Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

m-Bop, a Repressor Protein Essential for Cardiogenesis, Interacts with skNAC, a Heart- and Muscle-specific Transcription Factor

2002; Elsevier BV; Volume: 277; Issue: 29 Linguagem: Inglês

10.1074/jbc.m204121200

ISSN

1083-351X

Autores

Robert J. Sims, Elizabeth Weihe, Li Zhu, Sean O’Malley, June V. Harriss, Paul Gottlieb,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

The m-Bop protein encoded by the mouseBop gene is strongly expressed in heart and skeletal muscle, and recent studies with Bop knockout mice have demonstrated that m-Bop is essential for cardiogenesis in vivo and can act as a HDAC-dependent repressorin vitro. In the present studies, m-Bop was observed to interact with skNAC, a reported transcriptional activator specific to heart and skeletal muscle. The amino-terminal S region of the split S-ET domain of m-Bop as well as the MYND domain were required for interaction with skNAC in both the two-hybrid system and in coimmunoprecipitation experiments from cultured mammalian cells. As shown previously for interaction of the MYND domain-containing transcriptional corepressor, BS69, with several viral and cellular oncoproteins, a PXLXP motif in skNAC was required for interaction with m-Bop. Similar kinetics of induction and localization of m-Bop and skNAC during the induction of myogenesis in cultured C2C12 cells suggests a possible associated role for these proteins during this process. The m-Bop protein encoded by the mouseBop gene is strongly expressed in heart and skeletal muscle, and recent studies with Bop knockout mice have demonstrated that m-Bop is essential for cardiogenesis in vivo and can act as a HDAC-dependent repressorin vitro. In the present studies, m-Bop was observed to interact with skNAC, a reported transcriptional activator specific to heart and skeletal muscle. The amino-terminal S region of the split S-ET domain of m-Bop as well as the MYND domain were required for interaction with skNAC in both the two-hybrid system and in coimmunoprecipitation experiments from cultured mammalian cells. As shown previously for interaction of the MYND domain-containing transcriptional corepressor, BS69, with several viral and cellular oncoproteins, a PXLXP motif in skNAC was required for interaction with m-Bop. Similar kinetics of induction and localization of m-Bop and skNAC during the induction of myogenesis in cultured C2C12 cells suggests a possible associated role for these proteins during this process. histone deacetylase nascent polypeptide-associated complex tetramethylrhodamine isothiocyanate whole cell extracts The control of skeletal and cardiac maturation is ordered in a complex cascade of transcriptional activation and repression. The MyoD and MEF2 families of transcription factors dictate important developmental events that result in the formation of mature skeletal muscle. The MyoD family is comprised of the basic helix-loop-helix proteins MyoD, Myf5, MRF4, and myogenin. These myogenic regulators form heterodimers with the ubiquitous basic helix-loop-helix E proteins that subsequently activate key elements needed for the myogenic program (1Olson E.N. Klein W.H. Genes Dev. 1994; 8: 1-8Crossref PubMed Scopus (606) Google Scholar). The MEF2 family is defined by the MADS domain and consists of MEF2A, MEF2B, MEF2C, and MEF2D. Members of the MEF2 family are expressed in tissues besides skeletal muscle including cardiac tissue, neurons, and T cells (2Naya F.J. Olson E. Curr. Opin. Cell Biol. 1999; 11: 683-688Crossref PubMed Scopus (253) Google Scholar). Together, the MyoD and MEF2 families cooperate directly and indirectly to transduce the requisite signals for proper skeletal muscle formation.The discovery of chamber-specific transcription factors, such as the Hand1 and Hand2 proteins, has revealed the complex nature of cardiac transcriptional regulation (3Srivastava D. Thomas T. Lin Q. Kirby M.L. Brown D. Olson E.N. Nat. Genet. 1997; 16: 154-160Crossref PubMed Scopus (556) Google Scholar). The list of genes that correspond to specific cardiac defects continues to grow, whereas the molecular nature of these defects remains largely elusive (4Srivastava D. Olson E.N. Nature. 2000; 407: 221-226Crossref PubMed Scopus (523) Google Scholar). To further understand the molecular underpinnings of cardiac development, it is critical to identify the relationships of transcriptional regulators shown to be important in cardiac morphogenesis.The Bop gene encodes distinct proteins expressed in skeletal and cardiac muscle, as well as in cytotoxic T lymphocytes. The Bop proteins found in skeletal muscle (m-Bop) and cytotoxic T lymphocytes (t-Bop) are identical over 90% of their primary amino acid sequence, differing only at their extreme amino terminus (5Hwang I. Gottlieb P.D. J. Immunol. 