcriptional program via Pask activation and phosphorylation of Wdr5. It remains unknown how Pask activity and the Pask-Wdr5 interaction are stimulated by differentiation cues, but these data show that these phenomena are likely central components of this signaling pathway.

In vitro and in situ phosphorylation of Wdr5. For in vitro phosphorylation of Wdr5, His- or GST-tagged Wdr5 proteins purified from E. coli were incubated with WT or KD Pask in the presence or absence of BioE-1197 in kinase reaction buffer containing [γ-32Ρ]ΑΤΡ (Kikani et al., 2010). Kinase reactions were terminated after 10 min by addition of SDS buffer. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose and exposed to autoradiographic film. For in situ phosphorylation of Wdr5, metabolic 32P labeling was performed as described (Kikani et al, 2010) from cells co- expressing WT Pask with WT or S49A Wdr5. Incorporation of phosphate into Wdr5 was detected by purifying the Pask-Wdr5 complex from cells and determining

phosphorylation by autoradiography.

Methods and materials used are described herein and above.

Example 5: Wdr5S49E restores Myog expression and myogenesis in the absence of Pask activity.

Having established Wdr5 as a Pask substrate and given the published role of Wdr5 -containing complexes in myoblast differentiation via the regulation of myogenic gene expression (Asp et al, 2011 ; McKinnell et al, 2008; Segales et al, 2014), the next set of experiments were carried out to determine whether Wdr5 phosphorylation is the mechanism through which Pask regulates Myog expression and myocyte differentiation. If so, one might expect that expression of a Wdr5 variant that constitutively mimicked the phosphorylated form would reverse the effects of Pask inactivation and rescue the differentiation defect. Indeed, it was found that expression of Wdr5S49E, but not Wdr5WT or Wdr5S49A, rescued the morphological features of C2C12 differentiation, including multinucleated myofibers (FIG. 17). C2C12 myoblasts were transduced with retrovirus carrying WT, S49A or S49E mutants of Wdr5 and were selected with 3μg/ml puromycin for 48hrs. Pask (or control) was knocked down at 70% cell density in these populations by pooled Pask siRNA. 24hrs after siRNA treatment, differentiation was initiated with 2% horse serum-containing media. Next, the ability of Wdr5WT,

Wdr5S49A and Wdr5S49E to rescue the expression of MyoG and its targets upon Pask knockdown was examined. Wdr5WT and Wdr5S49A had minimal effect on Pax7, Myog, Mylpf, or Actal mRNA abundance in Pask-siRNA cells (FIG. 5A). qRT-PCR analysis was performed for the indicated mRNA on day 3 of differentiation. 18S rRNA was used as normalizer. On the other hand, the Wdr5S49E mutant either partially or completely rescued the defects in expression of each of these genes caused by Pask knockdown. Wdr5S49E caused a modest increase in Pask expression.

Using microscopy of co-immunostained cells, the ability of GFP, Wdr5WT,

Wdr5S49A or Wdr5 S49E to rescue the defect in progression from the Pax7+ to MyoG+ state in Pask-siRNA cells was examined. GFP, Wdr5WT, and Wdr5S49A all had no significant effect on the number of Pax7+ cells (FIG. 5B; quantified in 5C). Wdr5 S49E, on the other hand, completely reversed the aberrant increase in Pax7+ cells caused by Pask knockdown. Importantly, this decrease in Pax7 positivity was present in those cells in the population that expressed Wdr5S49E (S49E+; FIG. 5B, indicated by arrows), and not in those cells in the population that were uninfected (S49E-). After 24 h, cells were stained for Pax7 at Day 0 of differentiation. This demonstrates that these are cell autonomous effects that are directly and specifically related to expression of the phospho- mimetic Wdr5 mutant. Those cells expressing Wdr5S49E also exhibited an almost complete rescue of MyoG expression, while those expressing GFP and Wdr5S49A were unaffected (FIG. 5D; quantified in 5E). In this case, Wdr5WT also caused a significant increase in the fraction of MyoG+ cells. As before, MyoG expression was unaffected in those cells in the Wdr5WT and Wdr5S49E-expressing populations that were uninfected. The defect in MHC expression and myotube formation caused by Pask knockdown was also robustly rescued in Wdr5S49E-expressing cells and weakly rescued in Wdr5WT- expressing cells (FIG. 5F, G).

