Showing papers on "Histone H4 published in 2000"

The language of covalent histone modifications.

Abstract: Histone proteins and the nucleosomes they form with DNA are the fundamental building blocks of eukaryotic chromatin. A diverse array of post-translational modifications that often occur on tail domains of these proteins has been well documented. Although the function of these highly conserved modifications has remained elusive, converging biochemical and genetic evidence suggests functions in several chromatin-based processes. We propose that distinct histone modifications, on one or more tails, act sequentially or in combination to form a 'histone code' that is, read by other proteins to bring about distinct downstream events.

Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase

Abstract: Yeast Sir2 is a heterochromatin component that silences transcription at silent mating loci, telomeres and the ribosomal DNA, and that also suppresses recombination in the rDNA and extends replicative life span. Mutational studies indicate that lysine 16 in the amino-terminal tail of histone H4 and lysines 9, 14 and 18 in H3 are critically important in silencing, whereas lysines 5, 8 and 12 of H4 have more redundant functions. Lysines 9 and 14 of histone H3 and lysines 5, 8 and 16 of H4 are acetylated in active chromatin and hypoacetylated in silenced chromatin, and overexpression of Sir2 promotes global deacetylation of histones, indicating that Sir2 may be a histone deacetylase. Deacetylation of lysine 16 of H4 is necessary for binding the silencing protein, Sir3. Here we show that yeast and mouse Sir2 proteins are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases, which deacetylate lysines 9 and 14 of H3 and specifically lysine 16 of H4. Our analysis of two SIR2 mutations supports the idea that this deacetylase activity accounts for silencing, recombination suppression and extension of life span in vivo. These findings provide a molecular framework of NAD-dependent histone deacetylation that connects metabolism, genomic silencing and ageing in yeast and, perhaps, in higher eukaryotes.

Structure and Function of a Human TAFII250 Double Bromodomain Module

Abstract: TFIID is a large multiprotein complex that initiates assembly of the transcription machinery. It is unclear how TFIID recognizes promoters in vivo when templates are nucleosome-bound. Here, it is shown that TAFII250, the largest subunit of TFIID, contains two tandem bromodomain modules that bind selectively to multiply acetylated histone H4 peptides. The 2.1 angstrom crystal structure of the double bromodomain reveals two side-by-side, four-helix bundles with a highly polarized surface charge distribution. Each bundle contains an Nepsilon-acetyllysine binding pocket at its center, which results in a structure ideally suited for recognition of diacetylated histone H4 tails. Thus, TFIID may be targeted to specific chromatin-bound promoters and may play a role in chromatin recognition.

Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation on lysines 8 and 12.

Abstract: We have investigated the ability of dexamethasone to regulate interleukin-1beta (IL-1beta)-induced gene expression, histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity. Low concentrations of dexamethasone (10(-10) M) repress IL-1beta-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression and fail to stimulate secretory leukocyte proteinase inhibitor expression. Dexamethasone (10(-7) M) and IL-1beta (1 ng/ml) both stimulated HAT activity but showed a different pattern of histone H4 acetylation. Dexamethasone targeted lysines K5 and K16, whereas IL-1beta targeted K8 and K12. Low concentrations of dexamethasone (10(-10) M), which do not transactivate, repressed IL-1beta-stimulated K8 and K12 acetylation. Using chromatin immunoprecipitation assays, we show that dexamethasone inhibits IL-1beta-enhanced acetylated K8-associated GM-CSF promoter enrichment in a concentration-dependent manner. Neither IL-1beta nor dexamethasone elicited any GM-CSF promoter association at acetylated K5 residues. Furthermore, we show that GR acts both as a direct inhibitor of CREB binding protein (CBP)-associated HAT activity and also by recruiting HDAC2 to the p65-CBP HAT complex. This action does not involve de novo synthesis of HDAC protein or altered expression of CBP or p300/CBP-associated factor. This mechanism for glucocorticoid repression is novel and establishes that inhibition of histone acetylation is an additional level of control of inflammatory gene expression. This further suggests that pharmacological manipulation of of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.

