Histone methylation is known to be associated with both transcriptionally active and repressive chromatin states. Recent studies have identified SET domain-containing proteins such as SUV39H1 and Clr4 as mediators of H3 lysine 9 (Lys9) methylation and heterochromatin formation. Interestingly, H3 Lys9 methylation is not observed from bulk histones isolated from asynchronous populations of Saccharomyces cerevisiae or Tetrahymena thermophila. In contrast, H3 lysine 4 (Lys4) methylation is a predominant modification in these smaller eukaryotes. To identify the responsible methyltransferase(s) and to gain insight into the function of H3 Lys4 methylation, we have developed a histone H3 Lys4 methyl-specific antiserum. With this antiserum, we show that deletion of SET1, but not of other putative SET domain-containing genes, in S. cerevisiae, results in the complete abolishment of H3 Lys4 methylation in vivo. Furthermore, loss of H3 Lys4 methylation in a set1⌬ strain can be rescued by SET1. Analysis of histone H3 mutations at Lys4 revealed a slow-growth defect similar to a set1⌬ strain. Chromatin immunoprecipitation assays show that H3 Lys4 methylation is present at the rDNA locus and that Set1-mediated H3 Lys4 methylation is required for repression of RNA polymerase II transcription within rDNA. Taken together, these data suggest that Set1-mediated H3 Lys4 methylation is required for normal cell growth and transcriptional silencing.

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.KEY WORDS: Skeletal muscle growth, Myogenic bHLH transcription factors, Myogenin, Conditional knockout mice Development 133,[601][602][603][604][605][606][607][608][609][610]

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.The Tup1-Ssn6 corepressor complex mediates repression of a large and diverse set of genes in Saccharomyces cerevisiae (reviewed in Ref. 1). Examples of gene classes regulated by this co-repressor complex are genes that are repressed by glucose (e.g. SUC2), genes that respond to hypoxia (e.g. ANB1), genes induced by DNA damage (e.g. RNR2), and cell type-specific genes (e.g. STE6). Tup1-Ssn6 does not bind directly to DNA but is recruited to target genes by interactions with DNA-bound repressor proteins. The molecular mechanism by which Tup1-Ssn6 inhibits transcription is not fully understood, but Tup1-Ssn6 probably uses both interactions with chromatin and interactions with the general transcription machinery to achieve repression. Many subunits of the mediator complex that is associated with the C-terminal domain of the largest subunit of RNA polymerase II interact both genetically and physically with Tup1-Ssn6 (2-5).Tup1-Ssn6 also directly interacts with histones and influences the organization of chromatin. Certain repressed genes under Tup1-Ssn6 control are packaged into highly positioned nucleosomes during repression (6 -9). Tup1 binds preferentially to underacetylated H3 and H4 amino-terminal histone tails in vitro, and combined mutation of the H3 and H4 tails leads to a large derepression of Tup1-Ssn6-regulated genes in vivo (10, 11). Chromatin immunoprecipitation experiments indicate that Tup1 binding in vivo is associated with decreased acetylation of H3 and H4 (12-14). Accordingly, histone deacetylase activities are required for Tup1-Ssn6 repression (15, 16). Combined loss of three class I histone deacetylases, Rpd3, Hos1, and Hos2, completely abolishes Tup1-Ssn6 repression at all genes examined (15). Mutations in the class II deacetylase, Hda1, shows partial derepression of ENA1, another Tup1-Ssn6-regulated gene (16). Interactions between Tup1-Ssn6 and Rpd3, Hos2, and Hda1 have been detected using a combination of in vitro and in vivo techniques. HA 1 -Hos2 interacts with both a LexA-Ssn6 construct in vivo and a GST-Ssn6 construct in vitro. In vitro translated Hda1 interacts with GST-Tup1 in vitro. However, only Rpd3 has heretofore been shown to interact with native Tup1-Ssn6 in vivo.In this work, we demonstrate that native Tup1-Ssn6 interacts with multiple...

