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Title:
METHODS FOR IDENTIFYING THERAPEUTIC AGENTS MODULATING ACTIVITY OF THE IGF2 REPRESSOR ZBED6
Document Type and Number:
WIPO Patent Application WO/2010/093322
Kind Code:
A1
Abstract:
The present invention relates to methods for identifying therapeutic agents capable of modulating the activity of the IGF2 repressor ZBED6 for use in growth promotion,wound healing, angiogenesis, inhibition of muscle wasting, and treatment of cancer,tumours and other malignancies, muscle atrophy, muscle dystrophy, neuropathy,osteoporosis, cachexia, diabetes, obesity, acromegaly, polycystic ovary syndrome, and retinopathy.

Inventors:
ANDERSSON LEIF BERTIL (SE)
ANDERSSON GOERAN (SE)
HJAELM GOERAN (SE)
LINDBLAD-TOH KERSTIN (US)
CARR STEVEN A (US)
JAFFE JACOB D (US)
GNIRKE ANDREAS (US)
MIKKELSEN TARJEI S (US)
Application Number:
PCT/SE2010/050161
Publication Date:
August 19, 2010
Filing Date:
February 11, 2010
Export Citation:
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Assignee:
ANDERSSON LEIF BERTIL (SE)
ANDERSSON GOERAN (SE)
HJAELM GOERAN (SE)
LINDBLAD-TOH KERSTIN (US)
CARR STEVEN A (US)
JAFFE JACOB D (US)
GNIRKE ANDREAS (US)
MIKKELSEN TARJEI S (US)
International Classes:
C07K14/47; A61K38/17; A61K48/00; A61P3/04; A61P21/00; G01N33/68; C07K14/65
Other References:
VAN LAERE A-S: "From QTL to QTN, Identification of a Quantitative Trait Nucleotide Influencing Muscle Development and Fat Deposition in Pig", 26 January 2005 (2005-01-26), pages 26, XP003025664, Retrieved from the Internet [retrieved on 20100311]
VAN LAERE A-S. ET AL.: "A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig", NATURE, vol. 425, 2003, pages 832 - 836, XP002279572
JUNGERIUS B.J. ET AL.: "The IGF2-intron3-G3072A substitution explains a major imprinted QTL effect on backfat thickness in a Meishan x European white pig intercross", GENET. RES. CAMB., vol. 84, 2004, pages 95 - 101, XP003025665
MARKLJUNG ELLEN ET AL.: "ZBED6, a Novel Transcription Factor Derived from a Domesticated DNA Transposon Regulates IGF2 Expression and Muscle Growth", PLOS BIOL., vol. 7, no. 12, 2009, pages 1 - 13, XP003025666
MARKLJUNG ELLEN: "QTL Analysis in the Pig From the Identification of Quantitative Trait Loci to the Understanding of Molecular Mechanisms", 20 February 2009 (2009-02-20), pages 36 - 44, XP003025667, Retrieved from the Internet [retrieved on 20100303]
Attorney, Agent or Firm:
HYNELL PATENTTJÄNST AB (Patron Carls väg 2, Uddeholm, SE)
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Claims:
Claims

1. A method for identifying an agent potentially capable of modulating the activity of ZBED6 which method comprises:

(i) contacting a candidate compound modulator with a ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 80% sequence identity to SEQ ID NO:2, or a functionally active fragment thereof; and

(ii) determining the binding of the candidate compound modulator to the ZBED6 polypeptide.

2. A method for identifying an agent capable of modulating the activity of ZBED6 which method comprises:

(i) contacting a candidate compound modulator with an ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 80% sequence identity to SEQ ID NO:2 or a functionally active fragment thereof; and

(ii) determining an effect of the candidate compound modulator on the activity of the ZBED6 polypeptide to thereby identify the compound as a ZBED6 modulator.

3. A method for identifying an agent capable of modulating the activity of ZBED6, which method comprises:

(i) contacting a candidate compound modulator with a host-cell which expresses a polynucleotide sequence encoding a ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 80% sequence identity to SEQ ID NO:2 or a functionally active fragment thereof; and

(ii) determining an effect of the candidate compound modulator on the activity of ZBED6 to thereby identify the compound as a ZBED6 modulator.

4. The method according to claim 3 which comprises the use of a reporter-gene assay system.

5. A method for gene therapy comprising the use of a polynucleotide sequence encoding a ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least about 80% identity to SEQ ID NO:2 or a functionally fragment thereof.

6. A vector for the use in gene therapy comprising a polynucleotide sequence encoding a ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least about 80% identity to SEQ ID NO:2 or a functionally fragment thereof.

7. A pharmaceutical composition comprising a vector according to claim 6.

8. An RNAi molecules comprising a nucleotide sequence complementary to a part of a polynucleotide sequence selected from, a) the sequence SEQ ID NO : 1 , b) a variant of SEQ ID NO:1 having at least 80%, preferably at least 90%, such as at least 95%, sequence identity to SEQ ID NO:1, and c) a sequence complementary to the sequences a) and b).