The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase Gcn5p

Abstract: The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

Genomewide studies of histone deacetylase function in yeast

Abstract: The trichostatin A (TSA)-sensitive histone deacetylase (HDAC) Rpd3p exists in a complex with Sin3p and Sap30p in yeast that is recruited to target promoters by transcription factors including Ume6p. Sir2p is a TSA-resistant HDAC that mediates yeast silencing. The transcription profile of rpd3 is similar to the profiles of sin3, sap30, ume6, and TSA-treated wild-type yeast. A Ume6p-binding site was identified in the promoters of genes up-regulated in the sin3 strain. Two genes appear to participate in feedback loops that modulate HDAC activity: ZRT1 encodes a zinc transporter and is repressed by RPD3 (Rpd3p is zinc-dependent); BNA1 encodes a nicotinamide adenine dinucleotide (NAD)-biosynthesis enzyme and is repressed by SIR2 (Sir2p is NAD-dependent). Although HDACs are transcriptional repressors, deletion of RPD3 down-regulates certain genes. Many of these are down-regulated rapidly by TSA, indicating that Rpd3p may also activate transcription. Deletion of RPD3 previously has been shown to repress (“silence”) reporter genes inserted near telomeres. The profiles demonstrate that 40% of endogenous genes located within 20 kb of telomeres are down-regulated by RPD3 deletion. Rpd3p appears to activate telomeric genes sensitive to histone depletion indirectly by repressing transcription of histone genes. Rpd3p also appears to activate telomeric genes repressed by the silent information regulator (SIR) proteins directly, possibly by deacetylating lysine 12 of histone H4. Finally, bioinformatic analyses indicate that the yeast HDACs RPD3, SIR2, and HDA1 play distinct roles in regulating genes involved in cell cycle progression, amino acid biosynthesis, and carbohydrate transport and utilization, respectively.

Chromodomains are protein–RNA interaction modules

Abstract: In Drosophila, compensation for the reduced dosage of genes located on the single male X chromosome involves doubling their expression in relation to their counterparts on female X chromosomes1. Dosage compensation is an epigenetic process involving the specific acetylation of histone H4 at lysine 16 by the histone acetyltransferase MOF2,3,4,5. Although MOF is expressed in both sexes, it only associates with the X chromosome in males. Its absence causes male-specific lethality6. MOF is part of a chromosome-associated complex comprising male-specific lethal (MSL) proteins and at least one non-coding roX RNA7. How MOF is integrated into the dosage compensation complex is unknown. Here we show that association of MOF with the male X chromosome depends on its interaction with RNA. MOF specifically binds through its chromodomain to roX2 RNA in vivo. In vitro analyses of the MOF and MSL-3 chromodomains indicate that these chromodomains may function as RNA interaction modules. Their interaction with non-coding RNA may target regulators to specific chromosomal sites.

The drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation.

Abstract: In Drosophila, dosage compensation-the equalization of most X-linked gene products in males and females-is achieved by a twofold enhancement of the level of transcription of the X chromosome in males relative to each X chromosome in females. A complex consisting of at least five gene products preferentially binds the X chromosome at numerous sites in males and results in a significant increase in the presence of a specific histone isoform, histone 4 acetylated at lysine 16. Recently, RNA transcripts (roX1 and roX2) encoded by two different genes have also been found associated with the X chromosome in males. We have partially purified a complex containing MSL1, -2, and -3, MOF, MLE, and roX2 RNA and demonstrated that it exclusively acetylates H4 at lysine 16 on nucleosomal substrates. These results demonstrate that the MSL complex is responsible for the specific chromatin modification characteristic of the X chromosome in Drosophila males.