Gamma interferon (IFN-␥) is an inflammatory cytokine that has complex effects on myogenesis. Here, we show that the IFN-␥-induced inhibition of myogenesis is mediated by the major histocompatibility complex (MHC) class II transactivator, CIITA, which binds to myogenin and inhibits its activity. In IFN-␥-treated myoblasts, the inhibition of muscle-specific genes includes the expression of myogenin itself, while in myotubes, myogenin expression is unaffected. Thus, CIITA appears to act by both repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. Stimulation by IFN-␥ in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. The IFN-␥-mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-␥. Through overexpression studies, we confirm that the expression of CIITA, independent of IFN-␥, is sufficient to inhibit myogenesis. Through knockdown studies, we also demonstrate that CIITA is necessary for the IFN-␥-mediated inhibition of myogenesis. Finally, we show that CIITA, which lacks DNA binding activity, is recruited to muscle-specific promoters coincident with reductions in RNA polymerase II recruitment. Thus, this work reveals how IFN-␥ modulates myogenesis and demonstrates a key role for CIITA in this process.Gamma interferon (IFN-␥) is an inflammatory cytokine that was first identified as an antiviral factor. IFN-␥ is a pleiotropic cytokine that regulates different immune responses and influences many physiological processes. Many studies have also shown that IFN-␥ influences skeletal muscle homeostasis and repair. Transient administration of exogenous IFN-␥ has been shown to improve healing of skeletal muscle and limit fibrosis (14). Endogenous IFN-␥ is required for efficient muscle regeneration, as mice lacking IFN-␥ show impaired muscle regeneration following cardiotoxin-induced damage (6). Expression of IFN-␥ is robust in proliferating C2C12 cells, but expression is diminished in differentiated C2C12 cells (6). Exogenous IFN-␥ influences the proliferation and differentiation of cultured myoblasts and appears to have a direct role on gene expression (25,26,28,48).Myoblasts have been shown to express immunological properties such as the complement component of both the classical and alternative pathways and major histocompatibility complex (MHC) genes. Exogenous IFN-␥ treatment has been shown to increase the expression of MHC class II genes, complement C components, intracellular adhesion molecule (Icam1), chemokine (C-C motif) ligand 5 (Ccl5; RANTES), chemokine (C-C motif) ligand 2 (Ccl2), and chemokine (C-X-C motif) ligand 10 (Cxcl10; Ip10) (15,25,28,48). It is not currently known how IFN-␥ mediates these transcriptional effects in myoblasts.The positive role for IFN-␥ established in muscle healing and repair suggests that this cytokine plays an important role in muscle biology. However, IFN-␥ signaling is likely to be tightly regulated, as negative effects of IFN-␥ have been observed as well. Whe...

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.Proper regulation of gene expression requires not only gene activation but also active gene repression. As with activation, mechanisms of repression appear varied and may be multistep. The yeast Tup1-Ssn6 corepressor complex has provided a useful model for dissecting possible modes of repression. Tup1-Ssn6 mediates the repression of diverse classes of genes, including those controlled by mating type, DNA damage, glucose, and anaerobic stress (10, 48). The Tup1-Ssn6 complex does not directly bind DNA but is brought to target genes by interactions with specific DNA binding factors such as ␣2 (22, 41) and Crt1 (19), regulators of a cell-specific genes and DNA damage-inducible genes, respectively. Once recruited, Tup1-Ssn6 appears to interact with the transcription machinery. Mutations in several components of the RNA polymerase II holoenzyme, including Sin4 (7), Srb10/11 (25,47), Med3 (35), and Srb7 (13), decrease repression by Tup1-Ssn6. Moreover, direct physical interactions have been observed between Tup1-Ssn6 and Med3 (35), Srb7 (13), and Srb10/11 (55). Additionally, a modest amount of repression can be established on a nonnucleosomal template in vitro (36), indicating that Tup1-Ssn6 may directly target multiple aspects of the transcription machinery for repression.In eukaryotes, gene regulation occurs within the context of chromatin, and repression mediated by the Tup1-Ssn6 complex clearly has a chromatin component. Chromatin is composed of nucleosomal subunits, which can be folded into progressively higher-order structures. Each nucleosome consists of an octamer of histone proteins and two turns (147 bp) of DNA spooled around...