9. Use of an RNAi molecules according to claim 8 which is an inhibitor of ZBED6 activity as a therapeutic agent in growth promotion, muscle growth promotion, inhibition of muscle wasting, wound healing, angiogenesis, and treatment of diseases such as, muscle atrophy, muscle dystrophy, neuropathy, osteoporosis, cachexia, diabetes, obesity, polycystic ovary syndrome, and retinopathy.

10. A pharmaceutical composition comprising an RNAi molecule according to claim 8.

Description:
METHODS FOR IDENTIFYING THERAPEUTIC AGENTS MODULATING ACTIVITY OF THE IGF2 REPRESSOR ZBED6

FIELD OF THE INVENTION

The present invention relates to fields of molecular biology, pathology and medicine. In particular, the present invention relates to methods for identifying therapeutic agents capable of modulating the activity of the IGF2 repressor ZBED6. Such agents will find use in growth promotion, wound healing, angiogenesis, inhibition of muscle wasting, and treatment of cancer, tumours and other malignancies, muscle atrophy, muscle dystrophy, neuropathy, osteoporosis, cachexia, diabetes, obesity, acromegaly, polycystic ovary syndrome, and retinopathy.

RELATED ART

The Quantitative Trait Nucleotide (QTN) in the porcine insulin- like growth factor 2 (IGF2) gene is one of the rare examples where a single base substitution underlying a complex trait has been identified and where the mechanism of action is understood. Elevated paternal expression from the mutant allele in pigs increases the amount of skeletal muscle and thus meat production by 3-4%. The favourable allele has gone through a selective sweep and is close to fixation in pig breeds widely used for commercial meat production in the Western world. The mutation is believed to disrupt the interaction with a nuclear factor, a putative repressor, regulating IGF2 mRNA expression in a tissue- specific fashion. Pigs carrying the favourable allele at the paternal chromosome show higher expression from the IGF2 P2, P3 and P4 promoters in skeletal and cardiac muscle, but not in liver (Van Laere et al. 2003 Nature 425: 832-836). The repressor has remained to be identified.

SUMMARY OF THE INVENTION

The present inventors have identified and characterized a previously unknown IGF2 repressor designated ZBED6 (zinc-finger bed domain containing protein 6). Gene silencing of Zbedό in mouse myoblasts abolished ZBED6 mediated repression of IGF2 mRNA expression and protein production, and led to faster formation of myotubes and a faster wound healing process.

Accordingly, the present invention provides methods for identifying agents capable of modulating the activity of the IGF2 repressor ZBED6.

It is an object of the present invention to provide methods of utilizing the identified agents capable of modulating the activity of ZBED6 to provide a means of preventing and treating disease conditions and modulating growth in animals and human patients. The conditions are for example but not limited to growth promotion, wound healing, angiogenesis, and treatment of cancer, tumours and other malignancies, muscle atrophy, muscle dystrophy, neuropathy, osteoporosis, cachexia, diabetes, obesity, acromegaly, polycystic ovary syndrome, and retinopathy.

One aspect of the present invention provides a method for identifying an agent potentially capable of modulating the activity of ZBED6 which method comprises:

(i) contacting a candidate compound modulator with a ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 80% sequence identity to SEQ ID NO:2, or a functionally active fragment thereof; and

(ii) determining the binding of the candidate compound modulator to the ZBED6 polypeptide.

ZBED6 binders are indentified as potential modulators of ZBED6 activity.

In particular, the ZBED6 polypeptide comprises the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 90%, such at least 95%, sequence identity to SEQ ID NO:2, or a functionally active fragment thereof.

Another aspect of the present invention provides a method for identifying an agent capable of modulating the activity of ZBED6 which method comprises:

(i) contacting a candidate compound modulator with an ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 80% sequence identity to SEQ ID NO:2 or a functionally active fragment thereof;

(ii) determining an effect of the candidate compound modulator on the activity of the ZBED6 polypeptide to thereby identify the compound as a ZBED6 modulator.

In particular, the ZBED6 polypeptide comprises the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 90%, such at least 95%, sequence identity to SEQ ID NO:2, or a functionally active fragment thereof.

It will be appreciated that there are many procedures known in the art which may be employed to perform the present invention. Examples of suitable procedures which may be used to identify a ZBED6 modulator include rapid filtration of equilibrium binding mixtures, enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and fluorescence resonance energy transfer assays (FRET), scintillation proximity assay (SPA), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation analysis (ChIP), surface plasmon resonance (SPR) assays.

Cellular assay systems may be used to further identify ZBED6 modulators.

Therefore a further aspect of the invention provides a method for identifying an agent capable of modulating the activity of ZBED6, which method comprises: (i) contacting a candidate compound modulator with a host-cell which expresses a polynucleotide sequence encoding a ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 80% sequence identity to SEQ ID NO:2 or a functionally active fragment thereof;

(ii) determining an effect of the candidate compound modulator on the activity of ZBED6 to thereby identify the compound as a ZBED6 modulator.

In particular, the ZBED6 polypeptide comprises the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 90%, such at least 95%, sequence identity to SEQ ID NO:2, or a functionally active fragment thereof.

