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Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-Iβ. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-Iβ protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-Iβ depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-Iβ and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4.

Pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase complex (PDC) regulate production of acetyl-CoA, which functions as an acetyl donor in diverse enzymatic reactions, including histone acetylation. However, the mechanism by which the acetyl-CoA required for histone acetylation is ensured in a gene context-dependent manner is not clear. Here we show that PKM2, the E2 subunit of PDC and histone acetyltransferase p300 constitute a complex on chromatin with arylhydrocarbon receptor (AhR), a transcription factor associated with xenobiotic metabolism. All of these factors are recruited to the enhancer of AhR-target genes, in an AhR-dependent manner. PKM2 contributes to enhancement of transcription of cytochrome P450 1A1 (CYP1A1), an AhR-target gene, acetylation at lysine 9 of histone H3 at the CYP1A1 enhancer. Site-directed mutagenesis of PKM2 indicates that this enhancement of histone acetylation requires the pyruvate kinase activity of the enzyme. Furthermore, we reveal that PDC activity is present in nuclei. Based on these findings, we propose a local acetyl-CoA production system in which PKM2 and PDC locally supply acetyl-CoA to p300 from abundant PEP for histone acetylation at the gene enhancer, and our data suggest that PKM2 sensitizes AhR-mediated detoxification in actively proliferating cells such as cancer and fetal cells.

Replication-dependent histone genes are up-regulated during the G1/S phase transition to meet the requirement for histones to package the newly synthesized DNA. In mammalian cells, this increment is achieved by enhanced transcription and 3' end processing. The non-polyadenylated histone mRNA 3' ends are generated by a unique mechanism involving the U7 small ribonucleoprotein (U7 snRNP). By using affinity purification methods to enrich U7 snRNA, we identified FUS/TLS as a novel U7 snRNP interacting protein. Both U7 snRNA and histone transcripts can be precipitated by FUS antibodies predominantly in the S phase of the cell cycle. Moreover, FUS depletion leads to decreased levels of correctly processed histone mRNAs and increased levels of extended transcripts. Interestingly, FUS antibodies also co-immunoprecipitate histone transcriptional activator NPAT and transcriptional repressor hnRNP UL1 in different phases of the cell cycle. We further show that FUS binds to histone genes in S phase, promotes the recruitment of RNA polymerase II and is important for the activity of histone gene promoters. Thus, FUS may serve as a linking factor that positively regulates histone gene transcription and 3' end processing by interacting with the U7 snRNP and other factors involved in replication-dependent histone gene expression.

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. To identify genes regulated by hypoxia at the transcriptional level, we pulse-labeled HUVEC cells with 4-thiouridine and sequenced nascent transcripts. Then, we searched genome-wide binding profiles from the ENCODE project for factors that correlated with changes in transcription and identified binding of several components of the Sin3A co-repressor complex, including SIN3A, SAP30 and HDAC1/2, proximal to genes repressed by hypoxia. SIN3A interference revealed that it participates in the downregulation of 75% of the hypoxia-repressed genes in endothelial cells. Unexpectedly, it also blunted the induction of 47% of the upregulated genes, suggesting a role for this corepressor in gene induction. In agreement, ChIP-seq experiments showed that SIN3A preferentially localizes to the promoter region of actively transcribed genes and that SIN3A signal was enriched in hypoxia-repressed genes, prior exposure to the stimulus. Importantly, SINA3 occupancy was not altered by hypoxia in spite of changes in H3K27ac signal. In summary, our results reveal a prominent role for SIN3A in the transcriptional response to hypoxia and suggest a model where modulation of the associated histone deacetylase activity, rather than its recruitment, determines the transcriptional output., Ministerio de Ciencia e Innovacion (Spanish Ministry of Science and Innovation, MICINN) [SAF2011 24225 to L.d.P., SAF2014–53819-R to L.d.P., B.J.]; Canadian Institutes of Health Research (CIHR) [MOP-82875 to W.W.).]; Natural Sciences and Engineering Research Council of Canada (NSERC) [RGPIN355532–10 to W.W.W.]; National Institutes of Health [1R01GM084875 to W.W.W.]; CIHR Fellowship (to R.W.H.); Michael Smith Foundation for Health Research Fellowship (to R.W.H.); Caja Madrid Foundation for Visiting Professor Fellowship (to L.d.P). Funding for open access charge: Spanish Ministry of Science and Innovation, MICINN, [SAF2014-53819-R].

