Alterations in sperm long RNA contribute to the epigenetic inheritance of the effects of postnatal trauma.

Psychiatric diseases have a strong heritable component known to not be restricted to DNA sequence-based genetic inheritance alone but to also involve epigenetic factors in germ cells. Initial evidence suggested that sperm RNA is causally linked to the transmission of symptoms induced by traumatic experiences. Here, we show that alterations in long RNA in sperm contribute to the inheritance of specific trauma symptoms. Injection of long RNA fraction from sperm of males exposed to postnatal trauma recapitulates the effects on food intake, glucose response to insulin and risk-taking in adulthood whereas the small RNA fraction alters body weight and behavioural despair. Alterations in long RNA are maintained after fertilization, suggesting a direct link between sperm and embryo RNA.

Differential localization of HDAC4 orchestrates muscle differentiation.

The class II histone deacetylases HDAC4 and HDAC5 interact specifically with the myogenic MEF2 transcription factor and repress its activity. Here we show that HDAC4 is cytoplasmic during myoblast differentiation, but relocates to the nucleus once fusion has occurred. Inappropriate nuclear entry of HDAC4 following overexpression suppresses the myogenic programme as well as MEF2-dependent transcription. Activation of the Ca(2+)/calmodulin signalling pathway via constitutively active CaMKIV prevents nuclear entry of HDAC4 and HDAC4-mediated inhibition of differentiation. Consistent with a role of phosphorylation in HDAC4 cytoplasmic localisation, HDAC4 binds to 14-3-3 proteins in a phosphorylation-dependent manner. Together these data establish a role for HDAC4 in muscle differentiation. Recently, HDAC5 has also been implicated in muscle differentiation. However, despite the functional similarities of HDAC4 and HDAC5, their intracellular localisations are opposed, suggesting a distinct role for these enzymes during muscle differentiation.

Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.

Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homologue of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 methylate specifically lysine 9 of histone H3 (ref. 6). Here we show that HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a 'methyl marker' on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state.

Regulation of gene expression by transcription factor acetylation.

In the nucleus, DNA is tightly packaged into higher-order structures, generating an environment that is highly repressive towards DNA processes such as gene transcription. Acetylation of lysine residues within proteins has recently emerged as a major mechanism used by the cell to overcome this repression. Acetylation of non-histone proteins, including transcription factors, as well as histones, appears to be involved in this process. Like phosphorylation, acetylation is a dynamic process that can regulate protein-DNA and protein-protein interactions. Moreover, a conserved domain, the bromodomain, has been implicated in the binding of acetylated peptides, suggesting a role for acetylation in intracellular signalling.

Acetylation of importin-alpha nuclear import factors by CBP/p300.

Histone acetylases were originally identified because of their ability to acetylate histone substrates [1] [2] [3]. Acetylases can also target other proteins such as transcription factors [4] [5] [6] [7]. We asked whether the acetylase CREB-binding protein (CBP) could acetylate proteins not directly involved in transcription. A large panel of proteins, involved in a variety of cellular processes, were tested as substrates for recombinant CBP. This screen identified two proteins involved in nuclear import, Rch1 (human importin-alpha) and importin-alpha7, as targets for CBP. The acetylation site within Rch1 was mapped to a single residue, Lys22. By comparing the context of Lys22 with the sequences of other known substrates of CBP and the closely related acetylase p300, we identified G/SK (in the single-letter amino acid code) as a consensus acetylation motif. Mutagenesis of the glycine, as well as the lysine, severely impaired Rch1 acetylation, supporting the view that GK is part of a recognition motif for acetylation by CBP/p300. Using an antibody raised against an acetylated Rch1 peptide, we show that Rch1 was acetylated at Lys22 in vivo and that CBP or p300 could mediate this reaction. Lys22 lies within the binding site for a second nuclear import factor, importin-beta. Acetylation of Lys22 promoted interaction with importin-beta in vitro. Collectively, these results demonstrate that acetylation is not unique to proteins involved in transcription. Acetylation may regulate a variety of biological processes, including nuclear import.

The co-repressor mSin3A is a functional component of the REST-CoREST repressor complex.

The repressor REST/NRSF restricts expression of a large set of genes to neurons by suppressing their expression in non-neural tissues. We find that REST repression involves two distinct repressor proteins. One of these, CoREST, interacts with the COOH-terminal repressor domain of REST (Andres, M. E., Burger, C., Peral-Rubio, M. J., Battaglioli, E., Anderson, M. E., Grimes, J., Dallmanm J., Ballas, N. , and Mandel, G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9873-9878). Here we show that the co-repressor mSin3A also interacts with REST. The REST-mSin3A association involves the NH(2)-terminal repressor domain of REST and the paired amphipathic helix 2 domain of mSin3A. REST forms complexes with endogenous mSin3A in mammalian cells, and both mSin3A and CoREST interact with REST in intact mammalian cells. REST repression is blocked in yeast lacking Sin3 and rescued in its presence. In mammalian cells, repression by REST is reduced when binding to mSin3A is inhibited. In mouse embryos, the distribution of mSin3A and REST transcripts is largely coincident. The pattern of CoREST gene expression is more restricted, suggesting that mSin3A is required constitutively for REST repression, whereas CoREST is recruited for more specialized repressor functions.