1997; 158: 1165-1174PubMed Google Scholar). Bop contains the evolutionarily conserved MYND and SET domains found in transcriptional regulators linked to development, chromatin stability, and cancer. The MYND domains in the transcriptional regulators ETO (MTG8) and BS69 function as protein-protein interaction domains (6Wang J. Hoshino T. Redner R.L. Kajigaya S. Liu J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10860-10865Crossref PubMed Scopus (454) Google Scholar, 7Lutterbach B. Westendorf J.J. Linggi B. Patten A. Moniwa M. Davie J.R. Huynh K.D. Bardwell V.J. Lavinsky R.M. Rosenfeld M.G. Glass C. Seto E. Hiebert S.W. Mol. Cell. Biol. 1998; 18: 7176-7184Crossref PubMed Scopus (369) Google Scholar, 8Gelmetti V. Zhang J. Fanelli M. Minucci S. Pelicci P.G. Lazar M.A. Mol. Cell. Biol. 1998; 18: 7185-7191Crossref PubMed Scopus (428) Google Scholar, 9Masselink H. Bernards R. Oncogene. 2000; 19: 1538-1546Crossref PubMed Scopus (70) Google Scholar). The MYND of ETO has been implicated in the recruitment of HDACs1 (6Wang J. Hoshino T. Redner R.L. Kajigaya S. Liu J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10860-10865Crossref PubMed Scopus (454) Google Scholar, 7Lutterbach B. Westendorf J.J. Linggi B. Patten A. Moniwa M. Davie J.R. Huynh K.D. Bardwell V.J. Lavinsky R.M. Rosenfeld M.G. Glass C. Seto E. Hiebert S.W. Mol. Cell. Biol. 1998; 18: 7176-7184Crossref PubMed Scopus (369) Google Scholar, 8Gelmetti V. Zhang J. Fanelli M. Minucci S. Pelicci P.G. Lazar M.A. Mol. Cell. Biol. 1998; 18: 7185-7191Crossref PubMed Scopus (428) Google Scholar), and in BS69 the MYND domain is required for interaction with proteins bearing the PXLXP sequence (10Ansieau S. Leutz A. J. Biol. Chem. 2002; 277: 4906-4910Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Deleted in proofGoogle Scholar). The SET domains of several proteins, including SUV39H1, G9a, and Clr4, have been shown to function as histone methyltransferases (12Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2152) Google Scholar, 13Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). The significance of chromatin modifications in transcriptional regulation is currently the topic of great interest (14Cheung P. Allis C.D. Sassone-Corsi P. Cell. 2000; 103: 263-271Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar, 15Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar), and the presence in Bop of both MYND and SET domains implicated in histone deacetylation and methylation, respectively, suggests a role for Bop in chromatin regulation. Indeed, earlier studies have shown that m-Bop functions as a histone deacetylase-dependent transcriptional repressor (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar). Targeted inactivation of the Bop gene in mice resulted in embryonic death of Bop−/− null mice at approximately day E10.5 accompanied by failure of right ventricular development and accumulation of excessive extracellular matrix (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar).Bop-null mice failed to express the basic helix-loop-helix transcriptional regulator Hand2 in the heart, but not in the lateral plate mesoderm, suggesting that m-Bop plays an important specific role in the cardiac transcriptional cascade that leads to a functional adult heart (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar).The current studies demonstrate that m-Bop associates with the skeletal muscle- and heart-specific transcription factor skNAC. skNAC is an isoform of the ubiquitous protein, α-nascent polypeptide-associated complex (NAC) that was initially isolated as a heterodimeric complex that binds newly synthesized polypeptides emerging from ribosomes (17Wiedmann B. Sakai H. Davis T.A. Wiedmann M. Nature. 1994; 370: 434-440Crossref PubMed Scopus (319) Google Scholar). Further studies suggested that skNAC functions as a transcriptional coactivator (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). skNAC has been implicated in controlling myogenic differentiation, and skNAC transcripts are expressed at high levels only in skeletal and cardiac muscle (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar, 19Munz B. Wiedmann M. Lochmuller H. Werner S. J. Biol. Chem. 1999; 274: 13305-13310Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). skNAC mRNA is also induced in myoblasts of the panniculus carnosus within 12 h following skin injury, a finding that may reflect an early role for skNAC in muscle repair (19Munz B. Wiedmann M. Lochmuller H. Werner S. J. Biol. Chem. 1999; 274: 13305-13310Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We report here that m-Bop and skNAC interact in vitro and in vivo and display similar kinetics of expression during skeletal myogenesis in culture, suggesting an associated role for m-Bop and skNAC in striated muscle myogenesis. As observed for BS69 and related MYND domain-containing proteins, interaction of m-Bop and skNAC requires an