The S. cerevisiae FLO1 gene encodes a cell wall protein that imparts cell-cell adhesion. FLO1transcription is regulated via the antagonistic activities of the Tup1-Cyc8 co-repressor and Swi-Snfco-activator complexes. Tup1-Cyc8 represses transcription through the organisation of stronglypositioned, hypoacetylated nucleosomes across gene promoters. Swi-Snf catalyses remodelling ofthese nucleosomes in a mechanism involving histone acetylation that is poorly understood. Here,we show that FLO1 de-repression is accompanied by Swi-Snf recruitment, promoter histone eviction,and Sas3 and Ada2(Gcn5)-dependent histone H3K14 acetylation. In the absence of H3K14acetylation, Swi-Snf recruitment and histone eviction proceed, but transcription is reduced,suggesting these processes, while essential, are not sufficient for de-repression. Further analysis inthe absence of H3K14 acetylation reveals RNAP II recruitment at the FLO1 promoter still occurs, butRNAP II is absent from the gene-coding region, demonstrating Sas3 and Ada2-dependent histone H3acetylation is required for transcription elongation. Analysis of the transcription kinetics at othergenes reveals shared mechanisms coupled to a distinct role for histone H3 acetylation, essential atFLO1, downstream of initiation. We propose histone H3 acetylation in the coding region providesrate-limiting control during the transition from initiation to elongation which dictates whether thegene is permissive for transcription.

In this work, we developed a database WERAM (http://weram.biocuckoo.org/) for histone acetyltransferases, histone deacetylases, histone methyltransferases, histone demethylases and acetyl- or methyl-binding proteins, which catalyze, remove and recognize histone acetylation and methylation sites as 'writers', 'erasers' and 'readers', and synergistically determine the 'histone code'. From the scientific literature, we totally collected over 580 experimentally identified histone regulators from eight model organisms, including Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe and Saccharomyces cerevisiae We also collected ∼900 site-specific regulator-histone relations from the eight species. According to the experimental evidence, known histone regulators were classified into distinct families. To computationally detect more proteins in eukaryotes, we constructed hidden Markov model (HMM) profiles for histone regulator families. For families without HMM profiles, we also conducted orthologous searches. Totally, WERAM database contained more than 20 thousand non-redundant histone regulators from 148 eukaryotes. The detailed annotations and classification information of histone regulators were provided, together with site-specific histone substrates if available.

Epigenetic regulation of gene expression is important in maintaining self-renewal of embryonic stem (ES) and trophoblast stem (TS) cells. Histone deacetylases (HDACs) negatively control histone acetylation by removing covalent acetylation marks from histone tails. Because histone acetylation is a known mark for active transcription, HDACs presumably associate with inactive genes. Here, we used genome-wide chromatin immunoprecipitation to investigate targets of HDAC1 in ES and TS cells. Through evaluation of genes associated with acetylated histone H3 marks, and global expression analysis of Hdac1 knockout ES and trichostatin A-treated ES and TS cells, we found that HDAC1 occupies mainly active genes, including important regulators of ES and TS cells self-renewal. We also observed occupancy of methyl-CpG binding domain protein 3 (MBD3), a subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex, at a subset of HDAC1-occupied sequences in ES cells, including the pluripotency regulators Oct4, Nanog and Kfl4. By mapping HDAC1 targets on a global scale, our results describe further insight into epigenetic mechanisms of ES and TS cells self-renewal.

Genomic imprinting is an epigenetic inheritance system characterized by parental allele-specific gene expression. Allele-specific DNA methylation and chromatin composition are two epigenetic modification systems that control imprinted gene expression. To get a general assessment of histone lysine acetylation at imprinted genes we measured allele-specific acetylation of a wide range of lysine residues, H3K4, H3K18, H3K27, H3K36, H3K79, H3K64, H4K5, H4K8, H4K12, H2AK5, H2BK12, H2BK16 and H2BK46 at 11 differentially methylated regions (DMRs) in reciprocal mouse crosses using multiplex chromatin immunoprecipitation SNuPE assays. Histone acetylation marks generally distinguished the methylation-free alleles from methylated alleles at DMRs in mouse embryo fibroblasts and embryos. Acetylated lysines that are typically found at transcription start sites exhibited stronger allelic bias than acetylated histone residues in general. Maternally methylated DMRs, that usually overlap with promoters exhibited higher levels of acetylation and a 10% stronger allele-specific bias than paternally methylated DMRs that reside in intergenic regions. Along the H19/Igf2 imprinted domain, allele-specific acetylation at each lysine residue depended on functional CTCF binding sites in the imprinting control region. Our results suggest that many different histone acetyltransferase and histone deacetylase enzymes must act in concert in setting up and maintaining reciprocal parental allelic histone acetylation at DMRs.