Apicidin, a Histone Deacetylase Inhibitor, Inhibits Proliferation of Tumor Cells via Induction of p21WAF1/Cip1 and Gelsolin

Abstract: Apicidin [cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl -L-2-amino-8-oxodecanoyl)] is a fungal metabolite shown to exhibit antiparasitic activity by the inhibition of histone deacetylase (HDAC). In this study, we evaluated apicidin as a potential antiproliferative agent. Apicidin showed a broad spectrum of antiproliferative activity against various cancer cell lines, although with differential sensitivity. The antiproliferative activity of apicidin on HeLa cells was accompanied by morphological changes, cell cycle arrest at G1 phase, and accumulation of hyperacetylated histone H4 in vivo as well as inhibition of partially purified HDAC in vitro. In addition, apicidin induced selective changes in the expression of p21WAF1/Cip1 and gelsolin, which control the cell cycle and cell morphology, respectively. Consistent with increased induction of p21WAF1/Cip1, phosphorylation of Rb protein was markedly decreased, indicating the inhibition of cyclin-dependent kinases, which became bound to p21WAF1/Cip1. The effects of apicidin on cell morphology, expression of gelsolin, and HDAC1 activity in vivo and in vitro appeared to be irreversible, because withdrawal of apicidin did not reverse those effects, whereas the induction of p21WAF1/Cip1 by apicidin was reversible. Taken together, the results suggest that induction of histone hyperacetylation by apicidin is responsible for the antiproliferative activity through selective induction of genes that play important roles in the cell cycle and cell morphology.

Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression

Abstract: BACKGROUND—Sodium butyrate, a product of colonic bacterial fermentation, is able to inhibit cell proliferation and to stimulate cell differentiation of colonic epithelial cell lines. It has been proposed that these cellular effects could be linked to its ability to cause hyperacetylation of histone through the inhibition of histone deacetylase. AIM—To analyse the molecular mechanisms of butyrate action on cell proliferation/differentiation and to compare them with those of trichostatin A, a well known inhibitor of histone deacetylase. METHODS—HT-29 cells were grown in the absence or presence of butyrate or trichostatin A. Cell proliferation and cell cycle distribution were studied after DNA staining by crystal violet and propidium iodide respectively. Cell cycle regulatory proteins were studied by western blot and reverse transcription-polymerase chain reaction. Cell differentiation was followed by measuring brush border enzyme activities. Histone acetylation was studied by acid/urea/Triton acrylamide gel electrophoresis. RESULTS—Butyrate blocked cells mainly in the G1 phase of the cell cycle, whereas trichostatin A was inhibitory in both G1 and G2 phases. Butyrate inhibited the mRNA expression of cyclin D1 without affecting its protein expression and stimulated the protein expression of cyclin D3 without affecting its mRNA expression. Trichostatin A showed similar effects on cyclin D1 and D3. Butyrate and trichostatin A stimulated p21 expression both at the mRNA and protein levels, whereas their effects on the expression of cyclin dependent kinases were slightly different. Moreover, butyrate strongly stimulated the activity of alkaline phosphatase and dipeptidyl peptidase IV, whereas trichostatin A had no effect. Finally, a six hour exposure to butyrate or trichostatin A induced histone H4 hyperacetylation. At 15 and 24 hours, histone H4 remained hyperacetylated in the presence of butyrate, whereas it returned to control levels in the presence of trichostatin A. CONCLUSIONS—The data may explain how butyrate acts on cell proliferation/differentiation, and they show that trichostatin A does not reproduce every effect of butyrate, mainly because of its shorter half life. Keywords: butyrate; cyclin D; p21; trichostatin A; colonic epithelial cells; histone acetylation

The expression of D-cyclin genes defines distinct developmental zones in snapdragon apical meristems and is locally regulated by the Cycloidea gene.

Abstract: Three D-cyclin genes are expressed in the apical meristems of snapdragon (Antirrhinum majus). The cyclin D1 and D3b genes are expressed throughout meristems, whereas cyclin D3a is restricted to the peripheral region of the meristem, especially the organ primordia. During floral development, cyclin D3b expression is: (a) locally modulated in the cells immediately surrounding the base of organ primordia, defining a zone between lateral organs that may act as a developmental boundary; (b) locally modulated in the ventral petals during petal folding; and (c) is specifically repressed in the dorsal stamen by the cycloidea gene. Expression of both cyclin D3 genes is reduced prior to the cessation of cell cycle activity, as judged by histone H4 expression. Expression of all three D-cyclin genes is modulated by factors that regulate plant growth, particularly sucrose and cytokinin. These observations may provide a molecular basis for understanding the local regulation of cell proliferation during plant growth and development.