A particular cellular assay system for use in the method of the invention is a reporter-gene assay system. The reporter-gene system utilizes a promoter element which interacts with ZBED6 functionally coupled to a reporter gene, such as a gene encoding luciferase, aequorin or a fluorescent protein, such as the green fluorescent protein (GFP) or the yellow fluorescent protein (YFP). Preferably, the promoter element is selected from the IGF2 P2, P3 and P4 promoters.

ZBED6 modulators have the ability to influence the binding of ZBED6 to a target polynucleotide sequence, e.g. an IGF2 promoter element, more specifically the ability to influence the expression of IGF2, thereby mediating cell processes related to cellular growth and differentiation, such as muscle growth, inhibition of muscle wasting, wound healing, angiogenesis, cancer, tumours and other malignancies, muscle atrophy, muscle dystrophy, neuropathy, osteoporosis, cachexia, diabetes, obesity, acromegaly, polycystic ovary syndrome, and retinopathy.

Modulation of the activity of ZBED6 comprises either stimulation or inhibition. Thus a agent capable of modulating the activity of ZBED6 is an agent that either stimulates or inhibits the activity of ZBED6. The terms "modulator of ZBED6 activity" and "ZBED6 modulator" are also used herein to refer to an agent that either stimulates or inhibits the activity of ZBED6.

ZBED6 modulators which are inhibitors of ZBED6 activity have utility as therapeutic agents in growth promotion, wound healing, angiogenesis, and treatment of diseases such as, muscle atrophy, neuropathy, osteoporosis, cachexia, muscle atrophy, muscle dystrophy, neuropathy, osteoporosis, diabetes, obesity, polycystic ovary syndrome, and retinopathy.

ZBED6 modulators which are inhibitors of ZBED6 activity further have utility in promoting in vitro production of muscle proteins using cell culturing, especially myocyte cell culturing.

ZBED6 modulators which are stimulators of ZBED6 activity have utility as therapeutic agents in treatment of diseases such as cancer, tumours and other malignancies and acromegaly.

Candidate compounds which may be tested in the methods according to the invention include simple organic molecules, commonly known as "small molecules", for example those having a molecular weight of less than 2000 Daltons. The screen may also be used to screen compound libraries such as peptide libraries, including synthetic peptide libraries and peptide phage libraries. Other suitable molecules include polynucleotide sequences and any other molecules which modulate the activity of ZBED6.

Once an inhibitor or stimulator of ZBED6 activity is identified then medicinal chemistry techniques can be applied to further refine its properties, for example to enhance efficacy and/or reduce side effects.

Yet another aspect of the invention provides methods for gene therapy comprising the use of a polynucleotide sequence encoding a ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least about 80% identity to SEQ ID NO:2 or a functionally fragment thereof. Preferably, the polynucleotide sequence encodes a ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 90%, such at least 95%, sequence identity to SEQ ID NO:2, or a functionally active fragment thereof.

The polynucleotide sequence can be the sequence SEQ ID NO: 1 or variant of SEQ ID NO: 1 having at least about 80%, preferably at least 90%, such as at least 95%, sequence identity to SEQ ID NO:1.

Another related aspect of the invention is a vector for the use in gene therapy comprising a polynucleotide sequence encoding a ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least about 80% identity to SEQ ID NO:2 or a functionally fragment thereof. In particular, the polynucleotide sequence encodes a ZBED6 polypeptide comprising the amino acid sequence SEQ ID NO:2 or a variant of SEQ ID NO:2 having at least 90%, such at least 95%, sequence identity to SEQ ID NO:2, or a functionally active fragment thereof. The polynucleotide sequence can be the sequence SEQ ID NO: 1 or variant of SEQ ID NO: 1 having at least about 80%, preferably at least 90%, such as at least 95%, sequence identity to SEQ ID NO:1.

One aspect of the invention is a pharmaceutical composition comprising a vector according to the invention. The vector can be a viral vector, such as an adenoviral vector.

Yet another aspect of the invention provides RNAi molecules comprising nucleotide sequences complementary to a part of a polynucleotide sequence selected from, a) the sequence SEQ ID NO : 1 , b) a variant of SEQ ID NO: 1 having at least 80%, preferably at least 90%, such as at least 95%, sequence identity to SEQ ID NO:1, and/or c) a sequence complementary to the sequences a) and b). Such RNAi molecules are potential inhibitors of ZBED6 activity.

Yet another aspect of the invention provides use of RNAi molecules which are inhibitors of ZBED6 activity as therapeutic agents in growth promotion, muscle growth promotion, inhibition of muscle wasting, wound healing, angiogenesis, and treatment of diseases such as, muscle atrophy, muscle dystrophy, neuropathy, osteoporosis, cachexia, diabetes, obesity, polycystic ovary syndrome, and retinopathy. One aspect of the invention is a pharmaceutical composition comprising an RNAi molecule according to the invention.

By "a functionally fragment" of a ZBED6 polypeptide is meant a polypeptide that can bind to an IGF2 promoter element, such as the IGF2 P2, P3 and P4 promoters, thereby acting as a suppressor of IGF 2 expression.