Oxidized DNA bases, particularly 7,8-dihydro-8-oxoguanine (8-oxoG), are endogenously generated in cells, being a cause of carcinogenic mutations and possibly interfering with gene expression. We found that expression of an oxidatively damaged plasmid DNA is impaired after delivery into human host cells not only due to decreased retention in the transfected cells, but also due to selective silencing of the damaged reporter gene. To test whether the gene silencing was associated with a specific change of the chromatin structure, we determined the levels of histone modifications related to transcriptional activation (acetylated histones H3 and H4) or repression (methylated K9 and K27 of the histone H3, and histone H1) in the promoter region and in the downstream transcribed DNA. Acetylation of histone H4 was found to be specifically decreased by 25% in the proximal promoter region of the damaged gene, while minor quantitative changes in other tested chromatin components could not be proven as significant. Treatment with an inhibitor of histone deacetylases, trichostatin A, partially restored expression of the damaged DNA, suggesting a causal connection between the changes of histone acetylation and persistent gene repression. Based on these findings, we propose that silencing of the oxidatively damaged DNA may occur in a chromatin-mediated mechanism.

Plant homeodomain (PHD) fingers are often present in chromatin-binding proteins and have been shown to bind histone H3 N-terminal tails. Mutations in the autoimmune regulator (AIRE) protein, which harbours two PHD fingers, cause a rare monogenic disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE activates the expression of tissue-specific antigens by directly binding through its first PHD finger (AIRE-PHD1) to histone H3 tails non-methylated at K4 (H3K4me0). Here, we present the solution structure of AIRE-PHD1 in complex with H3K4me0 peptide and show that AIRE-PHD1 is a highly specialized non-modified histone H3 tail reader, as post-translational modifications of the first 10 histone H3 residues reduce binding affinity. In particular, H3R2 dimethylation abrogates AIRE-PHD1 binding in vitro and reduces the in vivo activation of AIRE target genes in HEK293 cells. The observed antagonism by R2 methylation on AIRE-PHD1 binding is unique among the H3K4me0 histone readers and represents the first case of epigenetic negative cross-talk between non-methylated H3K4 and methylated H3R2. Collectively, our results point to a very specific histone code responsible for non-modified H3 tail recognition by AIRE-PHD1 and describe at atomic level one crucial step in the molecular mechanism responsible for antigen expression in the thymus.

Giant chromatin-modifying complexes regulate gene transcription in eukaryotes by acting on chromatin substrates and 'setting' the histone code. The histone deacetylase (HDAC)-associated mammalian Sin3 corepressor complex regulates a wide variety of genes involved in all aspects of cellular physiology. The recruitment of the corepressor complex by transcription factors to specific regions of the genome is mediated by Sin3 as well as 10 distinct polypeptides that comprise the corepressor complex. Here we report the solution structure of a novel CCCH zinc finger (ZnF) motif in the SAP30 polypeptide, a key component of the corepressor complex. The structure represents a novel fold comprising two beta-strands and two alpha-helices with the zinc organizing center showing remote resemblance to the treble clef motif. In silico analysis of the structure revealed a highly conserved surface that is dominated by basic residues. NMR-based analysis of potential ligands for the SAP30 ZnF motif indicated a strong preference for nucleic acid substrates. We propose that the SAP30 ZnF functions as a double-stranded DNA-binding motif, thereby expanding the known functions of both SAP30 and the mammalian Sin3 corepressor complex. Our results also call into question the common assumption about the exclusion of DNA-binding core subunits within chromatin-modifying/remodeling complexes.

In plants, the histone H3.1 lysine 27 (H3K27) mono-methyltransferases ARABIDOPSIS TRITHORAX RELATED PROTEIN 5 and 6 (ATXR5/6) regulate heterochromatic DNA replication and genome stability. Our initial studies showed that ATXR5/6 discriminate between histone H3 variants and preferentially methylate K27 on H3.1. In this study, we report three regulatory mechanisms contributing to the specificity of ATXR5/6. First, we show that ATXR5 preferentially methylates the R/F-K*-S/C-G/A-P/C motif with striking preference for hydrophobic and aromatic residues in positions flanking this core of five amino acids. Second, we demonstrate that post-transcriptional modifications of residues neighboring K27 that are typically associated with actively transcribed chromatin are detrimental to ATXR5 activity. Third, we show that ATXR5 PHD domain employs a narrow binding pocket to selectively recognize unmethylated K4 of histone H3. Finally, we demonstrate that deletion or mutation of the PHD domain reduces the catalytic efficiency (kcat/Km of AdoMet) of ATXR5 up to 58-fold, highlighting the multifunctional nature of ATXR5 PHD domain. Overall, our results suggest that several molecular determinants regulate ATXR5/6 methyltransferase activity and epigenetic inheritance of H3.1 K27me1 mark in plants.