intact m-Bop MYND domain and the presence of a PXLXP motif in skNAC.DISCUSSIONMYND and SET domains similar to those present in m-Bop are found in proteins linked to transcriptional regulation and are involved in various cellular processes including differentiation, proliferation, and chromatin stability. The Bop gene is expressed in skeletal muscle, heart, and cytotoxic T cells. MYND and SET domains of other proteins have been shown, respectively, to recruit HDACs and to have histone methyltransferase activity, and we have shown previously that m-Bop itself recruits HDACs and can act as a repressor of reporter gene expression in mammalian cells (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar).Bop-null mice died in utero at approximately day E10.5 with abnormal hearts having the following characteristics at day E9.5: absence of a right ventricle, poor trabeculation of the left ventricle, and accumulation of an abnormally large amount of extracellular matrix (cardiac jelly) (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar). Additionally, Hand2 expression in the developing heart was shown to be dependent on an intact Bop gene (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar). Furthermore, the homeobox protein IRX4, shown previously to be Hand2-dependent (22Bruneau B.G. Bao Z.Z. Tanaka M. Schott J.J. Izumo S. Cepko C.L. Seidman J.G Seidman C.E. Dev. Biol. 2000; 217: 266-277Crossref PubMed Scopus (155) Google Scholar), was down-regulated in Bop-null mice, suggesting that IRX4 is regulated, in part, by a m-Bop-Hand2 regulatory cascade (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar).In the present studies, the yeast two-hybrid system was employed to identify m-Bop-interacting proteins that are expressed in mouse skeletal muscle. Although m-Bop is expressed equally strongly in heart, skeletal muscle was chosen for study initially because of the availability of mouse cell lines such as C2C12 and 10T1/2 that can be induced to undergo myogenesis in tissue culture. These systems would allow the possibility of overexpression of wild type or mutant m-Bop, its interaction partners, and their fragments to assess effects on myotube formation.The muscle-specific transcription factor skNAC was isolated from a skeletal muscle library screen using GAL4-m-Bop2 as bait (Table I). skNAC has been implicated in activating transcription in a sequence-specific manner and controlling skeletal muscle myogenesis (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar), although its function is poorly understood. Previous studies reported that skNAC activates the myoglobin promoter in a sequence-specific manner, suggesting that it is a sequence-specific transcriptional activator (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). It was further reported that both skNAC and αNAC bind to the same consensus DNA sequence, thus ascribing the DNA-binding properties of skNAC to residues common with αNAC (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). However, a recent report demonstrated that αNAC binds nucleic acids non-specifically (23Beatrix B. Sakai H. Wiedmann M. J. Biol. Chem. 2000; 275: 37838-37845Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and αNAC has also been reported to bind to four-way junction DNA (24Whitby M.C. Dixon J. J. Mol. Biol. 2001; 306: 703-716Crossref PubMed Scopus (12) Google Scholar), reminiscent of HMG-box proteins and the SWI/SNF complex (25Zlatanova J. Van Holde K. FASEB J. 1998; 12: 421-431Crossref PubMed Scopus (85) Google Scholar).The skNAC fragments isolated using the two-hybrid system associated with m-Bop in 293T cells in coimmunoprecipitation assays (Fig. 2). Whereas, both m-Bop1 and m-Bop2 associated with skNAC, t-Bop, which lacks the heart- and skeletal muscle-specific amino-terminal S region (Fig. 3 A), failed to interact with skNAC in the two-hybrid system and in animal cells (Table I; Fig. 3 B). Because we were unable to obtain expression of full-length skNAC in mammalian cells, m-Bop interaction with in vitro translated full-length skNAC was examined. Recombinant m-Bop2–6XHis was found to interact with in vitro translated full-length skNAC in a specific manner (Fig. 2).Structural mapping revealed that an intact MYND domain and amino-terminal S sequences of m-Bop were required for interaction with skNAC in mammalian cells (Fig. 3). It is likely that these m-Bop domains cooperate with one another to create a structural motif not present in t-Bop, which contains only the MYND domain. The m-Bop interaction domain of skNAC was found to be located in a carboxyl terminal region of skNAC (Fig. 4, A and B). Truncation and site-directed mutagenesis studies demonstrated that a PXLXP sequence (PPLIP) encoded near the end of the large skNAC-specific exon was necessary for m-Bop association (Fig.4, C and D). The PXLXP motif was previously shown to be