Furthermore, it was noticed that ectopic expression of Wdr5S49E, like WT Pask, was sufficient to induce expression of MyoG even in the absence of differentiation stimuli (FIG. 5H). Expression of kinase-dead (KD) Pask, Wdr5WT or Wdr5S49A failed to induce MyoG expression (FIG. 5H) in the same growth media condition. Cells were lysed after selection and abundance of the indicated proteins was determined by Western blotting. Taken together, these data show that Wdr5 phosphorylation is a major mechanism whereby Pask promotes MyoG expression and myotube formation.

Methods and materials used are described herein and above. Example 6: Pask phosphorylation of Wdr5 promotes transcriptional derepression of the Myog promoter via H3K4mel to H3K4me3 conversion and MyoD recruitment.

At the onset of differentiation, the Myog promoter is remodeled with H3K4me3 modification resulting in its transcriptional activation (Asp et al, 2011; Cheng et al., 2014). Because transcriptional induction of Myog is dependent on Pask kinase activity or phospho-mimetic Wdr5S49E, experiments were carried out to test whether Pask might act on the Myog promoter through regulating H3K4me3 accumulation. The Myog locus contains a region of H3K4me3 approximately -150bp upstream of the transcriptional start site (TSS), which overlaps with the peak of MyoD binding marked by E-Box sequences (FIG. 6A). In control cells, H3K4me3 abundance at two sites within this region of the promoter (Myog_b and Myog_c) was strongly increased on Day 1 of differentiation, while a region more distal to the TSS showed little H3K4me3 accumulation (Myog a, FIG. 6B). Because the b amplicon showed the most significant enrichment of H3K4me3 in control samples, it was selected for future studies. This increase in H3K4me3 was markedly blunted (Myog b) or abolished (Myog c) by Pask knockdown or inhibition (FIG. 6B). In contrast, Pask knockdown had no effect on H3K4me3 abundance on the Myod promoter (FIG. 6B).

To assess whether these Pask knockdown phenotypes were related to Wdr5 phosphorylation at Ser49, the ability of WT or kinase dead (KD) hPask as well as WT, S49A or S49E variants of Wdr5 to rescue these effects was tested. As previously observed in microscopy experiments, expression of WT, but not KD, hPask completely rescued H3K4me3 occupancy of the Myog promoter (FIG. 6C). Moreover, expression of Wdr5S49E restored H3K4me3 occupancy, whereas Wdr5S49A and Wdr5WT had no or modest effects, respectively (FIG. 6C). One potential mechanism whereby Wdr5 phosphorylation might stimulate H3K4me3 occupancy of the Myog promoter is through enhanced Wdr5 promoter recruitment. Flag-Wdr5 ChlP-qPCR analysis was performed on these same samples to determine Flag-Wdr5 occupancy on the Myog promoter. It was found that Wdr5WT modestly occupied the Myog promoter and this was decreased for the S49A mutant and increased for the S49E mutant (FIG. 6D). Neither WT nor KD Pask occupied the Myog promoter (FIG. 6D), consistent with our observation that Pask is not a stable member of any Wdr5 -containing complexes. The Pask-dependence of endogenous Wdr5 occupancy of the Myog promoter was also examined and found that the increased binding of Wdr5 to the Myog promoter in response to differentiation cues observed in control cells was blunted in Pask-siRNA cells (FIG. 6E). Wdr5 binding to the Myod promoter, on the other hand, was neither significantly induced upon differentiation, nor was it affected by Pask knockdown (FIG. 6E), consistent with the observation that Pask is not required for expression of MyoD (FIG. 2B).