The percent identity between two amino acid sequences is determined as follows. First, an amino acid sequence is compared to, for example, SEQ ID NO:2 using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from the U.S. Government's National Center for Biotechnology Information web site at http://www.ncbi.nlm.nih.gov. Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options of B12seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seql.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share sequence similarity, then the designated output file will present those regions of similarity as aligned sequences. If the two compared sequences do not share sequence similarity, then the designated output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.

The percent identity is determined by dividing the number of matches by the length of the sequence set forth in an identified sequence followed by multiplying the resulting value by 100. For example, if a sequence is compared to the sequence set forth in SEQ ID NO:2 (the length of the sequence set forth in SEQ ID NO:2 is 979) and the number of matches is 890, then the sequence has a percent identity of 90.9 (i.e., 890 ÷ 979 * 100 = 90.9) to the sequence set forth in SEQ ID NO:2.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention. Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1. Identification of Zbedό. Mass spectrometric quantification of Zbedό-enrichment using SILAC; the core sequences of the wild-type q and the mutant Q oligonucleotides used for protein capture are shown on top. Zbedό was identified by six unique peptides in the SILAC-heavy q sample. Two peptides were simultaneously observed in the SILAC-light Q sample and were used as the basis for SILAC quantification. A representative peptide is shown where the area under the curve corresponds to the amount of Zbedό enriched by the q probe (grey) and the Q probe (white). The average overall enrichment of Zbedό by q was 9.0-fold (±1.2-fold, n=2 experiments).

Figure 2. Genome organization of Zbedό and Zc3hlla in mouse. Adapted from the UCSC Genome Browser (http://genome-test.cse.ucsc.edu/). Non-translated and translated exons of Zc3hl Ia are indicated by black and grey bars, respectively. A mammalian conservation track from the UCSC browser is included at the bottom.

Figure 3. Schematic representation of the six ZBED-family members present in human.

Figure 4. Northern blot analysis of Zbedό expression in mouse tissue. Multiple tissue and Developmental skeletal muscle Northern blot analysis of Zbedό and Zc3hlla; β-actin (Acbt) probe was used as control. The Zbedό and Zc3hlla probes apparently hybridized to the same transcript, while Zc3hlla also hybridized to shorter alternative transcripts lacking Zbedό. B- brain (whole), S-stomach (whole), I-intestine (whole), C-colon, Li-liver, Lu-lung, K-kidney, H-heart, O-ovary, SM-skeletal muscle, Sp-spleen, Te-testis, Th-thymus, U-uterus (non-pregnant), P-placenta (late pregnancy). The estimated molecular weights are indicated. Figure 5. Real-time-PCR analysis of Zbedό mRNA expression in mouse tissues. RNA was isolated from tissue samples from six C57BL/6 mice (three males and three females) either by RNeasy mini kit (Qiagen) or acidic phenol extraction, all samples were subjected to reverse transcription and mRNA transcripts were measured by quantitative PCR analysis. Zbedό mRNA expression was normalized using 18S-rRNA.

Figure 6A. Western blot analysis. Western blot analysis of recombinant protein (Zbedόa and Zbedόb) and C2C12 protein extracts using the polyclonal antibody raised against the Zbedό BED- domains. Two isoforms, Zbedόa and Zbedόb, with apparent molecular weights of 122kDa and 11 όkDa (calculated molecular weights 109 kDa and 104 kDa, respectively) corresponding to two predicted alternative translation start sites were detected in C2C12 protein extracts.

Figure 6B. Nuclear localization of Zbedό in C2C12 cells. Constructs containing Zbedό BEDl/2-domains fused to GFP compared with GFP alone.

Figure 7. Electrophoretic mobility shift assay (EMSA). EMSA showing binding of recombinant Zbedό BED 1/2 domains to wild-type (q) but not to mutant (Q) probe (left panel). Likewise, endogenous Zbedό in nuclear extracts (NE) from C2C12 cells forms a complex with wild- type probe that is competed by q but not Q probe (right panel). The complex is supershifted by the anti-Zbedό antibody and competition confirmed the specific interaction with Zbedό.

Figures 8A-C. RNAi-mediated Zbedό silencing in C2C12 cells. (Fig 8A).

Immunocytochemical staining of cytospins of C2C12 cells with anti-Zbedό antibody and nuclear counter-stain. (Fig 8B). Luciferase assays of reporter constructs containing the wild-type q or mutant Q sequence of pig IGF2 intron 3 and the pig IGF2 P3 promoter. Firefly reporter levels in relation to control Renilla luciferase level. (Fig 8C). Ig/2 mRNA expression day 2, 3 and 6 after transfection. Black bars scrambled siRNA, grey bars Zbedό siRNA. Assays were performed 48 h after transfection. At least six transfections were performed for each siRNA. Error bars, s.e.m. (P<0.05, *; P< 0.01, **; P<0.001, ***).

Figure 9A-B. Wound healing assay. (Fig 9A). Monolayer scratch wound healing. (Fig 9B). Cell number in scratches. Assays were performed 72 h after transfection. Black bars scrambled siRNA, grey bars Zbedό siRNA. At least six transfections were performed for each siRNA. Error bars, s.e.m. (PO.05, *; P< 0.01, **; P<0.001, ***).

