H2A Variant Research Articles

Histone H3 lysine 4 trimethylation regulates cotranscriptional H2A variant exchange by Tip60 complexes to maximize gene expression

Histone H3 lysine 4 trimethylation (H3K4me3) and the acetylated H2A variant, H2A.Z/v (H2Avac), are enriched at promoters of highly transcribed loci including the stress response genes. Using the inducible Drosophila hsp70 loci as a model, we study here the roles of the dSet1 and dTip60 complexes in the generation of these two chromatin modifications. We find that Heat Shock Factor recruits the dTip60 complex to the hsp70 loci in cells treated with salicylate, which triggers chromatin remodeling at these loci without transcription activation. Under these conditions, H2Avac or H3K4me3 are not enriched at the hsp70 promoter. By contrast, heat shock-induced hsp70 transcription induces dSet1-dependent H3K4me3 and H2Avac deposition by the dTip60 complex. The loss of dSet1 or dTip60 abolishes H2Avac incorporation, impairs Pol II release from the hsp70 promoter, and causes a stalling of mRNA production during phases of transcription maximization. Biochemical assays confirm that nucleosomal H3K4me3 stimulates the histone acetyltransferase and H2Av exchange activities of dTip60 complexes. H2Avac contributes to nucleosome destabilization at promoters, and H3K4me3 restricts its incorporation to phases of acute transcription. The process uncouples cotranscriptional chromatin remodeling by dTip60 complexes from their role in the activation of PARP, which is responsible for the removal of transcription-incompatible or damaged chromatin during the initial stress response. The control of the multifunctional dTip60 complex by H3K4me3 ensures optimal stress response and cell survival by mediating the rapid maximization of hsp70 expression. Furthermore, this mechanism prevents the accumulation of epigenetic noise caused by random complex-nucleosome collisions.

SIRT1-metabolite binding histone macroH2A1.1 protects hepatocytes against lipid accumulation

Non-alcoholic-fatty-liver-disease (NAFLD) encompasses conditions associated to fat deposition in the liver, which are generally deteriorated during the aging process. MacroH2A1, a variant of histone H2A, is a key transcriptional regulator involved in tumorigenic processes and cell senescence, and featuring two alternatively splicing isoforms, macroH2A1.1 and macroH2A1.2. MacroH2A1.1 binds with high affinity O-acetyl ADP ribose, a small metabolite produced by the reaction catalysed by NAD+-dependent deacetylase SIRT1, whereas macroH2A1.2 is unable to do so. The functional significance of this binding is unknown. We previously reported that the hepatic levels of macroH2A1.1 and macroH2A1.2 are differentially expressed in mice models of NAFLD. Here we show that over-expression of macroH2A1.1, but not of macroH2A1.2, is able to protect hepatocytes against lipid accumulation. MacroH2A1.1 over-expressing cells display ameliorated glucose metabolism, reduced expression of lipidogenic genes and fatty acids content. SIRT1/macroH2A1.1-dependent epigenetic regulation of lipid metabolism may be relevant to NAFLD development.

H2A.B facilitates transcription elongation at methylated CpG loci

H2A.B is a unique histone H2A variant that only exists in mammals. Here we found that H2A.B is ubiquitously expressed in major organs. Genome-wide analysis of H2A.B in mouse ES cells shows that H2A.B is associated with methylated DNA in gene body regions. Moreover, H2A.B-enriched gene loci are actively transcribed. One typical example is that H2A.B is enriched in a set of differentially methylated regions at imprinted loci and facilitates transcription elongation. These results suggest that H2A.B positively regulates transcription elongation by overcoming DNA methylation in the transcribed region. It provides a novel mechanism by which transcription is regulated at DNA hypermethylated regions.

The histone variant composition of centromeres is controlled by the pericentric heterochromatin state during the cell cycle.