essential for interaction of the closely related MYND domains of the transcriptional corepressor protein and potential tumor suppressor, BS69, and the Caenorhabditis elegans proteins, Bra-1 and Bra-2, with viral oncoproteins and the Myc-related cellular protein, MGA (10Ansieau S. Leutz A. J. Biol. Chem. 2002; 277: 4906-4910Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The present results further support a commonality in the interactions of MYND-containing proteins with their protein partners. A second PXLXP motif (PPLEP) is present near the amino terminus of the skNAC-specific exon, suggesting a second potential m-Bop-binding motif. This may explain m-Bop2 binding to premature transcription/translation skNAC fragmentsin vitro (Fig. 2 C). Additional sequences of skNAC may contribute to m-Bop association because FLAG-N5 weakly bound to m-Bop2–6XHis in vitro, although to a substantially lower extent than FLAG-N4 (data not shown).During skeletal muscle differentiation, distinct transcriptional programs direct the proper formation of mature myotubes. In previous studies, skNAC was reported to adversely affect myogenesis when overexpressed in differentiating myotubes (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). In the present studies, Western blots demonstrate that m-Bop and skNAC are induced within the first 24 h of myogenesis observed in cell culture (Fig. 5). Both proteins continue to be expressed at high levels up to 6 days after induction of differentiation. While m-Bop protein levels remain high, skNAC expression is reduced dramatically 2 weeks after the onset of myogenesis (Fig. 5). Immunofluorescence studies indicated that m-Bop and skNAC were localized in the nucleus early in C2C12 differentiation, followed by greatly enhanced expression in the cytoplasm as myogenesis progressed (Fig. 5). Studies in progress with adenovirus vectors expressing skNAC fragments with the wild type (PPLIPP) or mutant (PPAIPP) m-Bop interaction motifs should yield information on the functional significance of m-Bop/skNAC interaction during skeletal muscle myogenesis. Because m-Bop and skNAC are expressed in the heart and recent studies suggest that they are colocalized in the embryonic heart, 2S. Pierce, R. Sims, D. Srivastava, and P. Gottlieb, unpublished studies. the present studies of skeletal muscle myogenesis should shed light on the role of these proteins in cardiogenesis as well. The control of skeletal and cardiac maturation is ordered in a complex cascade of transcriptional activation and repression. The MyoD and MEF2 families of transcription factors dictate important developmental events that result in the formation of mature skeletal muscle. The MyoD family is comprised of the basic helix-loop-helix proteins MyoD, Myf5, MRF4, and myogenin. These myogenic regulators form heterodimers with the ubiquitous basic helix-loop-helix E proteins that subsequently activate key elements needed for the myogenic program (1Olson E.N. Klein W.H. Genes Dev. 1994; 8: 1-8Crossref PubMed Scopus (606) Google Scholar). The MEF2 family is defined by the MADS domain and consists of MEF2A, MEF2B, MEF2C, and MEF2D. Members of the MEF2 family are expressed in tissues besides skeletal muscle including cardiac tissue, neurons, and T cells (2Naya F.J. Olson E. Curr. Opin. Cell Biol. 1999; 11: 683-688Crossref PubMed Scopus (253) Google Scholar). Together, the MyoD and MEF2 families cooperate directly and indirectly to transduce the requisite signals for proper skeletal muscle formation. The discovery of chamber-specific transcription factors, such as the Hand1 and Hand2 proteins, has revealed the complex nature of cardiac transcriptional regulation (3Srivastava D. Thomas T. Lin Q. Kirby M.L. Brown D. Olson E.N. Nat. Genet. 1997; 16: 154-160Crossref PubMed Scopus (556) Google Scholar). The list of genes that correspond to specific cardiac defects continues to grow, whereas the molecular nature of these defects remains largely elusive (4Srivastava D. Olson E.N. Nature. 2000; 407: 221-226Crossref PubMed Scopus (523) Google Scholar). To further understand the molecular underpinnings of cardiac development, it is critical to identify the relationships of transcriptional regulators shown to be important in cardiac morphogenesis. The Bop gene encodes distinct proteins expressed in skeletal and cardiac muscle, as well as in cytotoxic T lymphocytes. The Bop proteins found in skeletal muscle (m-Bop) and cytotoxic T lymphocytes (t-Bop) are identical over 90% of their primary amino acid sequence, differing only at their extreme amino terminus (5Hwang I. Gottlieb P.D. J. Immunol. 