Cloning and characterization of a novel human histone deacetylase, HDAC8.

Abstract: Histone deacetylases (HDACs) are a growing family of enzymes implicated in transcriptional regulation by affecting the acetylation state of core histones in the nucleus of cells. HDACs are known to have key roles in the regulation of cell proliferation [Brehm, Miska, McCance, Reid, Bannister and Kouzarides (1998) Nature (London) 391, 597–600], and aberrant recruitment of an HDAC complex has been shown to be a key step in the mechanism of cell transformation in acute promyelocytic leukaemia [Grignani, De Matteis, Nervi, Tomassoni, Gelmetti, Cioce, Fanelli, Ruthardt, Ferrara, Zamir et al. (1998) Nature (London) 391, 815–818; Lin, Nagy, Inoue, Shao, Miller and Evans (1998), Nature (London) 391, 811–814]. Here we present the complete nucleotide sequence of a cDNA clone, termed HDAC8, that encodes a protein product with similarity to the RPD3 class (I) of HDACs. The predicted 377-residue HDAC8 product contains a shorter C-terminal extension relative to other members of its class. After expression in two cell systems, immunopurified HDAC8 is shown to possess trichostatin A- and sodium butyrate-inhibitable HDAC activity on histone H4 peptide substrates as well as on core histones. Expression profiling reveals the expression of HDAC8 to various degrees in every tissue tested and also in several tumour lines. Mutation of two adjacent histidine residues within the predicted active site severely decreases activity, confirming these residues as important for HDAC8 enzyme activity. Finally, linkage analysis after radiation hybrid mapping has localized HDAC8 to chromosomal position Xq21.2–Xq21.3. These results confirm HDAC8 as a new member of the HDAC family.

Histone H4 Acetylation of Euchromatin and Heterochromatin Is Cell Cycle Dependent and Correlated with Replication Rather Than with Transcription

Abstract: Reversible acetylation of nucleosomal histones H3 and H4 generally is believed to be correlated with potential transcriptional activity of eukaryotic chromatin domains. Here, we report that the extent of H4 acetylation within euchromatin and heterochromatic domains is linked with DNA replication rather than with transcriptional activity, whereas H3 acetylation remains fairly constant throughout the cell cycle. Compared with euchromatin, plant nucleolus organizers were more strongly acetylated at H4 during mitosis but less acetylated during S phase, when the nucleolus appeared to be (at least transiently) devoid of nucleosomes. Deposition-related acetylation of lysines 5 and 12 of H4 seems to be conserved in animals and plants and extended to K16 in plants. A possibly species-specific above-average acetylation at lysines 9/18 and 14 of H3 appeared in 4′,6-diamidino-2-phenylindole (DAPI)–stained heterochromatin fractions. These results were obtained by combining immunodetection of all acetylatable isoforms of H3 and H4 on mitotic chromosomes and nuclei in G1, early S, mid-S, late S, and G2 phases of the field bean with identification of specific chromatin domains by fluorescence in situ hybridization or DAPI staining. In addition, the histone acetylation patterns of distinct domains were compared with their replication and transcription patterns.

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33ING1 Are Associated with Histone Acetyltransferase Activities