Histone chaperones are thought to be important for maintaining the physiological activity of histones; however, their exact roles are not fully understood. The physiological function of template activating factor (TAF)-I, one of the histone chaperones, also remains unclear; however, its biochemical properties have been well studied. By performing microarray analyses, we found that TAF-I stimulates the transcription of a sub-set of genes. The transcription of endogenous genes that was up-regulated by TAF-I was found to be additively stimulated by histone acetylation. On performing an experiment with a cell line containing a model gene integrated into the chromosome, TAF-I was found to stimulate the model gene transcription in a histone chaperone activity-dependent manner additively with histone acetylation. TAF-I bound to the core histones and remodeled the chromatin structure independent of the N-terminal histone tail and its acetylation level in vitro. These results suggest that TAF-I remodel the chromatin structure through its interaction with the core domain of the histones, including the histone fold, and this mechanism is independent of the histone acetylation status.

The null mutation of the SIR2 gene in Saccharomyces cerevisiae has been associated with a series of different phenotypes including loss of transcriptional silencing, genome instability and replicative aging. Thus, the SIR2 gene product is an important constituent of the yeast cell. SIR2 orthologues and paralogues have been discovered in organisms ranging from bacteria to man, underscoring the pivotal role of this protein. Here we report that a plasmid introduced into sir2Delta cells accumulates more negative supercoils compared to the same plasmid introduced into wild-type (WT) cells. This effect appears to be directly related to SIR2 expression as shown by the reduction of negative supercoiling when SIR2 is overexpressed, and does not depend on the number or positioning of nucleosomes on plasmids. Our results indicate that this new phenotype is due to the lack of Sir2p histone deacetylase activity in the sir2Delta strain, because only the H4-K16 residue of the histone octamer undergoes an alteration of its acetylation state. A model proposing interference with the replication machinery is discussed.

Histone acetylation is important for regulating chromatin structure and gene expression. Three classes of mammalian histone deacetylases have been identified. Among class II, there are five known members, namely HDAC4, HDAC5, HDAC6, HDAC7 and HDAC9. Here we describe the identification and characterization of a novel class II member termed HDAC10. It is a 669 residue polypeptide with a bipartite modular structure consisting of an N-terminal Hda1p-related putative deacetylase domain and a C-terminal leucine-rich domain. HDAC10 is widely expressed in adult human tissues and cultured mammalian cells. It is enriched in the cytoplasm and this enrichment is not sensitive to leptomycin B, a specific inhibitor known to block the nuclear export of other class II members. The leucine-rich domain of HDAC10 is responsible for its cytoplasmic enrichment. Recombinant HDAC10 protein possesses histone deacetylase activity, which is sensitive to trichostatin A, a specific inhibitor for known class I and class II histone deacetylases. When tethered to a promoter, HDAC10 is able to repress transcription. Furthermore, HDAC10 interacts with HDAC3 but not with HDAC4 or HDAC6. These results indicate that HDAC10 is a novel class II histone deacetylase possessing a unique leucine-rich domain.

Histone biogenesis is tightly controlled at multiple steps to maintain the balance between the amounts of DNA and histone protein during the cell cycle. In particular, translation and degradation of replication-dependent histone mRNAs are coordinately regulated. However, the underlying molecular mechanisms remain elusive. Here, we investigate remodeling of stem-loop binding protein (SLBP)-containing histone mRNPs occurring during the switch from the actively translating mode to the degradation mode. The interaction between a CBP80/20-dependent translation initiation factor (CTIF) and SLBP, which is important for efficient histone mRNA translation, is disrupted upon the inhibition of DNA replication or at the end of S phase. This disruption is mediated by competition between CTIF and UPF1 for SLBP binding. Further characterizations reveal hyperphosphorylation of UPF1 by activated ATR and DNA-dependent protein kinase upon the inhibition of DNA replication interacts with SLBP more strongly, promoting the release of CTIF and eIF3 from SLBP-containing histone mRNP. In addition, hyperphosphorylated UPF1 recruits PNRC2 and SMG5, triggering decapping followed by 5′-to-3′ degradation of histone mRNAs. The collective observations suggest that both inhibition of translation and recruitment of mRNA degradation machinery during histone mRNA degradation are tightly coupled and coordinately regulated by UPF1 phosphorylation.

The product of the retinoblastoma susceptibility gene, the Rb protein, functions partly through transcriptional repression of E2F-regulated genes. Repression by Rb is mediated, at least in part, by a histone deacetylase complex, whose enzymatic activity relies on HDAC1, HDAC2 or HDAC3. Recently, we have shown that the Rb-associated histone deacetylase complex contains RbAp48 protein, which interacts with HDAC1 and HDAC2. RbAp48 could favour the deacetylation of histones since it binds directly to histone H4. In agreement with that, we show that transcriptional repression of E2F activity requires the presence of RbAp48. HDAC3 was thought not to interact with RbAp48. However, we found that it shared with HDAC1 the ability to favour the recruitment of RbAp48 to Rb. This latter effect was unlikely to be due to activation of Rb function, since HDAC3 did not increase Rb–E2F1 interaction. Rather, we found, surprisingly, that HDAC3 could physically interact with RbAp48 both in vitro and in living cells. Taken together, our data suggest a model in which Rb mediates the recruitment to E2F-regulating promoters of a repressive complex containing either HDAC1, HDAC2 or HDAC3 and the histone-binding protein RbAp48.