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Methods

Proteomics. Two sets of murine C2C12 myoblast cells (ATCC CRL- 1772) were used. Cultures were grown according to standard cell culture procedures in SILAC-light and SILAC-heavy labeled Dulbecco's Modified Eagle Medium (DMEM, ThermoFisher) containing 10% dialyzed fetal bovine serum (FBS) and 13 C6-L-Arg and 13 C O , 15 N 2 -L-LyS in the 'heavy' formulation, (see Ong & Mann 2006. Nat Protoc 1, 2650-2660 for more details). 'Light' and 'Heavy' nuclear extracts were prepared using a commercially available kit (ActiveMotif). The (+)-strand sequences of the wild-type q and mutant Q oligonucleotides were as follows:

IGF2-q: 5'-Biotin-GATCCTTCGCCTAGGCTCGCAGCGCGGGAGCGA-3' (SEQ ID NO:3) IGF2-Q: S'-Biotin-GATCCTTCGCCTAGGCTCACAGCGCGGGAGCGA-S' (SEQ ID NO:4)

The complementary (-)-strands were also synthesized, and the pairs were annealed prior to use. One pmol of ds-q oligonucleotide was mixed with 1.4 mg of "heavy" nuclear extract and one pmol of ds-Q oligonucleotide was mixed with 1.4 mg of "light" nuclear extract in binding buffer (50 mM Tris pH 8, 150 mM NaCl, 0.25 mM EDTA, 0.5 mM DTT, 0.1% Tween-20, 0.5 mg/mL BSA, 200 μg/mL poly-dl/dC) in a total volume of 700 μl. Mixtures were incubated for 45 min at room temperature on a rotator. Ten μl of Streptavidin-coated magnetic beads (Dynal) was added to each tube, and the mixture was further incubated for 30 min at room temperature on a rotator. Beads were spun for 5 s at lOOOg and then captured using a magnetic pull-down system. Beads were washed 3 x 700 μL in binding buffer without poly-dl/dC and then 4 x 700 μl in binding buffer without poly-dl/dC or BSA. The supernatant was discarded and proteins were eluted by boiling in Laemmli buffer (+10 mM DTT). Protein eluates were mixed 1 :1 by volume.

Proteins were separated on an SDS-PAGE gel (4-12% NuPage), stained with Coomassie Blue, and the entire lane was cut into -20 bands. Each band was reduced, alkylated, digested with trypsin according to standard proteomics practices (Kinter & Sherman. Protein sequencing and identification using tandem mass spectrometry. John Wiley, 2000), and the resulting peptides were analyzed by LCMS on an Orbitrap (ThermoFisher) mass spectrometer as described (Jaffe et al. 2008. MoI Cell Proteomics 7, 1952-1962). Database searching was performed by Mascot against the REFSEQ database of mouse proteins as of June 2006. SILAC quantification was performed using msQuant (Andersen et al. 2005. Nature 433, 77-83).

Expression of recombinant protein and antibody production. RT-PCR, using poly-A enriched RNA extracted from C2C12 cells, was used to obtain nucleotides 1-2943 and 139-2943 of mouse Zbedό transcripts (named Zbedόa and Zbedόb, respectively; the two constructs begin at the two alternative start codons. These constructs, containing a Kozak sequence for efficient initiation of translation, were cloned into pcDNA3 (Invitrogen) and verified by DNA sequencing.

PCR was used to subclone a fragment encoding the two BED-domains, amino acid residues 90-384 of Zbed6a, into pGEX-5X-3 (GE Healthcare). GST and GST-BED1/2 fusion protein was purified from BL21(DE3)pLysS bacteria using gluthathione Sepharose 4B or GSTrap FF columns (GE Healthcare), according to manufacturer's instructions.

Polyclonal antibody production was performed by Agrisera AB (Umea, Sweden). Shortly, GST-BED 1/2 was used to immunize one rabbit. Polyclonal anti-Zbed6 antibodies were affinity- purified by first passing serum over a HiTrap NHS-activated HP column (GE Healthcare) coupled with GST, where after the flow-through was applied to a column coupled with GST-BED 1/2. Anti- Zbedό antibodies were eluted with 0.2 M glycine (pH 2.5) and dialyzed against 20 mM HEPES (pH 7.4) and 15O mM NaCl.