Abstract: Several observations suggest that mammalian p33ING1 is involved in the regulation of cell proliferation and apoptosis (18, 21, 28). NIH 3T3 cells transformed by infection with a retrovirus containing a region of the Ing1 cDNA in the antisense orientation exhibit anchorage-independent growth in soft agar, and they form tumors in nude mice. Furthermore, microinjection of constructs that express Ing1 in the sense orientation results in inhibition of DNA synthesis and cell cycle progression in human diploid fibroblasts. Ing1 levels are also increased upon the induction of apoptosis in P19 cells by serum deprivation, and overexpression of Ing1 in P19 and rodent fibroblasts enhances Myc-dependent apoptosis (28). Evidence indicates that expression of Ing1 is repressed in a majority of breast and lymphoid cancer cell lines and glioblastomas and is mutated in some neuroblastoma cell lines, breast cancers, and brain tumors (21, 52, 74). Together, these observations suggest that Ing1 acts as a tumor suppressor and that it is involved in regulating apoptosis. This is further supported by reports that Ing1 and the p53 tumor suppressor form a complex and functionally cooperate to control cell growth (20, 83). The carboxyl-terminal 70 amino acid residues of Ing1 contain the Cys4-His-Cys3 sequence of a PHD finger domain. This evolutionarily conserved domain is predicted to chelate two Zn2+ ions and is similar to, but distinct from, other zinc binding motifs such as the RING finger (Cys3-His-Cys4) and LIM domain (Cys2-His-Cys5). PHD finger domains have been found in many different proteins, including transcription factors and other proteins implicated in chromatin-mediated transcriptional regulation (1). In particular, PHD fingers are found in the Drosophilia melanogaster polycomb (Pc-G) and trithorax (trx-G) group proteins, which are thought to reside in large multiprotein complexes. Pc-G and trx-G are required for the expression of homeotic genes, and evidence suggests that they exert their effects through chromatin modification or interaction. Thus, it has been proposed that PHD finger domains may be involved in complex formation or recognition of nuclear targets related to chromatin structure and chromatin regulation (1). In eukaryotes, DNA metabolism is strongly influenced by the packaging of DNA into higher-order chromatin. In general, chromatin structure is repressive to transcription (53, 54), and gene activation or silencing often requires remodeling of nucleosomes in promoter regions (38, 56). Covalent modifications, including acetylation, of core histones have been known for some time to be correlated with the activity of genetic loci (9, 75). Lysines in the amino-terminal extensions of histones are the targets of histone acetyltransferases (HATs) and histone deacetylases (HDACs). It has been hypothesized that neutralization of the positively charged histone N-terminal tails by acetylation lower their affinity for DNA, alter chromatin structure, and/or increase the interaction of histones with transcription factors (8, 31, 41, 44, 79). Several previously identified transcriptional coactivators or corepressors have been shown to possess the ability to acetylate or deacetylate histones (38, 56, 72). In Saccharomyces cerevisiae, proteins that possess HAT activity include Hat1 (36), Gcn5 (11), and Esa1 (68). Hat1 is localized in both the cytosol and nucleus and acetylates primarily newly synthesized histone H4 prior to its assembly into nucleosomes (36, 55, 60, 78). Gcn5 is a nuclear HAT that preferentially acetylates H3, and to a lesser extent it acetylates H2B and H4 (25). Gcn5 is not essential, but it is required for transcriptional regulation of some genes (22), and mutations that impair Gcn5 HAT activity correlate with decreased transcriptional activity (39, 81). Esa1 is an essential gene that was recently shown to possess HAT activity with a preference for H2A and H4 (13). Several mammalian transcription regulators have also been shown