Histone acetylation alters the chromatin structure and activates the genes that are repressed by histone deacetylation. This investigation demonstrates that treating P3HR1 cells with trichostatin A (TSA) activates the Epstein-Barr virus (EBV) lytic cycle, allowing the virus to synthesize three viral lytic proteins-Rta, Zta and EA-D. Experimental results indicate that TSA and 12-O:-tetradecanoylphorbol-13-acetate synergistically activate the transcription of BRLF1, an immediate-early gene of EBV. Chromatin immunoprecipitation assay reveals that histone H4 at the BRLF1 promoter is acetylated after P3HR1 cells are treated with TSA, suggesting that histone acetylation activates BRLF1 transcription. Furthermore, results in this study demonstrate that mutation of a YY1-binding site in the BRLF1 promoter activates BRLF1 transcription 1.6- and 2.3-fold in P3HR1 cells and C33A cells, respectively. Real time PCR analysis reveals that the mutation also increases the histone acetylation level of the nucleosomes at the BRLF1 promoter 1. 64- and 3.08-fold in P3HR1 and C33A cells, respectively. Results presented herein suggest that histone deacetylation plays an important role in maintaining the viral latency and histone acetylation at the BRLF1 promoter allows the virus to express Rta and to activate the viral lytic cycle.

Purified histone acetyltransferase B (HAT-B) from maize consists of two subunits, p50 and p45. Cloning of the cDNA and genomic DNA encoding the catalytic subunit p50 revealed a consensus motif reminiscent of other acetyltransferases. Internal peptide sequences and immunological studies identified p45 as a protein related to the Retinoblastoma associated protein Rbap. Antibodies against recombinant p50 were able to immunoprecipitate the enzymatic activity of p50 as well as p45. Consistent with the idea that HAT-B is involved in acetylation of newly synthesized histone H4 during DNA replication, mRNA and protein levels are correlated with S-phases during embryo germination. Inhibition of histone deacetylases by HC toxin or Trichostatin A caused a decrease of the in vivo expression of HAT-B mRNA. Regardless of its predominant cytoplasmic localization, a significant proportion of HAT-B-p50 is present in nuclei, irrespective of the cell cycle stage, suggesting an additional nuclear function.

Searches of several sequence databases reveal that human HD1, yeast HDA1, yeast RPD3 and other eukaryotic histone deacetylases share nine motifs with archaeal and eubacterial enzymes, including acetoin utilization protein (acuC) and acetylpolyamine amidohydrolase. Histone deacetylase and acetylpolyamine amidohydrolase also share profound functional similarities in that both: (i) recognize an acetylated aminoalkyl group; (ii) catalyze the removal of the acetyl group by cleaving an amide bond; (iii) increase the positive charge of the substrate. Stabilization of nucleosomal DNA-histone interaction brought about by the change in charge has been implicated as the underlying cause for histone deacetylase-mediated transcriptional repression. We speculate that the eukaryotic histone deacetylases originated from a prokaryotic enzyme similar to the acetylpolyamine amidohydrolases that relied on reversible acetylation and deacetylation of the aminoalkyl group of a DNA binding molecule to achieve a gene regulatory effect.

Dynamic acetylation of all lysine-4-trimethylated histone H3 is a complex phenomenon involved in Immediate-early gene induction in metazoan eukaryotes. Higher eukaryotes express repeated copies of three closely related H3 variants, inaccessible to genetic analysis. We demonstrate conservation of these phenomena in Dictyostelium which has three single-copy H3 variant genes. Although dynamic acetylation is targeted to two H3 variants which are K4-trimethylated, K9-acetylation is preferentially targeted to one. In cells lacking Set1 methyltransferase and any detectable K4-trimethylation, dynamic acetylation is lost demonstrating a direct link between the two. Gene replacement to express mutated H3 variants reveals a novel interaction between K4-trimethylation on different variants. Cells expressing only one variant show defects in growth, and in induction of a UV-inducible gene, demonstrating the functional importance of variant expression. These studies confirm that dynamic acetylation targeted to H3K4me3 arose early in evolution and reveal a very high level of specificity of histone variant utilization in a simple multicellular eukaryote.