1997; 158: 1165-1174PubMed Google Scholar). Bop contains the evolutionarily conserved MYND and SET domains found in transcriptional regulators linked to development, chromatin stability, and cancer. The MYND domains in the transcriptional regulators ETO (MTG8) and BS69 function as protein-protein interaction domains (6Wang J. Hoshino T. Redner R.L. Kajigaya S. Liu J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10860-10865Crossref PubMed Scopus (454) Google Scholar, 7Lutterbach B. Westendorf J.J. Linggi B. Patten A. Moniwa M. Davie J.R. Huynh K.D. Bardwell V.J. Lavinsky R.M. Rosenfeld M.G. Glass C. Seto E. Hiebert S.W. Mol. Cell. Biol. 1998; 18: 7176-7184Crossref PubMed Scopus (369) Google Scholar, 8Gelmetti V. Zhang J. Fanelli M. Minucci S. Pelicci P.G. Lazar M.A. Mol. Cell. Biol. 1998; 18: 7185-7191Crossref PubMed Scopus (428) Google Scholar, 9Masselink H. Bernards R. Oncogene. 2000; 19: 1538-1546Crossref PubMed Scopus (70) Google Scholar). The MYND of ETO has been implicated in the recruitment of HDACs1 (6Wang J. Hoshino T. Redner R.L. Kajigaya S. Liu J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10860-10865Crossref PubMed Scopus (454) Google Scholar, 7Lutterbach B. Westendorf J.J. Linggi B. Patten A. Moniwa M. Davie J.R. Huynh K.D. Bardwell V.J. Lavinsky R.M. Rosenfeld M.G. Glass C. Seto E. Hiebert S.W. Mol. Cell. Biol. 1998; 18: 7176-7184Crossref PubMed Scopus (369) Google Scholar, 8Gelmetti V. Zhang J. Fanelli M. Minucci S. Pelicci P.G. Lazar M.A. Mol. Cell. Biol. 1998; 18: 7185-7191Crossref PubMed Scopus (428) Google Scholar), and in BS69 the MYND domain is required for interaction with proteins bearing the PXLXP sequence (10Ansieau S. Leutz A. J. Biol. Chem. 2002; 277: 4906-4910Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Deleted in proofGoogle Scholar). The SET domains of several proteins, including SUV39H1, G9a, and Clr4, have been shown to function as histone methyltransferases (12Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2152) Google Scholar, 13Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). The significance of chromatin modifications in transcriptional regulation is currently the topic of great interest (14Cheung P. Allis C.D. Sassone-Corsi P. Cell. 2000; 103: 263-271Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar, 15Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar), and the presence in Bop of both MYND and SET domains implicated in histone deacetylation and methylation, respectively, suggests a role for Bop in chromatin regulation. Indeed, earlier studies have shown that m-Bop functions as a histone deacetylase-dependent transcriptional repressor (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar). Targeted inactivation of the Bop gene in mice resulted in embryonic death of Bop−/− null mice at approximately day E10.5 accompanied by failure of right ventricular development and accumulation of excessive extracellular matrix (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar).Bop-null mice failed to express the basic helix-loop-helix transcriptional regulator Hand2 in the heart, but not in the lateral plate mesoderm, suggesting that m-Bop plays an important specific role in the cardiac transcriptional cascade that leads to a functional adult heart (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar). The current studies demonstrate that m-Bop associates with the skeletal muscle- and heart-specific transcription factor skNAC. skNAC is an isoform of the ubiquitous protein, α-nascent polypeptide-associated complex (NAC) that was initially isolated as a heterodimeric complex that binds newly synthesized polypeptides emerging from ribosomes (17Wiedmann B. Sakai H. Davis T.A. Wiedmann M. Nature. 1994; 370: 434-440Crossref PubMed Scopus (319) Google Scholar). Further studies suggested that skNAC functions as a transcriptional coactivator (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). skNAC has been implicated in controlling myogenic differentiation, and skNAC transcripts are expressed at high levels only in skeletal and cardiac muscle (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar, 19Munz B. Wiedmann M. Lochmuller H. Werner S. J. Biol. Chem. 1999; 274: 13305-13310Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). skNAC mRNA is also induced in myoblasts of the panniculus carnosus within 12 h following skin injury, a finding that may reflect an early role for skNAC in muscle repair (19Munz B. Wiedmann M. Lochmuller H. Werner S. J. Biol. Chem. 1999; 274: 13305-13310Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We report here that m-Bop and skNAC interact in vitro and in vivo and display similar kinetics of expression during skeletal myogenesis in culture, suggesting an associated role for m-Bop and skNAC in striated muscle myogenesis. As observed for BS69 and related MYND domain-containing proteins, interaction of m-Bop and skNAC requires an intact m-Bop MYND domain and the presence of a PXLXP motif in skNAC. DISCUSSIONMYND and SET domains similar to those present in m-Bop are found in proteins linked to transcriptional regulation and are involved in