Electrophoretic mobility shift assay (EMSA). Nuclear extracts from C2C12 cells were prepared using the NucBuster Protein Extraction kit (Novagen). EMSAs were performed as previously described with minor modifications (Van Laere et al. 2003. Nature 425, 832-836). The following oligonucleotides were annealed in lxNEB2 buffer (NEB):

q/Q-fwd: AGATCCTTCGCCTAGGCTC(G/A)CAGCGCGGGAGCGA (SEQ ID NO:5) and q/Q-rev: AGATCTCGCTCCCGCGCTG(C/T)GAGCCTAGGCGAAG. (SEQ ID NO:6)

Twenty ng of purified GST-BED1/2 protein or 10 μg C2C12 nuclear extracts were pre- incubated on ice for 20 min in binding buffer (15 mM Hepes-KOH pH 7.65 at room temperature, 30.1 mM KCl, 2 mM MgCl 2 , 0.1 mM EDTA, 0.063% NP-40, 7.5% Glycerol, 1.3 mM dithiothreitol, 2 mM spermidine, O.lμg/μl Poly(dI-dC)*Poly(dI-dC)). Competition reactions were supplemented with 4000 frnol (100-fold excess) unlabelled ds-oligonucleotide. After the addition of 40 frnol end-labeled P- dCTP ds-oligonucleotide, reactions were incubated at room temperature for 30 min. In EMSAs including supershift reaction, incubation for 20 min at room temperature preceded the addition of 2 μl of purified polyclonal anti-ZBED6 antibody (0.3 μg/μl) and incubation then continued for an additional 20 min at room temperature. Complexes were separated on a 1.5 mm 5% native 29:1 polyacrylamide gel in 0.5XTBE at 70V for 3-4 h.

Cell culture. The C2C12 mouse myoblast cell line (ATCC-CLR- 1772) was cultured at 37 0 C in a humidified atmosphere of 5% CO 2 using DMEM (ATCC-30-2002) supplemented with 10% FBS (Invitrogen) and Ix Antibiotic- Antimycotic solution (Invitrogen). The cultures were split every two to three days. C2C12 cells were differentiated by growing the cells in differentiation media, DMEM with 2% horse serum.

GFP-BED 1/2 transfection. BED 1/2 (amino acid residues 90-384 of Zbed6a) was cloned into pcDNA3 containing an N-terminal enhanced green fluorescent protein (GFP) coding sequence. 5x10 4 cells were plated the day before transfection in 12-well plates. Two μg DNA of either GFP or GFP- BED 1/2 was transfected using 6 μl Lipofectamine 2000 reagent (Invitrogen) in Opti-MEM (Gibco). Media was changed to growth media without antibiotics four hours post transfection and cells were analyzed the following day. Photographs were taken using a Nikon Eclipse TSlOO microscope and a super plan fluor 6OX objective.

Gene silencing using siRNA. 5x10 4 C2C12 cells or 30-50% confluent cells were transfected with 50 pmol of negative control siRNAs (Ambion) or Zbedό siRNAs (Ambion) with 6 μl Lipofectamine 2000 Reagent (Invitrogen) in 1.5 ml Opti-MEM I (Invitrogen) per well in 6-well plates. The following pooled Silencer Select siRNA sequences (Ambion) were used to silence Zbedό expression in C2C12 cells: duplex 1 sense, 5' -CUUCAAC ACUUC AACGACAtt-3' (SEQ ID NO:7); duplex 2 sense, S'-UGUGGUACAUGCAAUCAAAtt-S^SEQ ID NO:8); duplex 3 sense, 5'-GGGCUGUUGCCAACAAAGAtt-3'(SEQ ID NO:9).

After 24 h, medium was changed to fresh DMEM with 10% FBS. Biological triplicates were used for each siRNA treatment.

Luciferase assay. Silencing was performed two days prior to transient transfection with luciferase reporters. Previously described (Van Laere et al. 2003. Nature 425, 832-836) constructs containing the porcine IGF2 QTN region, the porcine IGF2 P3 promoter and the firefly luciferase reporter gene (P3, q+P3 and Q+P3) were used. ZBED6-silenced C2C12 cells grown in 12-well plates were transfected with a total of 2 μg DNA and 6 μl Lipofectamine 2000 Reagent (Invitrogen). One μg firefly luciferase construct and 20 ng Renilla luciferase vector as control (ph-RG, Promega) and empty pcDNA3 vector (Invitrogen) up to 2 μg DNA were used. Transfections were performed in opti-MEM (Gibco) and media changed to growth media (DMEM supplemented with 10% FBS) after 4 h. Cells were harvested 24 h post transfection and firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) and an Infinite M200 luminometer (Tecan).

Cell proliferation assay. 48 h-post siRNA transfection, the cells were incubated 3-4 h with media containing 10% Alamar blue (Invitrogen). The reduction of Alamar blue was measured on a Tecan Sunrise Absorbance Plate Reader (Oxidized/Reduced: 600/570 nm). Scratch wound assay. 2 h-post siRNA transfection, cells reached almost confluence and medium was replaced with fresh DMEM supplemented with 0.1% FBS. A surface wound was created by scraping a pipette tip across the confluent cell monolayer. 24 h after scraping, the number of cells in the scratch was counted and cells treated with negative control siRNAs and Zbedό siRNAs were compared. Statistical analysis was performed using a Student's t-test.