to possess HAT activity, including Gcn5 and Esa1 homologs (8), p300 and CREB-binding protein (5, 51), pCAF (82), ACTR (12), Src-1 (69), and TAFII250 (48). HATs function as components of large, evolutionarily conserved macromolecular assemblies, five of which have been identified in S. cerevisiae (16, 24, 25, 63). These include the 1.8-MDa SAGA (Spt-Ada-Gcn5-acetyltransferase), 0.8-MDa ADA, NuA3, 1.3-MDa NuA4 (nucleosomal H2A.H4), and the novel SLIK (SAGA-like) complexes. Esa1 was recently shown to be the HAT subunit of NuA4 (3), whereas Gcn5 is the catalytic HAT in the SAGA and ADA complexes (25). The HAT of the NuA3 complex has not been characterized aside from its substrate preference for histone H3 (25). Purified SAGA promotes acetyl coenzyme A (acetyl-CoA)-dependent transcription from nucleosomal promoter templates, but not free DNA, in vitro (70). This observation is consistent with the requirement for Gcn5 HAT activity for both promoter-directed histone acetylation and Gcn5-mediated transcriptional activation in vivo (39, 81). Furthermore, acidic activators such as Gcn4 and the VP16 activation domain can physically interact with purified native SAGA complex, and GAL4-VP16 targets acetylation and transcriptional stimulation by SAGA (76). Like SAGA, NuA4 is recruited to promoters by acidic activator proteins to promote histone acetylation and transcriptional stimulation (3, 13, 76). Unlike SAGA, which preferentially acetylates the N termini of histones H3 and H2B, NuA4 targets mainly the N termini of histone H4 and to a lesser extent H2A (3, 13). The SAGA complex contains at least four protein modules, including the Ada and Spt subgroups of transcription regulators, the histone-fold subgroup of TATA-binding protein-associated factors, and the essential 433-kDa Tra1 protein (24, 25). Tra1 has also been shown to be a component of the SLIK and NuA4 HAT complexes, and it also coelutes in a high-molecular-weight region distinct from the nucleosomal HAT complexes, indicating that it is present in uncharacterized protein complexes (24). Tra1 has been highly conserved among eukaryotes (47), and the mammalian homolog, TRRAP, is associated with the PCAF HAT complex (77). TRRAP was identified as a cofactor that interacts with c-Myc and E2F-1 and is required for transformation by c-Myc and E1A (47). The identification of TRRAP as an essential cofactor for these oncogenic transcription factors suggests that it regulates gene expression. Tra1 and TRRAP belong to the phosphatidyl inositol-3 (PI3) kinase family of serine/threonine protein kinases that includes mammalian DNA-PK, ATM, FRAP, Schizosaccharomyces pombe Rad3, and S. cerevisiae Vps34, Pik1, Stt4, Tor1, Tor2, Tel1, and Mec1 (63; reviewed in reference 8). These proteins appear to be involved in processes including cell cycle control, DNA repair, and transcription (35, 42). Although Tra1 and TRRAP are closely related to the PI3 kinases, they do not contain the DXXXXN and DFG motifs conserved in the catalytic site of PI3 kinases (47). The association of Tra1 and TRRAP with HAT complexes suggests that they regulate transcriptional activation through the recruitment of HAT activity to activator-bound promoters (24, 76). Although a scaffolding role of Tra1 has been suggested (8, 24), the molecular function of Tra1 and TRRAP are not known. Three proteins, Yng1, Yng2, and Pho23, in the budding yeast S. cerevisiae share significant sequence identity in their PHD finger domains with mammalian Ing1. We show that Yng2 is associated with Tra1, and we further demonstrate that Yng1, Yng2, and Pho23 are associated with HAT activities. We also provide strong evidence that the Yng2-associated HAT is Esa1, suggesting that Yng2 is a component of the NuA4 complex. Our results suggest that Yng1, Yng2, and Pho23 are involved in chromatin remodeling and possibly transcriptional regulation. We also report genetic and biochemical evidence suggesting that human and yeast Ing1 homologs have been functionally conserved.

