Judith K. Davie

Loss of myogenin in postnatal life leads to normal skeletal muscle but reduced body size

Although the mechanisms regulating the formation of embryonic skeletal muscle in vertebrates are well characterized, less is known about postnatal muscle formation even though the largest increases in skeletal muscle mass occur after birth. Adult muscle stem cells (satellite cells) appear to recapitulate the events that occur in embryonic myoblasts. In particular, the myogenic basic helix-loop-helix factors, which have crucial functions in embryonic muscle development, are assumed to have similar roles in postnatal muscle formation. Here, we test this assumption by determining the role of the myogenic regulator myogenin in postnatal life. Because Myog-null mice die at birth, we generated mice with floxed alleles of Myog and mated them to transgenic mice expressing Cre recombinase to delete Myog before and after embryonic muscle development. Removing myogenin before embryonic muscle development resulted in myofiber deficiencies identical to those observed in Myog-null mice. However, mice in which Myog was deleted following embryonic muscle development had normal skeletal muscle, except for modest alterations in the levels of transcripts encoding Mrf4 (Myf6) and Myod1 (MyoD). Notably, Myog-deleted mice were 30% smaller than control mice, suggesting that the absence of myogenin disrupted general body growth. Our results suggest that postnatal skeletal muscle growth is controlled by mechanisms distinct from those occurring in embryonic muscle development and uncover an unsuspected non-cell autonomous role for myogenin in the regulation of tissue growth.

Site-specific loss of acetylation upon phosphorylation of histone H3

Post-translational modification of histones is a central aspect of gene regulation. Emerging data indicate that modification at one site can influence modification of a second site. As one example, histone H3 phosphorylation at serine 10 (Ser10) facilitates acetylation of lysine 14 (Lys14) by Gcn5 in vitro (1, 2). In vivo, phosphorylation of H3 precedes acetylation at certain promoters. Whether H3 phosphorylation globally affects acetylation, or whether it affects all acetylation sites in H3 equally, is not known. We have taken a genetic approach to this question by mutating Ser10 in H3 to fix either a negative or a neutral charge at this position, followed by analysis of the acetylation states of the mutant histones using site-specific antibodies. Surprisingly, we find that conversion of Ser10 to glutamate (S10E) or aspartate (S10D) causes almost complete loss of H3 acetylation at lysine 9 (Lys9) in vivo. Acetylation of Lys9is also significantly reduced in cells bearing mutations in the Glc7 phosphatase that increase H3 phosphorylation levels. Mutation of Ser10 in H3 and the concomitant loss of Lys9acetylation has minimal effects on expression of a Gcn5-dependent reporter gene. However, synergistic growth defects are observed upon loss of GCN5 in cells bearing H3 Ser10 mutations that are reminiscent of delays in G2/M progression caused by combined loss ofGCN5 and acetylation site mutations. Together these results demonstrate that H3 phosphorylation directly causes site-specific and opposite changes in acetylation levels of two residues within this histone, Lys9 and Lys14, and they highlight the importance of these histone modifications to normal cell functions.

Tup1-Ssn6 interacts with multiple class I histone deacetylases in vivo

The Tup1-Ssn6 corepressor complex in Saccharomyces cerevisiae represses the transcription of a diverse set of genes. Chromatin is an important component of Tup1-Ssn6-mediated repression. Tup1 binds to underacetylated histone tails and requires multiple histone deacetylases (HDACs) for its repressive functions. Here, we describe physical interactions of the corepressor complex with the class I HDACs Rpd3, Hos2, and Hos1. In contrast, no in vivo interaction was observed between Tup-Ssn6 and Hda1, a class II HDAC. We demonstrate that Rpd3 interacts with both Tup1 and Ssn6. Rpd3 and Hos2 interact with Ssn6 independently of Tup1 via distinct tetratricopeptide domains within Ssn6, suggesting that these two HDACs may contact the corepressor at the same time.