These data show that Wdr5 phosphorylation affects H3K4 trimethylation activity at the Myog promoter. MyoD is recruited to the Myog promoter during differentiation and then in turn orchestrates the chromatin remodeling process to promote transcriptional activation of Myog (Palacios and Puri, 2006; Rampalli et al, 2007; Saccone and Puri, 2010). whether Pask plays a role in this process was examined by assessing whether MyoD occupancy on the Myog promoter during differentiation was affected by Pask knockdown. Indeed, it was found that Pask knockdown completely blocked the differentiation-stimulated increase in MyoD occupancy of the Myog promoter (FIG. 6F). This requirement for Pask is due to Wdr5 phosphorylation as expression of Wdr5S49E increased MyoD occupancy of the Myog promoter at Day 1 of differentiation, whereas Wdr5S49A blocked it (FIG. 6G). Interestingly, Wdr5S49E increased the MyoD occupancy of the Myog promoter even in the absence of differentiation cues (Day 0) to a level similar to that found in control cells at Day 1 of differentiation (FIG. 6G). This result is consistent with the observation that Wdr5S49E expression is sufficient to induce MyoG expression in the absence of differentiation cues (FIG. 5H). Thus, it appears that Pask and/or phospho-Wdr5 are necessary and sufficient to stimulate the myogenic transcriptional cascade, and perhaps that MyoD occupancy and H3K4me3 modification synergize to maximize expression from the Myog promoter during differentiation.

The Myog promoter was recently shown to have high H3K4mel occupancy in non-differentiating C2C12 cells (Cheng et al, 2014). These marks are converted to H3K4me3 marks in response to differentiation cues through an unknown mechanism (Cheng et al, 2014). Pask is activated by these same differentiation cues and expression of either WT Pask or phospho-mimetic Wdr5 is sufficient to bypass the requirement of differentiation cues. It was next tested whether Pask-Wdr5 signaling regulates H3K4mel to H3K4me3 conversion on the Myog promoter in response to differentiation cues. In control cells, H3K4mel marks were progressively depleted from the Myog promoter over the differentiation time-course (FIG. 7A), most likely due to further methylation to H3K4me3 (see FIG. 6B). Pask knockdown severely blunted that loss of H3K4mel, in concert with blunting the appearance of H3K4me3 (FIG. 7 A, 6B). This H3K4mel to H3K4me3 conversion was accompanied by a decrease in total histone H3 density at the Myog promoter in control cells (FIG. 7B), suggestive of a transition from a

transcriptionally inactive or closed (nucleosome rich) to a transcriptionally accessible or open (nucleosome depleted) state that might facilitate MyoD recruitment in control cells. This loss of H3 density during differentiation was abolished by Pask knockdown (FIG. 7B). Because H3K4mel is commonly found at enhancers, it was next tested whether Pask is required for H3K4mel occupancy at the Myog enhancer. However, unlike the proximal Myog promoter, H3K4mel to H3K4me3 conversion at the Myog upstream enhancer is not impaired by Pask knockdown, nor is the appearance of H3K27ac (FIG. 18B). MyoD recruitment to the enhancer during differentiation is impaired similarly to the proximal promoter by Pask knockdown, possibly due to the interdependence of the enhancer and promoter for optimal activation (Sanyal et al, 2012). Like Myog, the Mybph and Actal promoters, but not Myod were reported to undergo H3K4mel to

H3K4me3 conversion during differentiation in C2C12 myoblasts (Cheng et al, 2014) and this is also Pask-dependent (FIG. 18C, D).