Nickel Compounds Are Novel Inhibitors of Histone H4 Acetylation

Abstract: Environmental factors influence carcinogenesis by interfering with a variety of cellular targets. Carcinogenic nickel compounds, although generally inactive in most gene mutation assays, induce chromosomal damage in heterochromatic regions and cause silencing of reporter genes when they are located near telomere or heterochromatin in either yeast or mammalian cells. We studied the effects of nickel on the lysine acetylation status of the NH2-terminal region of histone H4. At nontoxic levels, nickel decreased the levels of histone H4 acetylation in vivo in both yeast and mammalian cells, affecting only lysine 12 in mammalian cells and all of the four lysine residues in yeast. In yeast, lysine 12 and 16 were more greatly affected than lysine 5 and 8. Interestingly, a histidine Ni2+ anchoring site is found at position 18 from the NH2-terminal tail of H4. Nickel was also found to inhibit the acetylation of H4 in vitro using purified recombinant histone acetyltransferase. To our knowledge, this is the first agent shown to decrease histone H4 acetylation at nontoxic levels.

Type B histone acetyltransferase Hat1p participates in telomeric silencing.

Abstract: Hat1p and Hat2p are the two subunits of a type B histone acetyltransferase from Saccharomyces cerevisiae that acetylates free histone H4 on lysine 12 in vitro. However, the role for these gene products in chromatin function has been unclear, as deletions of the HAT1 and/or HAT2 gene displayed no obvious phenotype. We have now identified a role for Hat1p and Hat2p in telomeric silencing. Telomeric silencing is the transcriptional repression of telomere-proximal genes and is mediated by a special chromatin structure. While there was no change in the level of silencing on a telomeric gene when the HAT1 or HAT2 gene was deleted, a significant silencing defect was observed when hat1Δ or hat2Δ was combined with mutations of the histone H3 NH2-terminal tail. Specifically, when at least two lysine residues were changed to arginine in the histone H3 tail, a hat1Δ-dependent telomeric silencing defect was observed. The most dramatic effects were seen when one of the two changes was in lysine 14. In further analysis, we found that a single lysine out of the five in the histone H3 tail was sufficient to mediate silencing. However, K14 was the best at preserving silencing, followed by K23 and then K27; K9 and K18 alone were insufficient. Mutational analysis of the histone H4 tail indicated that the role of Hat1p in telomeric silencing was mediated solely through lysine 12. Thus, in contrast to other histone acetyltransferases, Hat1p activity was required for transcriptional repression rather than gene activation.

Molecular mechanism for silencing virally transduced genes involves histone deacetylation and chromatin condensation

Abstract: Virally transduced genes are often silenced after integration into the host genome. Chromatin immunoprecipitation and nuclease sensitivity experiments now demonstrate that silencing of the transgene is characterized by deacetylation of histone H4 lysines and chromatin condensation. Trichostatin A treatment results in dramatic reactivation of gene expression that is preceded by histone acetylation and chromatin decondensation. Analysis of individual histone H4 lysines demonstrate that chromatin domain opening is coincident with rapid acetylation of histone H4 K5, K12, and K16 and that maintenance of the open domain is correlated with acetylation of histone H4 K8. Removal of trichostatin A results in rapid deacetylation of histone H4 K8, chromatin condensation, and transcription silencing. The results suggest that deacetylation of histone H4 lysines and coincident chromatin condensation are critically involved in the silencing of virally transduced genes.

Telomere folding is required for the stable maintenance of telomere position effects in yeast.

Abstract: Yeast telomeres reversibly repress the transcription of adjacent genes, a phenomenon called telomere position effect (TPE). TPE is thought to result from Rap1 and Sir protein-mediated spreading of heterochromatin-like structures from the telomeric DNA inwards. Because Rap1p is associated with subtelomeric chromatin as well as with telomeric DNA, yeast telomeres are proposed to form fold-back or looped structures. TPE can be eliminated in trans by deleting SIR genes or in cis by transcribing through the C(1-3)A/TG(1-3) tract of a telomere. We show that the promoter of a telomere-linked URA3 gene was inaccessible to restriction enzymes and that accessibility increased both in a sir3 strain and upon telomere transcription. We also show that subtelomeric chromatin was hypoacetylated at histone H3 and at each of the four acetylatable lysines in histone H4 and that histone acetylation increased both in a sir3 strain and when the telomere was transcribed. When transcription through the telomeric tract occurred in G(1)-arrested cells, TPE was lost, demonstrating that activation of a silenced telomeric gene can occur in the absence of DNA replication. The loss of TPE that accompanied telomere transcription resulted in the rapid and efficient loss of subtelomeric Rap1p. We propose that telomere transcription disrupts core heterochromatin by eliminating Rap1p-mediated telomere looping. This interpretation suggests that telomere looping is critical for maintaining TPE.

Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development

Abstract: Amphibian metamorphosis is marked by dramatic, thyroid hormone (TH)-induced changes involving gene regulation by TH receptor (TR). It has been postulated that TR-mediated gene regulation involves chromatin remodeling. In the absence of ligand, TR can repress gene expression by recruiting a histone deacetylase complex, whereas liganded TR recruits a histone acetylase complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving histone deacetylation. During metamorphosis, endogenous TH allows TR to activate gene expression through histone acetylation. Here using chromatin immunoprecipitation assay, we directly demonstrate TR binding to TH response genes constitutively in vivo in premetamorphic tadpoles. We further show that TH treatment leads to histone deacetylase release from TH response gene promoters. Interestingly, in whole animals, changes in histone acetylation show little correlation with the expression of TH response genes. On the other hand, in the intestine and tail, where TH response genes are known to be up-regulated more dramatically by TH than in most other organs, we demonstrate that TH treatment induces gene activation and histone H4 acetylation. These data argue for a role of histone acetylation in transcriptional regulation by TRs during amphibian development in some tissues, whereas in others changes in histone acetylation levels may play no or only a minor role, supporting the existence of important alternative mechanisms in gene regulation by TR.

Activation of the BRLF1 promoter and lytic cycle of Epstein–Barr virus by histone acetylation

Abstract: 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.

MSL1 plays a central role in assembly of the MSL complex, essential for dosage compensation in Drosophila

Abstract: In male Drosophila, histone H4 acetylated at Lys16 is enriched on the X chromosome, and most X-linked genes are transcribed at a higher rate than in females (thus achieving dosage compensation). Five proteins, collectively called the MSLs, are required for dosage compensation and male viability. Here we show that one of these proteins, MSL1, interacts with three others, MSL2, MSL3 and MOF. The latter is a putative histone acetyl transferase. Overexpression of either the N- or C-terminal domain of MSL1 has dominant-negative effects, i.e. causes male-specific lethality. The lethality due to expression of the N-terminal domain is reduced if msl2 is co-overexpressed. MSL2 co-purifies over a FLAG affinity column with the tagged region of MSL1, and both MSL3 and MOF co-purify with the FLAG-tagged MSL1 C-terminal domain. Furthermore, the MSL1 C-terminal domain binds specifically to a GST-MOF fusion protein and co-immunoprecipitates with HA-tagged MSL3. The MSL1 C-terminal domain shows similarity to a region of mouse CBP, a transcription co-activator. We conclude that a main role of MSL1 is to serve as the backbone for assembly of the MSL complex.

Targeting the chromatin-remodeling MSL complex of Drosophila to its sites of action on the X chromosome requires both acetyl transferase and ATPase activities.

Abstract: Dosage compensation in Drosophila is mediated by a multiprotein, RNA-containing complex that associates with the X chromosome at multiple sites. We have investigated the role that the enzymatic activities of two complex components, the histone acetyltransferase activity of MOF and the ATPase activity of MLE, may have in the targeting and association of the complex with the X chromosome. Here we report that MLE and MOF activities are necessary for complexes to access the various X chromosome sites. The role that histone H4 acetylation plays in this process is supported by our observations that MOF overexpression leads to the ectopic association of the complex with autosomal sites.

Isolation and use of a homologous histone H4 promoter and a ribosomal DNA region in a transformation vector for the oil-producing fungus Mortierella alpina.

Abstract: Mortierella alpina was transformed successfully to hygromycin B resistance by using a homologous histone H4 promoter to drive gene expression and a homologous ribosomal DNA region to promote chromosomal integration. This is the first description of transformation in this commercially important oleaginous organism. Two pairs of histone H3 and H4 genes were isolated from this fungus. Each pair consisted of one histone H3 gene and one histone H4 gene, transcribed divergently from an intergenic promoter region. The pairs of encoded histone H3 or H4 proteins were identical in amino acid sequence. At the DNA level, each histone H3 or H4 open reading frame showed 97 to 99% identity to its counterpart but the noncoding regions had little sequence identity. Unlike the histone genes from other filamentous fungi, all four M. alpina genes lacked introns. During normal vegetative growth, transcripts from the two histone H4 genes were produced at approximately the same level, indicating that either histone H4 promoter could be used in transformation vectors. The generation of stable, hygromycin B-resistant transformants required the incorporation of a homologous ribosomal DNA region into the transformation vector to promote chromosomal integration.