Histone-Dependent Association of Tup1-Ssn6 with Repressed Genes In Vivo

ABSTRACT The Tup1-Ssn6 complex regulates diverse classes of genes in Saccharomyces cerevisiae and serves as a model for corepressor functions in many organisms. Tup1-Ssn6 does not directly bind DNA but is brought to target genes through interactions with sequence-specific DNA binding factors. Full repression by Tup1-Ssn6 appears to require interactions with both the histone tails and components of the general transcription machinery, although the relative contribution of these two pathways is not clear. Here, we map Tup1 locations on two classes of Tup1-Ssn6-regulated genes in vivo via chromatin immunoprecipitations. Distinct profiles of Tup1 are observed on a cell-specific genes and DNA damage-inducible genes, suggesting that alternate repressive architectures may be created on different classes of repressed genes. In both cases, decreases in acetylation of histone H3 colocalize with Tup1. Strikingly, although loss of the Srb10 mediator protein had no effect on Tup1 localization, both histone tail mutations and histone deacetylase mutations crippled the association of Tup1 with target loci. Together with previous findings that Tup1-Ssn6 physically associates with histone deacetylase activities, these results indicate that the repressor complex alters histone modification states to facilitate interactions with histones and that these interactions are required to maintain a stable repressive state.

Genetic interactions between TFIIS and the Swi-Snf chromatin-remodeling complex.

ABSTRACT The eukaryotic transcript elongation factor TFIIS enables RNA polymerase II to read through blocks to elongation in vitro and interacts genetically with a variety of components of the transcription machinery in vivo. In Saccharomyces cerevisiae, the gene encoding TFIIS (PPR2) is not essential, and disruption strains exhibit only mild phenotypes and an increased sensitivity to 6-azauracil. The nonessential nature of TFIIS encouraged the use of a synthetic lethal screen to elucidate the in vivo roles of TFIIS as well as provide more information on other factors involved in the regulation of transcript elongation. Several genes were identified that are necessary for either cell survival or robust growth when the gene encoding TFIIS has been disrupted. These include UBP3,KEX2, STT4, and SWI2/SNF2. SWI1 andSNF5 disruptions were also synthetically lethal withppr2Δ, suggesting that the reduced ability to remodel chromatin confers the synthetic phenotype. The synthetic phenotypes show marked osmosensitivity and cytoskeletal defects, including a terminal hyperelongated bud phenotype with the Swi-Snf complex. These results suggest that genes important in osmoregulation, cell membrane synthesis and integrity, and cell division may require the Swi-Snf complex and TFIIS for efficient transcription. The detection of these genetic interactions provides another functional link between the Swi-Snf complex and the elongation machinery.

Transcriptional control: an activating role for arginine methylation.

A rare histone modification, arginine methylation, has been linked to activation of hormone-responsive genes. Interestingly, methylation of a lysine residue in the same histone is present prior to hormone activation, but is excluded from the active loci.

Alternative Splicing of MEF2C pre-mRNA Controls Its Activity in Normal Myogenesis and Promotes Tumorigenicity in Rhabdomyosarcoma Cells

Background: MEF2C is an important regulator of many developmental programs. Results: Alternative splicing of the α exon of MEF2C regulates myogenesis. Loss of SRPK3 in rhabdomyosarcoma cells inhibits this splicing and blocks differentiation. Conclusion: MEF2Cα2 promotes myogenesis, and restoration of MEF2Cα2 in rhabdomyosarcoma cells inhibits growth. Significance: Defining the function and deregulation of MEF2Cα2 enhances the understanding of normal myogenesis and RMS tumorigenesis. Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Many cellular disruptions contribute to the progression of this pediatric cancer, including aberrant alternative splicing. The MEF2 family of transcription factors regulates many developmental programs, including myogenesis. MEF2 gene transcripts are subject to alternate splicing to generate protein isoforms with divergent functions. We found that MEF2Cα1 was the ubiquitously expressed isoform that exhibited no myogenic activity and that MEF2Cα2, the muscle-specific MEF2C isoform, was required for efficient differentiation. We showed that exon α in MEF2C was aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 highly down-regulated in RMS cells compared with normal myoblasts. Compared with MEF2Cα2, MEF2Cα1 interacted more strongly with and recruited HDAC5 to myogenic gene promoters to repress muscle-specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increased myogenic activity and promoted differentiation in RMS cells. We also identified a serine protein kinase, SRPK3, that was down-regulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage-independent growth of RMS cells. Together, our findings indicate that the alternative splicing of MEF2C plays an important role in normal myogenesis and RMS development. An improved understanding of alternative splicing events in RMS cells will potentially reveal novel therapeutic targets for RMS treatment.