If the enhanced H3K4mel occupancy at the Myog promoter is responsible for the transcriptional repression observed upon loss of Pask activity, then silencing of M113 at the onset of differentiation, which is specific for deposition of H3K4mel marks might rescue the expression of Myog upon Pask knockdown. Indeed, it was found that M113 knockdown in Pask-siRNA cells rescued the impaired Myog expression and restored differentiation as indicated by Mylpf expression (FIG. 7C). Taken together, these results show that Pask is an important mediator whereby differentiation cues stimulate H3K4mel to H3K4me3 conversion and chromatin remodeling to facilitate MyoD recruitment to the Myog promoter. The requirement of Pask for loss of H3K4mel and total H3 from the Myog promoter appears to be related to Wdr5 phosphorylation since Wdr5S49E expression was sufficient to deplete H3K4mel marks (FIG. 7D) and total H3 occupancy (FIG. 7E) on the Myog promoter even in the absence of differentiation cues. In contrast, the depletion of H3K4mel and total H3 at Day 1 of differentiation was prevented by the expression of Wdr5S49A (FIG. 7D,E). These effects of Wdr5S49A and Wdr5S49E expression mirror those on MyoD occupancy of the Myog promoter (FIG. 6G). Thus, Wdr5

phosphorylation status may dictate MyoD recruitment to the Myog promoter, perhaps via H3K4mel to H3K4me3 conversion.

Role of Pask-pWdr 5 in H3K4mel to H3K4me3 conversion. H3K4mel was recently demonstrated to be a transcriptionally repressive mark at the promoters of myogenic genes (Cheng et al, 2014). In response to differentiation cues, H3K4mel is further methylated to H3K4me3 on these promoters to initiate their expression. H3K4 monomethylation is catalyzed by the Wdr5 -containing M113/M114 complexes (Hu et al, 2013), while the Wdr5 -containing M111/M112 complexes catalyze H3K4 trimethylation (Shilatifard, 2012). Thus, it has been predicted that differentiation cues promote a M113/M114 to M111/M112 switch on myogenic promoters via an unknown mechanism (Cheng et al, 2014). Pask silencing prevented the H3K4mel to H3K4me3 conversion on the Myog promoter in response to differentiation cues and silencing of M113 rescued the defect in the induction of Myog expression caused by Pask silencing. H3K4mel and H3K4me3 was unaffected at the Myod promoter or at the Myod and Myog enhancers during differentiation in Pask-silenced condition. This shows that Pask and Wdr5 phosphorylation are not required for the enzymatic activity of the Mlll/2 or M113/4 complexes, but for their activity on specific promoters like Myog, Actal and Mybph. These promoters are repressed during proliferation and required to be derepressed at the onset of differentiation. These data show that Pask mediates chromatin remodeling including H3K4mel to H3K4me3 conversion, resulting in enhanced MyoD binding and transcriptional activation during differentiation via Wdr5 phosphorylation. Future studies will determine whether Pask-mediated Wdr5 phosphorylation promotes switching of the M113/4 and Mlll/2 complexes at the Myog promoter.

Methods and materials used are described herein and above. Example 7: Pask inhibition is increases the expression of Pax7 in muscle stem cells.

Experiments were carried out testing whether PASK inhibition will change the the expression of markers for stem cells, such as Pax7 in muscle stem cells, to improve self-renewal and regenerative capacity.

Results. The results show that PASK inhibition by BioE-1197 stimulates increase in Pax7+ cell number in C2C12 myoblasts (FIG. 20). C2C12 myoblasts were treated with DMSO or BioE-1197 for 2 days. Pax7 was quantified (FIG. 20, right) using immunofluroscence microscopy using anti-Pax7 antibdoy. FIG. 21 shows that PASK phosphorylation of Wdr5 results in a decrease in Pax7 protein expression (Wdr5S49E lane) whereas unphosphorylable Wdr5 (Wdr5S49A) results in increased Pax7 expression, similar to PASK inhibition. FIG. 22 shows that BioE-1197 pretreatment restores Pax7 expression and differentiation capacity of differentiation defective myoblasts.

Differentiation defective cells were cultured in either DMSO or 100 μΜ BioE-1197 for up to 5 passages. This phase is called self-renewal phase. Next, differentiation was induced in the presence or absence of BioE-1197. The cells pre-treated with BioE-1197 differentiated in absence of it (FIG. 22C, dashed rectangle, lane 3) and showed remarkable improvement in both MyoG levels and MHC levels, which is indicative of restoration of differentiation defect as seen in DMSO pre-treated cells (FIG. 22C, lane 1).

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