RNA isolation and real-time (RT) PCR analysis. RNA was isolated from tissue samples from six C57BL/6 mice (three males and three females) either by RNeasy mini kit (Qiagen) or acidic phenol extraction as described (Nat Meth 3, 149-150, 2006). RNA from C2C12 cell samples was isolated using the RNeasy mini kit (Qiagen) then all samples were subjected to reverse transcription using cDNA high capacity kit (Applied Biosystems). mRNA transcripts were measured by quantitative PCR analysis using TaqMan Gene Expression master mix (Applied Biosystems) on a 7900HT Fast RT-PCR System (Applied Biosystems). Data were analyzed with a threshold set in the linear range of amplification, based on a standard curve of serial ten- fold dilutions for each primer set. The Zbedό data was normalized to the level of cDNA from two endogenous housekeeping genes (GAPDH and 18S rRNA) and plotted as mean fold change (± s.e.m.). Statistical analysis was performed using a Student's t-test.

Immunocvtochemical staining of cvtospins ofC2C12 cells. Cells were trypsinized by 0.25% trypsin EDTA (Invitrogen). After blocking with 10% FBS and washing twice in PBS (Invitrogen), 2-3 million cells were resuspended in 1 ml PBS with 20% FBS and centrifuged as cytospins for 3 min at 800 rpm. The spins were dried at room temperature overnight and then fixed with acetone for 10 min and hybridized with 250 μl of anti-Zbedό antibody (0.2 μg/ml) for 20 min, followed by staining protocol described by Human Protein Atlas (Mathivanan et al. 2008. Nat Biotechnol 26, 164-167).

Northern blot. A mouse mixed tissue northern blot panel 4 (MN-MT-I) and a mouse developmental tissue skeletal muscle panel (MN- 102-D) from Zyagen (San Diego, USA) was used. Partial Zbedό and Zc3hlla coding sequences were cloned into a vector and probe template amplified by PCR including either SP6 or T7 sequence from the vector. 200 ng of purified probe template was used for 32 P-labeled RNA probe synthesis using the MAXIscript Kit (Ambion). Mouse β-actin (Actb) was amplified by PCR from C2C12 cDNA, sequenced and used as template for P-labeled DNA probe synthesis using the Amersham Megaprime DNA labeling system (GE Healthcare). Hybridizations were done at 68 0 C (RNA probe) or 42°C (DNA probe) using the ULTRAhyb buffer (Ambion) followed by washes in 2XSSC + 0.1% SDS and O.lXSSC+0.1% SDS at 68 0 C (RNA probes) or 6O 0 C (DNA probe). Autoradiographs were exposed for a few hours to several days.

Example 1. Isolation of the IGF 2 repressor

Isolation of the IGF2 repressor was attempted with affinity capture using biotinylated oligonucleotides corresponding to the wild-type (q) and mutant (Q) sequence where only the former binds the repressor (Van Laere et al. 2003. Nature 425, 832-836). Two different nuclear extracts were prepared from mouse C2C12 myoblasts using stable isotope labelling of amino acids in culture (SILAC) technique (Ong et al. 2002. MoI Cell Proteomics 1, 376-386) where the 'heavy' extract proteins contained the stable-isotope labelled amino acids lysine and arginine while the 'light' extract proteins contained the natural versions of these amino acids. The wild-type and mutant oligonucleotides were incubated with 'heavy' and 'light' extracts, respectively. Captured 'heavy' and 'light' proteins were mixed and separated with SDS-PAGE. Gel slices were digested with trypsin, and the resulting peptides were analyzed by liquid chromatography mass spectrometry (LCMS). The acquired spectra were searched against the REFSEQ database containing mouse protein sequences to identify proteins present in the sample. Ratios of the amount of each protein enriched by the q and Q constructs were computed by comparing the mass spectral signals from the 'heavy' and 'light' versions of each identified peptide composing the protein. The protein demonstrating the highest fold enrichment by q (9. O± 1.2 fold; Figure 1) corresponded to a transcript annotated as an alternative splice form of the poorly characterized Zc3hlla gene. Zc3hl Ia belongs to a large family of zinc finger proteins with 58 members in mouse (Liang et al. 2006. PLoS ONE 3, e2880). However, a closer examination revealed that the captured peptide is encoded by a retrogene located in intron 1 of Zc3hlla (Figure 2). A retrogene is a gene that has been inserted in the genome via reverse transcription of an mRNA sequence and is often expressed from an existing promoter in the vicinity of the integration site (Vinckenbosch et al. 2006. Proc Natl Acad Sci USA 103, 3220-3225). This retrogene contains an open reading frame of more than 900 codons and encodes a protein with no sequence similarity to Zc3hlla. The encoded protein contains two BED domains and a hATC dimerization domain (Figure 3). The BED domain was first identified by a bioinformatic analysis using two chromatin-boundary-element-binding proteins from Drosophila, BEAF and DREF, as seeds for homology search (Aravind 2000. Trends Biochem Sci 25, 421-423). We named our protein ZBED6 because it is the sixth mammalian protein with one or more BED domains.

Example 2. Expression and tissue distribution

Northern blot analysis (Figure 4) and real-time PCR analysis (Figure 5) showed that Zbedό, like Ig/2, has a broad tissue distribution in mouse and is expressed in skeletal muscle consistent with it being the IGF2 repressor. Northern blot analysis (Figure 4) and RT-PCR amplification and sequencing (data not shown), revealed that Zbedό is co-expressed with Zc3hlla as a -13 kb splice variant of Zc3hlla, retaining the genomic region from exon 1 to exon 4 (with Zbedό located in intron 1) spliced to the remaining exons.