The replication dependent histone transcripts terminate with a highly conserved stem-loop structure. This feature distinguishes them from most other eukaryotic mRNAs which end with a poly(A) tail. The 3' terminus of histone mRNA is a main determinant for rapid turnover of these transcripts. In this study, we report the identification of two cytoplasmic protein complexes that interact in a sequence specific fashion with 3' terminal sequences of a mouse histone H4 and a human histone H2A mRNA. The binding activities are conserved from frog to man. At least a fraction of one of the protein complexes appears to be specifically associated with polysomes. The evidence for an involvement of the observed protein complexes in turnover of histone transcripts is discussed.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is produced by T cells, but not B cells, in response to immune signals. GM-CSF gene activation in response to T-cell stimulation requires remodelling of chromatin associated with the gene promoter, and these changes do not occur in B cells. While the CpG methylation status of the murine GM-CSF promoter shows no correlation with the ability of the gene to respond to activation, we find that the basal chromatin environment of the gene promoter influences its ability to respond to immune signals. In unstimulated T cells but not B cells, the GM-CSF promoter is selectively marked by enrichment of histone acetylation, and association of the chromatin-remodelling protein BRG1. BRG1 is removed from the promoter upon activation concomitant with histone depletion and BRG1 is required for efficient chromatin remodelling and transcription. Increasing histone acetylation at the promoter in T cells is paralleled by increased BRG1 recruitment, resulting in more rapid chromatin remodelling, and an associated increase in GM-CSF mRNA levels. Furthermore, increasing histone acetylation in B cells removes the block in chromatin remodelling and transcriptional activation of the GM-CSF gene. These data are consistent with a model in which histone hyperacetylation and BRG1 enrichment at the GM-CSF promoter, generate a chromatin environment competent to respond to immune signals resulting in gene activation.

Histone acetylation plays a key role in the regulation of gene expression. The chromatin structure and accessibility of genes to transcription factors is regulated by enzymes that acetylate and deacetylate histones. The Sin3A corepressor complex recruits histone deacetylases and in many cases represses transcription. Here, we report that SAP30L, a close homolog of Sin3-associated protein 30 (SAP30), interacts with several components of the Sin3A corepressor complex. We show that it binds to the PAH3/HID (Paired Amphipathic Helix 3/Histone deacetylase Interacting Domain) region of mouse Sin3A with residues 120–140 in the C-terminal part of the protein. We provide evidence that SAP30L induces transcriptional repression, possibly via recruitment of Sin3A and histone deacetylases. Finally, we characterize a functional nucleolar localization signal in SAP30L and show that SAP30L and SAP30 are able to target Sin3A to the nucleolus. peerReviewed

T-cells retain cell-type-specific programming for IL-2 inducibility through many rounds of division without being stimulated to transcribe the locus. To understand the layering of controls needed to poise this gene heritably for activation, we have used chromatin immunoprecipitation to map histone modifications across the murine IL2 locus, from -10.2 through +0.25 kb, in induction-competent and incompetent cells. In highly inducible EL4 T-lineage cells, stimulation with PMA/A23187 induced strong acetylation of histone H3 and H4, in parallel with transcriptional activation, from -4.6 through +0.25 kb. However, dimethylation of histone H3/K4 was already fully elevated across the same restricted domain before stimulation, with little change after stimulation. RNA polymerase II binding, in contrast, was only found at the known promoter region after stimulation. Similar patterns of histone modifications were seen also in normal IL-2-inducible T-lineage cells. However, neither acetylated histone H3, H4 nor dimethylated histone H3/K4 marking was detected, with or without stimulation, in expression-incompetent cells (NIH/3T3 or Scid.adh). These results identify a discrete new domain of IL2 regulatory sequence marked by dimethylated histone H3/K4 in expression-permissive T-cells even when they are not transcribing IL2, setting boundaries for histone H3 and H4 acetylation when the IL2 gene is transcriptionally activated.

The respective involvement of transcriptional and post-transcriptional mechanisms in coupling H3 and H4 histone gene expression to the S phase of the cell cycle has been studied in synchronized tobacco cells. Induction of histone gene expression at the G1/S transition is shown to be essentially directed by an increase in the transcription rate in response to cellular signals occurring at the initiation step of DNA replication. Histone gene induction thus precedes the burst of DNA synthesis. However, when the elongation step of DNA replication is ineffective or artificially arrested, feedback mechanisms apparently act at the translation level to avoid overproduction of histone proteins from their mRNAs. At the end of S phase, post-transcriptional mechanisms ensure a rapid degradation of histone mRNAs. Transcription factors are bound to the cis -elements of histone promoters throughout the cell cycle, thus suggesting a post-translational modification of some of them to trigger promoter activation at the G1/S transition. Based on these results, a model is proposed for histone gene transcriptional induction in connection with the components of the cell cycle machinery.

The methylated DNA binding protein-2-H1 (MDBP-2-H1), present in rooster liver, is a member of the histone H1 family which inhibits transcription by binding selectively to methylated promoters. Here we have determined the primary structure of MDBP-2-H1. A comparison between histone H1 and MDBP-2-H1 was achieved by analyzing reversed phase HPLC-purified and V8-digested proteins by mass spectrometry and/or microsequencing. In rooster liver the most abundant histone H1 subtypes are H1 01 and H1 11L. Similarly, MDBP-2-H1 contains the same subtypes of histone H1. The histone H1 subtype H1 01 in MDBP-2-H1 has 150 amino acids, whereas the full-size histone H1 01 is 218 amino acids. The difference in mass between the two proteins is explained by C-terminal truncation of histone H1 01.