various cellular processes including differentiation, proliferation, and chromatin stability. The Bop gene is expressed in skeletal muscle, heart, and cytotoxic T cells. MYND and SET domains of other proteins have been shown, respectively, to recruit HDACs and to have histone methyltransferase activity, and we have shown previously that m-Bop itself recruits HDACs and can act as a repressor of reporter gene expression in mammalian cells (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar).Bop-null mice died in utero at approximately day E10.5 with abnormal hearts having the following characteristics at day E9.5: absence of a right ventricle, poor trabeculation of the left ventricle, and accumulation of an abnormally large amount of extracellular matrix (cardiac jelly) (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar). Additionally, Hand2 expression in the developing heart was shown to be dependent on an intact Bop gene (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar). Furthermore, the homeobox protein IRX4, shown previously to be Hand2-dependent (22Bruneau B.G. Bao Z.Z. Tanaka M. Schott J.J. Izumo S. Cepko C.L. Seidman J.G Seidman C.E. Dev. Biol. 2000; 217: 266-277Crossref PubMed Scopus (155) Google Scholar), was down-regulated in Bop-null mice, suggesting that IRX4 is regulated, in part, by a m-Bop-Hand2 regulatory cascade (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar).In the present studies, the yeast two-hybrid system was employed to identify m-Bop-interacting proteins that are expressed in mouse skeletal muscle. Although m-Bop is expressed equally strongly in heart, skeletal muscle was chosen for study initially because of the availability of mouse cell lines such as C2C12 and 10T1/2 that can be induced to undergo myogenesis in tissue culture. These systems would allow the possibility of overexpression of wild type or mutant m-Bop, its interaction partners, and their fragments to assess effects on myotube formation.The muscle-specific transcription factor skNAC was isolated from a skeletal muscle library screen using GAL4-m-Bop2 as bait (Table I). skNAC has been implicated in activating transcription in a sequence-specific manner and controlling skeletal muscle myogenesis (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar), although its function is poorly understood. Previous studies reported that skNAC activates the myoglobin promoter in a sequence-specific manner, suggesting that it is a sequence-specific transcriptional activator (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). It was further reported that both skNAC and αNAC bind to the same consensus DNA sequence, thus ascribing the DNA-binding properties of skNAC to residues common with αNAC (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). However, a recent report demonstrated that αNAC binds nucleic acids non-specifically (23Beatrix B. Sakai H. Wiedmann M. J. Biol. Chem. 2000; 275: 37838-37845Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and αNAC has also been reported to bind to four-way junction DNA (24Whitby M.C. Dixon J. J. Mol. Biol. 2001; 306: 703-716Crossref PubMed Scopus (12) Google Scholar), reminiscent of HMG-box proteins and the SWI/SNF complex (25Zlatanova J. Van Holde K. FASEB J. 1998; 12: 421-431Crossref PubMed Scopus (85) Google Scholar).The skNAC fragments isolated using the two-hybrid system associated with m-Bop in 293T cells in coimmunoprecipitation assays (Fig. 2). Whereas, both m-Bop1 and m-Bop2 associated with skNAC, t-Bop, which lacks the heart- and skeletal muscle-specific amino-terminal S region (Fig. 3 A), failed to interact with skNAC in the two-hybrid system and in animal cells (Table I; Fig. 3 B). Because we were unable to obtain expression of full-length skNAC in mammalian cells, m-Bop interaction with in vitro translated full-length skNAC was examined. Recombinant m-Bop2–6XHis was found to interact with in vitro translated full-length skNAC in a specific manner (Fig. 2).Structural mapping revealed that an intact MYND domain and amino-terminal S sequences of m-Bop were required for interaction with skNAC in mammalian cells (Fig. 3). It is likely that these m-Bop domains cooperate with one another to create a structural motif not present in t-Bop, which contains only the MYND domain. The m-Bop interaction domain of skNAC was found to be located in a carboxyl terminal region of skNAC (Fig. 4, A and B). Truncation and site-directed mutagenesis studies demonstrated that a PXLXP sequence (PPLIP) encoded near the end of the large skNAC-specific exon was necessary for m-Bop association (Fig.4, C and D). The PXLXP motif was previously shown to be essential for interaction of the closely related MYND domains of the transcriptional corepressor protein and potential tumor suppressor, BS69, and the Caenorhabditis elegans proteins, Bra-1 and Bra-2, with viral oncoproteins