Nuclear receptor corepressors partner with class II histone deacetylases in a Sin3-independent repression pathway.

Transcriptional repression mediated by corepressors N-CoR and SMRT is a critical function of nuclear hormone receptors, and is dysregulated in human myeloid leukemias. At the present time, these corepressors are thought to act exclusively through an mSin3/HDAC1 complex. Surprisingly, however, numerous biochemical studies have not detected N-CoR or SMRT in mSin3- and HDAC1-containing complexes. Each corepressor contains multiple repression domains (RDs), the significance of which is unknown. Here we show that these RDs are nonredundant, and that one RD, which is conserved in N-CoR and SMRT, represses transcription by interacting directly with class II HDAC4 and HDAC5. Endogenous N-CoR and SMRT each associate with HDAC4 in a complex that does not contain mSin3A or HDAC1. This is the first example of a single corepressor utilizing distinct domains to engage multiple HDAC complexes. The alternative HDAC complexes may mediate specific repression pathways in normal as well as leukemic cells.

MEF-2 function is modified by a novel co-repressor, MITR.

The MEF-2 proteins are a family of transcriptional activators that have been detected in a wide variety of cell types. In skeletal muscle cells, MEF-2 proteins interact with members of the MyoD family of transcriptional activators to synergistically activate gene expression. Similar interactions with tissue or lineage-specific cofactors may also underlie MEF-2 function in other cell types. In order to screen for such cofactors, we have used a transcriptionally inactive mutant of Xenopus MEF2D in a yeast two-hybrid screen. This approach has identified a novel protein expressed in the early embryo that binds to XMEF2D and XMEF2A. The MEF-2 interacting transcription repressor (MITR) protein binds to the N-terminal MADS/MEF-2 region of the MEF-2 proteins but does not bind to the related Xenopus MADS protein serum response factor. In the early embryo, MITR expression commences at the neurula stage within the mature somites and is subsequently restricted to the myotomal muscle. In functional assays, MITR negatively regulates MEF-2-dependent transcription and we show that this repression is mediated by direct binding of MITR to the histone deacetylase HDAC1. Thus, we propose that MITR acts as a co-repressor, recruiting a specific deacetylase to downregulate MEF-2 activity.

HDAC4 deacetylase associates with and represses the MEF2 transcription factor.

The acetylation state of histones can influence transcription. Acetylation, carried out by acetyltransferases such as CBP/p300 and P/CAF, is commonly associated with transcriptional stimulation, whereas deacetylation, mediated by the three known human deacetylases HDAC1, 2 and 3, causes transcriptional repression. The known human deacetylases represent a single family and are homologues of the yeast RPD3 deacetylase. Here we identify and characterize HDAC4, a representative of a new human histone deacetylase family, which is homologous to the yeast HDA1 deacetylase. We show that HDAC4, unlike other deacetylases, shuttles between the nucleus and the cytoplasm in a process involving active nuclear export. In the nucleus, HDAC4 associates with the myocyte enhancer factor MEF2A. Binding of HDAC4 to MEF2A results in the repression of MEF2A transcriptional activation, a function that requires the deacetylase domain of HDAC4. These results identify MEF2A as a nuclear target for HDAC4-mediated repression and suggests that compartmentalization may be a novel mechanism for controlling the nuclear activity of this new family of deacetylases.

The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth.

E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways.

Retinoblastoma protein recruits histone deacetylase to repress transcription.

The retinoblastoma protein (Rb) silences specific genes that are active in the S phase of the cell cycle and which are regulated by E2F transcription factors. Rb binds to the activation domain of E2F and then actively represses the promoter by a mechanism that is poorly understood. Here we show that Rb associates with a histone deacetylase, HDAC1, through the Rb 'pocket' domain. Association with the deacetylase is reduced by naturally occurring mutations in the pocket and by binding of the human papilloma virus oncoprotein E7. We find that Rb can recruit histone deacetylase to E2F and that Rb cooperates with HDAC1 to repress the E2F-regulated promoter of the gene encoding the cell-cycle protein cyclin E. Inhibition of histone deacetylase activity by trichostatin A (TSA) inhibits Rb-mediated repression of a chromosomally integrated E2F-regulated promoter. Our results indicate that histone deacetylases are important for regulating the cell cycle and that active transcriptional repression by Rb may involve the modification of chromatin structure.