Expression of replication-dependent histone genes of all classes is up-regulated coordinately at the onset of DNA synthesis. The cellular signals involved in coordinate regulation of these genes are not known. Here we report identification of an alpha element, present within the mouse histone coding region activating sequence (CRAS). We show evidence that this element is present in histone genes from two classes, H2a and H3, in the mouse. This element has two biological functions in histone gene expression, i.e. the element interacts with nuclear proteins in regulation of gene expression, as well as encoding the amino acids of the histone proteins. We present both in vivo and in vitro evidence that interaction of nuclear proteins with this element is required for normal expression. The binding site for nuclear protein(s) has been precisely defined by means of synthetic oligonucleotides, as well as DNase I protection and methylation interference. It is interesting to note that the histone CRAS alpha element is mutated in a replication-independent H3.3 gene; 5 of 7 nt in the CRAS alpha box are changed in this gene.

The stem-loop structure at the 3' end of replication-dependent histone mRNA is required for efficient pre-mRNA processing, localization of histone mRNA to the polyribosomes, and regulation of histone mRNA degradation. A protein, the stem-loop binding protein (SLBP), binds the 3' end of histone mRNA and is thought to mediate some or all of these processes. A mutant histone mRNA with two nucleotide changes in the loop was constructed and found to be transported inefficiently to the cytoplasm. The mutant histone mRNA, unlike the wild-type histone mRNA, was not rapidly degraded when DNA synthesis is inhibited, and was not stabilized upon inhibition of protein synthesis. The stem-loop binding protein (SLBP) has between a 20-50 fold greater affinity for the wild type histone stem-loop structure than for the mutant stem-loop structure, suggesting that the alteration in the efficiency of transport and the normal degradation pathway in histone mRNA may be due to the reduced affinity of the mutant stem-loop for the SLBP.

It 1s likely that histone HI 1s Involved 1n the condensation of chromatin 1n eukaryotes. However, both the presence of histone HI 1n yeast and the extent of yeast chromatin condensation are controversial. A 20 kD protein copurifies with yeast chromatin and was shown by other Investigators to have characteristics of histone HI protein. In an attempt to obtain a positive Identification of the 20 kD protein, we purified the protein to homogeneity and raised antibodies against 1t. We show here by Immunofluores cence that the 20 kD protein does not localize to the nucleus but to cytoplasmic particles resembling mitochondria. Furthermore, we show by Western-blot analysis that ant1-20 kD protein antibodies react to protein Isolated from purified mitochondria. Finally, we present evidence based on size, charge, amino acid composition and Immunological cross reactivity to suggest that the yeast 20 kD protein 1s likely to be the mitochondrial DNA-b1nd1ng HM protein. This leaves no candidate for histone HI 1n yeast.

Zinc finger proteins (Zfp) are encoded by a large family of genes present in many organisms including yeast and human. Some of them are transcriptional activators and bind specifically to DNA by zinc mediated folded structures commonly known as zinc fingers. The Drosophila Krūppel (Kr) is a segmentation gene and encodes a zinc finger protein. Using a probe from the finger domain of Kr, we have isolated a structurally related gene Zfp-1 from the mouse. In this paper, we report the complete nucleotide sequence of two cDNA clones and the amino acid sequence deduced from them. The putative Zfp-1 protein contains in addition to 7 zinc fingers, two helix-turn-helix motifs. During murine embryogenesis, the Zfp-1 was found to express at a peak level in day 12 embryos. The ubiquitously expressed Zfp-1 gene is located in the 16q region on mouse chromosome 8, between the uvomorulin and the tyrosine amino transferase genes.

The DNA sequences of the entire structural H4, H3, H2A and H2B genes and of their 5' flanking regions have been determined in the histone DNA clone h19 of the sea urchin Psammechinus miliaris. In clone h19 the polarity of transcription and the relative arrangement of the histone genes is identical to that in clone h22 of the same species. The histone proteins encoded by h19 DNA differ in their primary structure from those encoded by clone h22 and have been compared to histone protein sequences of other sea urchin species as well as other eukaryotes. A comparative analysis of the 5' flanking DNA sequences of the structural histone genes in both clones revealed four ubiquitous sequence motifs; a pentameric element GATCC, followed at short distance by the Hogness box GTATAAATAG, a conserved sequence PyCATTCPu, in or near which the 5' ends of the mRNAs map in h22 DNA and lastly a sequence A, containing the initiation codon. These sequences are also found, sometimes in modified version, in front of other eukaryotic genes transcribed by polymerase II. When prelude sequences of isocoding histone genes in clone h19 and h22 are compared areas of homology are seen to extend beyond the ubiquitous sequence motifs towards the divergent AT-rich spacer and terminate between approximately 140 and 240 nucleotides away from the structural gene. These prelude regions contain quite large conservative sequence blocks which are specific for each type of histone genes.