and the Myc-related cellular protein, MGA (10Ansieau S. Leutz A. J. Biol. Chem. 2002; 277: 4906-4910Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The present results further support a commonality in the interactions of MYND-containing proteins with their protein partners. A second PXLXP motif (PPLEP) is present near the amino terminus of the skNAC-specific exon, suggesting a second potential m-Bop-binding motif. This may explain m-Bop2 binding to premature transcription/translation skNAC fragmentsin vitro (Fig. 2 C). Additional sequences of skNAC may contribute to m-Bop association because FLAG-N5 weakly bound to m-Bop2–6XHis in vitro, although to a substantially lower extent than FLAG-N4 (data not shown).During skeletal muscle differentiation, distinct transcriptional programs direct the proper formation of mature myotubes. In previous studies, skNAC was reported to adversely affect myogenesis when overexpressed in differentiating myotubes (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). In the present studies, Western blots demonstrate that m-Bop and skNAC are induced within the first 24 h of myogenesis observed in cell culture (Fig. 5). Both proteins continue to be expressed at high levels up to 6 days after induction of differentiation. While m-Bop protein levels remain high, skNAC expression is reduced dramatically 2 weeks after the onset of myogenesis (Fig. 5). Immunofluorescence studies indicated that m-Bop and skNAC were localized in the nucleus early in C2C12 differentiation, followed by greatly enhanced expression in the cytoplasm as myogenesis progressed (Fig. 5). Studies in progress with adenovirus vectors expressing skNAC fragments with the wild type (PPLIPP) or mutant (PPAIPP) m-Bop interaction motifs should yield information on the functional significance of m-Bop/skNAC interaction during skeletal muscle myogenesis. Because m-Bop and skNAC are expressed in the heart and recent studies suggest that they are colocalized in the embryonic heart, 2S. Pierce, R. Sims, D. Srivastava, and P. Gottlieb, unpublished studies. the present studies of skeletal muscle myogenesis should shed light on the role of these proteins in cardiogenesis as well. MYND and SET domains similar to those present in m-Bop are found in proteins linked to transcriptional regulation and are involved in various cellular processes including differentiation, proliferation, and chromatin stability. The Bop gene is expressed in skeletal muscle, heart, and cytotoxic T cells. MYND and SET domains of other proteins have been shown, respectively, to recruit HDACs and to have histone methyltransferase activity, and we have shown previously that m-Bop itself recruits HDACs and can act as a repressor of reporter gene expression in mammalian cells (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar).Bop-null mice died in utero at approximately day E10.5 with abnormal hearts having the following characteristics at day E9.5: absence of a right ventricle, poor trabeculation of the left ventricle, and accumulation of an abnormally large amount of extracellular matrix (cardiac jelly) (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar). Additionally, Hand2 expression in the developing heart was shown to be dependent on an intact Bop gene (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar). Furthermore, the homeobox protein IRX4, shown previously to be Hand2-dependent (22Bruneau B.G. Bao Z.Z. Tanaka M. Schott J.J. Izumo S. Cepko C.L. Seidman J.G Seidman C.E. Dev. Biol. 2000; 217: 266-277Crossref PubMed Scopus (155) Google Scholar), was down-regulated in Bop-null mice, suggesting that IRX4 is regulated, in part, by a m-Bop-Hand2 regulatory cascade (16Gottlieb P.D. Pierce S.A. Sims III, R.J. Weihe E.K. Harriss J.V. Maika S.D. Kuziel W.A. King H.L. Yamagishi H. Olson E.N. Nakagawa O. Srivastava D. Nat. Genet. 2002; 31: 25-32Crossref PubMed Scopus (259) Google Scholar). In the present studies, the yeast two-hybrid system was employed to identify m-Bop-interacting proteins that are expressed in mouse skeletal muscle. Although m-Bop is expressed equally strongly in heart, skeletal muscle was chosen for study initially because of the availability of mouse cell lines such as C2C12 and 10T1/2 that can be induced to undergo myogenesis in tissue culture. These systems would allow the possibility of overexpression of wild type or mutant m-Bop, its interaction partners, and their fragments to assess effects on myotube formation. The muscle-specific transcription factor skNAC was isolated from a skeletal muscle library screen using GAL4-m-Bop2 as bait (Table I). skNAC has been implicated in activating transcription in a sequence-specific manner and controlling skeletal muscle myogenesis (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar), although its function is poorly understood. Previous studies reported that