Example 3. Analysis of Zbedό isoforms

We developed a polyclonal anti-Zbedό antibody by immunizing rabbits with a recombinant mouse protein containing the two BED domains. Western blot analysis of proteins from mouse C2C12 cells revealed two different isoforms, denoted Zbedόa and Zbedόb, with apparent molecular weights of 122 and 116 kDa, respectively (Figure 6A). These isoforms correspond to the use of two alternative start codons in the open reading frame of Zbedό as demonstrated by co-migration with recombinant proteins representing the two forms (Figure 6A). Bioinformatic analysis revealed three bipartite nuclear localization signals, amino acid residues 61-78, 63-80 and 231-248, in ZBED6a. At least the 231-248 motif is functional, since a deletion construct encoding amino acid residues 90-384 expressed a GFP-fusion protein with a nuclear localization in C2C12 cells (Figure 6B). Interestingly, the fusion protein was primarily associated with granular structures in the nucleus, as previously reported for human ZBEDl (Yamashita, D. et al 2007. J Biol Chem 282, 7563-7575). Example 4. Electrophoretic mobility shift assay

To address if ZBED6 is the bonafide repressor binding the QTN region in IGF2 intron 3, we produced recombinant mouse Zbedό including the two DNA-binding BED domains and used this in an electrophoretic mobility shift assay (EMSA) with the q and Q oligonucleotides differing only at the QTN position. EMSA revealed a highly specific interaction with the wild-type q oligonucleotide and a 100-fold excess of mutant Q oligonucleotide could not compete out the interaction (Figure 7). A supershift was obtained when nuclear extracts from C2C12 cells were incubated with anti-Zbedό antibody, providing further support for Zbedό as the elusive IGF2 repressor (Figure 7).

Example 5. Zbedό gene silencing

Zbedό was silenced in C2C12 cells using siRNA to obtain further insight into its functional significance. Highly efficient silencing (>75%) was obtained (Figure 8A). Zbedό-silenced and control C2C12 cells were used to repeat our previously described Luciferase assay including a reporter construct containing the wild-type or mutant sequence of the QTN region fused with the IGF2 P3 promoter (Van Laere et al. 2003. Nature 425, 832-836). An assay based on C2C12 control cells transfected with scrambled oligonucleotide replicated our previous results since a construct containing the wild-type QTN region repressed luciferase expression in comparison with a construct containing P3 alone, whereas a construct including the mutant QTN region was associated with no or only minor repression (Figure 8B). In contrast, transfection experiments using three different silencing oligonucleotides directed against Zbedό mRNA completely abolished repression with the wild-type q construct (Figure 8B).

Zbedό function was further investigated by specific gene silencing in C2C12 cells that were induced to differentiate, subsequent to silencing, by changing from growth to differentiation media. Ig/2 mRNA expression was low in both control and Zbedό-silenced cells the first days after differentiation was induced (Figure 8C). However, at day 6, Ig/2 mRNA expression was significantly increased in silenced cells compared with controls (Figure 8C) and this was accompanied by increased cell proliferation and a faster formation of myotubes. Furthermore, silencing also led to a faster wound healing process observed after scratching the surface of growing C2C12 cells (Figure 9A-B). These results are all in line with the phenotype observed in mutant pigs involving increased IGF2 expression in skeletal muscle and increased muscle growth (Van Laere et al. 2003. Nature 425, 832-836).

Conclusions

The present inventors have provided conclusive evidence that ZBED6 is the repressor binding the QTN site in IGF2 intron 3. It is notable that a protein, which is so highly conserved across all mammals, has remained unknown still seven years after the release of the human genome sequence (Waterston et al. 2002. Nature 420, 520-56). The very high sequence conservation, nearly 100% identity in the BED domains among mammals, implies that ZBED6 has an essential role in mammalian biology. The mechanism by which ZBED6 acts as a repressor remains to be determined, but it is interesting that other members of the ZBED family are involved in regulating higher order chromatin structure (Hirose et al. 2002. MoI Cell Biol 22, 5182-5193; Hirose et al. 2001. MoI Cell Biol 21, 7231-7242; Hochheimer et al. 2005. Nature 420, 439-445). The previously reported phenotypic effects in mutant pigs and the data presented on mouse C2C12 cells establish that the interaction between ZBED6 and the IGF2 QTN region plays a prominent role for regulating IGF2 mRNA expression and thereby skeletal muscle development. However, the broad tissue distribution of ZBED6 implies that it may control IGF2 expression in many cell types. ZBED6 may in fact be a tumour suppressor because there is a well-established link between IGF2 overexpression, due to loss of imprinting, and tumour development (Kaneda & Feinberg 2005. Cancer Res 65, 11236-11240; Kaneda et al. 2007. Proc Natl Acad Sci USA 104, 20926-20931). The discovery of ZBED6 has potentially broad implications for epigenetic regulation of gene expression, developmental biology, tumour biology and the understanding of the dynamic regulation of skeletal muscle growth.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.




 
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