Histone modifications in corepressor functions.

Publisher Summary This chapter emphasizes histone modifications in corepressor functions. Histone modifications play a regulatory role in gene expression by altering higher order chromatin structures and by providing docking sites for non-histone regulatory proteins. Many corepressor complexes contain or recruit histone-modifying activities. A few examples of such interactions are described to illustrate the fundamental connections between transcriptional repression and the organization of chromatin––namely, (1) Tup1–Ssn6 and Groucho/ transducin-like enhancer of split (TLE) proteins, (2) silencing mediator of retinoic acid and thyroid hormone receptors (SMRT)/nuclear receptor corepressor (N-CoR), (3) Sin3, (4) C-terminal binding protein (CtBP), (5) retinoblastoma (Rb) protein, and (6) nucleosome remodeling and deacetylase (NuRD). Tup1–Ssn6 mediates repression of a large and diverse set of genes in Saccharomyces cerevisiae. Examples of gene classes regulated by this corepressor complex are genes that are repressed by glucose, genes that respond to hypoxia, genes induced by DNA damage, and cell type-specific genes.

CIITA is silenced by epigenetic mechanisms that prevent the recruitment of transactivating factors in rhabdomyosarcoma cells

Rhabdomyosarcomas (RMS) are highly malignant pediatric sarcomas. We have discovered that the gene encoding the major histocompatibilty complex class II transactivator, CIITA, is silenced in cells representing both major subtypes of RMS. Silencing of CIITA prevents the IFN‐γ inducible expression of MHC class II genes in these cells. Overexpression of CIITA in these cells can restore MHC expression. We have found that IFN‐γ signaling is intact in these cells, but pSTAT1 and IRF1 do not bind to the CIITA PIV promoter. The CIITA promoter is not hypermethylated in RD (ERMS) cells but does show a modestly enhanced methylation status in SJRH30 (ARMS) cells. We have found that histone acetylation, which normally increases on the CIITA PIV promoter following IFN‐γ treatment, is blocked in both types of RMS cells. In RD cells, treatment with a histone deacetylase inhibitor (TSA) reverses the silencing of CIITA. In SJRH30 cells, treatment with DNA methyltransferase inhibitors and TSA cooperatively restores CIITA expression. Surprisingly, we have also shown that the expression of two components of the immunoproteasome, which are embedded in the class II locus, is stimulated by IFN‐γ in certain RMS cells in the absence of stimulation by CIITA. CIITA overexpression can also activate the expression of these genes, indicating that the immunoproteasome genes LMP2 and LMP7 can be activated by both CIITA dependent and CIITA independent pathways.

TBX2 blocks myogenesis and promotes proliferation in rhabdomyosarcoma cells

Rhabdomyosarcomas (RMSs) are the most frequent soft tissue sarcomas in children that share many features of developing skeletal muscle. We have discovered that a T‐box family member, TBX2, is highly upregulated in tumor cells of both major RMS subtypes. TBX2 is a repressor that is often overexpressed in cancer cells and is thought to function in bypassing cell growth control, including repression of p14 and p21. The cell cycle regulator p21 is required for the terminal differentiation of skeletal muscle cells and is silenced in RMS cells. We have found that TBX2 interacts with the myogenic regulatory factors MyoD and myogenin and inhibits the activity of these factors. TBX2 is expressed in primary myoblasts and C2C12 cells, but is strongly downregulated upon differentiation. TBX2 recruits the histone deacetylase HDAC1 and is a potent inhibitor of the expression of muscle‐specific genes and the cell cycle regulators, p21 and p14. TBX2 promotes the proliferation of RMS cells and either depletions of TBX2 or dominant negative TBX2 upregulate p21‐ and muscle‐specific genes. Significantly, depletion or interference with TBX2 completely inhibits tumor growth in a xenograft assay, highlighting the oncogenic role of TBX2 in RMS cells. Thus, the data demonstrate that elevated expression of TBX2 contributes to the pathology of RMS cells by promoting proliferation and repressing differentiation‐specific gene expression. These results show that deregulated TBX2 serves as an oncogene in RMS, suggesting that TBX2 may serve as a new diagnostic marker or therapeutic target for RMS tumors.