skNAC activates the myoglobin promoter in a sequence-specific manner, suggesting that it is a sequence-specific transcriptional activator (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). It was further reported that both skNAC and αNAC bind to the same consensus DNA sequence, thus ascribing the DNA-binding properties of skNAC to residues common with αNAC (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). However, a recent report demonstrated that αNAC binds nucleic acids non-specifically (23Beatrix B. Sakai H. Wiedmann M. J. Biol. Chem. 2000; 275: 37838-37845Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and αNAC has also been reported to bind to four-way junction DNA (24Whitby M.C. Dixon J. J. Mol. Biol. 2001; 306: 703-716Crossref PubMed Scopus (12) Google Scholar), reminiscent of HMG-box proteins and the SWI/SNF complex (25Zlatanova J. Van Holde K. FASEB J. 1998; 12: 421-431Crossref PubMed Scopus (85) Google Scholar). The skNAC fragments isolated using the two-hybrid system associated with m-Bop in 293T cells in coimmunoprecipitation assays (Fig. 2). Whereas, both m-Bop1 and m-Bop2 associated with skNAC, t-Bop, which lacks the heart- and skeletal muscle-specific amino-terminal S region (Fig. 3 A), failed to interact with skNAC in the two-hybrid system and in animal cells (Table I; Fig. 3 B). Because we were unable to obtain expression of full-length skNAC in mammalian cells, m-Bop interaction with in vitro translated full-length skNAC was examined. Recombinant m-Bop2–6XHis was found to interact with in vitro translated full-length skNAC in a specific manner (Fig. 2). Structural mapping revealed that an intact MYND domain and amino-terminal S sequences of m-Bop were required for interaction with skNAC in mammalian cells (Fig. 3). It is likely that these m-Bop domains cooperate with one another to create a structural motif not present in t-Bop, which contains only the MYND domain. The m-Bop interaction domain of skNAC was found to be located in a carboxyl terminal region of skNAC (Fig. 4, A and B). Truncation and site-directed mutagenesis studies demonstrated that a PXLXP sequence (PPLIP) encoded near the end of the large skNAC-specific exon was necessary for m-Bop association (Fig.4, C and D). The PXLXP motif was previously shown to be essential for interaction of the closely related MYND domains of the transcriptional corepressor protein and potential tumor suppressor, BS69, and the Caenorhabditis elegans proteins, Bra-1 and Bra-2, with viral oncoproteins and the Myc-related cellular protein, MGA (10Ansieau S. Leutz A. J. Biol. Chem. 2002; 277: 4906-4910Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The present results further support a commonality in the interactions of MYND-containing proteins with their protein partners. A second PXLXP motif (PPLEP) is present near the amino terminus of the skNAC-specific exon, suggesting a second potential m-Bop-binding motif. This may explain m-Bop2 binding to premature transcription/translation skNAC fragmentsin vitro (Fig. 2 C). Additional sequences of skNAC may contribute to m-Bop association because FLAG-N5 weakly bound to m-Bop2–6XHis in vitro, although to a substantially lower extent than FLAG-N4 (data not shown). During skeletal muscle differentiation, distinct transcriptional programs direct the proper formation of mature myotubes. In previous studies, skNAC was reported to adversely affect myogenesis when overexpressed in differentiating myotubes (18Yotov W.V. St-Arnaud R. Genes Dev. 1996; 10: 1763-1772Crossref PubMed Scopus (73) Google Scholar). In the present studies, Western blots demonstrate that m-Bop and skNAC are induced within the first 24 h of myogenesis observed in cell culture (Fig. 5). Both proteins continue to be expressed at high levels up to 6 days after induction of differentiation. While m-Bop protein levels remain high, skNAC expression is reduced dramatically 2 weeks after the onset of myogenesis (Fig. 5). Immunofluorescence studies indicated that m-Bop and skNAC were localized in the nucleus early in C2C12 differentiation, followed by greatly enhanced expression in the cytoplasm as myogenesis progressed (Fig. 5). Studies in progress with adenovirus vectors expressing skNAC fragments with the wild type (PPLIPP) or mutant (PPAIPP) m-Bop interaction motifs should yield information on the functional significance of m-Bop/skNAC interaction during skeletal muscle myogenesis. Because m-Bop and skNAC are expressed in the heart and recent studies suggest that they are colocalized in the embryonic heart, 2S. Pierce, R. Sims, D. Srivastava, and P. Gottlieb, unpublished studies. the present studies of skeletal muscle myogenesis should shed light on the role of these proteins in cardiogenesis as well. We thank Dr. Clarence Chan for his helpful advice and for the generous use of his fluorescence microscope.

Referência(s)