A nuclear protein with affinity for the 5' flanking region of a cell cycle dependent human H4 histone gene has been partially purified from nuclear extracts of human HeLa S3 cells. The region involved in the binding of the protein has been localized to an upstream DNA segment using an electrophoretic mobility shift assay. This DNA segment is devoid of RNA polymerase II consensus sequences and contains both homopurinic and A/T rich tracts. Analogous experiments have identified a similar, and perhaps identical, factor that has affinity for a cell cycle dependent human H3 histone gene promoter. This protein appears to bind to a DNA segment containing A/T rich sequences that bear homology with the binding region of the H4 histone promoter. Cell synchronization experiments have shown that the overall affinity of the protein(s) for the H3 and H4 histone 5' flanking regions in vitro is not dramatically altered during the cell cycle. Although the rate of histone gene transcription is modulated during early S phase, transcription occurs throughout the cell cycle. Hence, the protein(s) we have detected here may play a role in the basal expression of these genes.

A human H4 histone gene was isolated and the nucleotide sequences of the mRNA coding as well as the 5' and 3' flanking regions were determined. No intervening sequences were found in this gene. A series of sequences which have been assigned putative regulatory roles in histone genes and/or in other genes were identified both upstream and downstream from the H4 histone protein coding region. Deletion mutants were constructed by BAL-31 nuclease digestion of sequences in the 5' flanking region of this H4 histone gene and were assayed in an in vitro transcription system. No regions upstream from the TATA box were required for site specific initiation in vitro. Data are presented which suggest that sequences located downstream from the 3' end of the coding region may influence the in vitro transcription of this human H4 histone gene.

We have purified HeLa histone mRNA from polysomes of S-phase cells which had been synchronized by hydroxyurea treatment. This mRNA was shown to direct the in vitro synthesis of all five histones which amount to at least 90-95% of its total translational activity. Polysomal histone mRNP was also purified and identified by cell-free translation and hybridization to a clone of histone DNA from E. esculentus. The protein moiety of this mRNP contained three prominent species of molecular weight 86,000, 73,000 and 53,000 daltons. The presence of the 73,000 species previously assessed to be bound to poly(A) is discussed in view of the fact that histone mRNA does not contain a poly(A) tail. As globin mRNA, histone mRNA as well as histone mRNP were translated with equal efficiency in cell-free extracts from either S-phase or hydroxyurea blocked HeLa cells.

The 5' region of a cell cycle regulated human H1 histone gene appears to contain at least six promoter DNA elements that are shared with some, but not all human core (H2A, H2B, H3 and H4) histone genes. We show that two of these elements represent separate binding sites for two distinct, partially purified factors. The first promoter domain contains A/T rich repeats and is involved in the binding of HiNF-A, a nuclear factor previously found to bind to A/T rich direct repeats in the promoters of human H4 and H3 histone genes. The second domain, containing the general promoter element 5' dACCAAT, acts as a binding site for a two component mosaic factor we have designated HiNF-B. These data suggest that coordinate transcriptional regulation of human H1 and core histone genes may involve two classes of trans-acting factors: those specific for histone gene promoters and those that act on a broad spectrum of human gene promoters.

We have cloned and characterized the gene encoding a sperm specific H2B-2 histone subtype from the sea urchin L. pictus. The gene is not clustered with any other histone genes. However, it resembles other histone genes in many respects. The gene contains no intervening sequences or polyadenylation signals. Like other histone genes it contains the conserved 3' hairpin loop sequence and CAAGAAAGA box necessary for the processing of the 3' end of the histone transcript. Upstream of the gene in addition to the TATA box, and two copies of inverted CCAAT Boxes is a conserved sequence element found in many H2B genes. Unlike most histone genes, the H2B-2 gene is expressed in a sex and tissue specific manner only in testis. The sperm H2B-2 subtype encoded by this gene is 142 amino acids in length. The larger size of this H2B protein is accounted for by residues in the N-terminus of the protein consisting of a series of pentapeptide repeats typical of this histone subtype.

The nucleotide sequence of an 8.2 kb BamHI fragment containing the entire chicken histone H2AF gene has been determined. Unlike the majority of histone genes, the coding region is interrupted by four intervening sequences. While sequencing the 8.2 kb BamHI fragment it was found that the promoter and first exon of an unidentified non-histone gene lies immediately downstream of the H2AF gene. Studies of H2AF gene transcription show that, unlike the major core and H1 histone genes, it is not coupled to DNA synthesis.