Correct chromosome segregation requires a unique chromatin environment at centromeres and in their vicinity. Here, we address how the deposition of canonical H2A and H2A.Z histone variants is controlled at pericentric heterochromatin (PHC). Whereas in euchromatin newly synthesized H2A and H2A.Z are deposited throughout the cell cycle, we reveal two discrete waves of deposition at PHC - during mid to late S phase in a replication-dependent manner for H2A and during G1 phase for H2A.Z. This G1 cell cycle restriction is lost when heterochromatin features are altered, leading to the accumulation of H2A.Z at the domain. Interestingly, compromising PHC integrity also impacts upon neighboring centric chromatin, increasing the amount of centromeric CENP-A without changing the timing of its deposition. We conclude that the higher-order chromatin structure at the pericentric domain influences dynamics at the nucleosomal level within centromeric chromatin. The two different modes of rearrangement of the PHC during the cell cycle provide distinct opportunities to replenish one or the other H2A variant, highlighting PHC integrity as a potential signal to regulate the deposition timing and stoichiometry of histone variants at the centromere.

Disparity Expression of GammaH2AX in Papillary Thyroid Cancer and Nodular Goiter

H2AX, one of the variants of histone H2A with conserved sequence, plays a vital role in maintaining genomic stability. DNA double-strand break (DSB) induces H2AX phosphorylation (γH2AX). Growing evidence indicated γH2AX was linked to thyroid disease. However, the precise expression of γH2AX in papillary thyroid carcinoma (PTC) and nodular goiter was unknown. In this study, the yH2AX expression in PTC and nodular goiter and the significance of the aberrant expression were investigated. The γH2AX expression level was evaluated by immunohistochemistry staining in 140 specimens with thyroid diseases (30 cases of nodular goiter, 30 cases of PTC, and 80 cases of PTC coexisting with nodular goiter). The possible relationships between γH2AX expression and patients' clinicopathological characteristics were analyzed. In the set of nodular goiter, 20.0% (6/30) of nodular goiter component and 10.0% (3/30) of normal adjacent tissues exhibited high expression of γH2AX (p > 0.05). In the set of PTC, 70.0% (21/30) of PTC component and 0.0% (0/30) of normal adjacent tissues showed high expression of γH2AX (p < 0.05). In the set of PTC coexisting with nodular goiter, 95.0% (76/80) of PTC component, 12.5% (10/80) of nodular goiter component, and 8.8% (7/80) of normal adjacent specimens showed high expression of γH2AX (p < 0.05). 85.5% (65/76) of PTC patients with lymph node metastasis were found to have a high expression level of γH2AX, and the proportion was lower than that in patients without lymph node metastasis (34/34, 100.0%) (p = 0.003). High γH2AX expression was also found in 76.2% (16/21) of stage IV patients and the proportion was lower than that in stage I - III patients (83/89, 93.3%) (p = 0.034). No statistically significant correlation was found between yH2AX expression level and other clinicopathological features, including age, gender, capsular invasion, tumor size, tumor focus number, distant metastasis, and T status. γH2AX expression was increased in PTC patients, while nodular goiter and normal adjacent tissues exhibited no statistically significant difference in γH2AX expression. yH2AX expression had a negative correlation with TNM stage and lymph node metastasis. γH2AX seems to play a more critical role in early-stage PTC rather than late-stage PTC and might inhibit lymph node metastasis.

Localization and expression of histone H2A variants during mouse oogenesis and preimplantation embryo development.

Epigenetic modifications of the genome, such as histone H2A variants, ensure appropriate gene activation or silencing during oogenesis and preimplantation embryo development. We examined global localization and expression of the histone H2A variants, including H2A.Bbd, H2A.Z and H2A.X, during mouse oogenesis and preimplantation embryo development. Immunocytochemistry with specific antibodies against various histone H2A variants showed their localization and changes during oogenesis and preimplantation development. H2A.Bbd and H2A.Z were almost absent from nuclei of growing oocytes (except 5-day oocyte), whereas H2A.X was deposited in nuclei throughout oogenesis and in preimplantation embryos. In germinal vesicle (GV) oocyte chromatin, H2A.Bbd was detected as a weak signal, whereas no fluorescent signal was detected in GV breakdown (GVBD) or metaphase II (MII) oocytes; H2A.Z showed intense signals in chromatin of GV, GVBD and MII oocytes. H2A. Bbd showed very weak signals in both pronucleus and 2-cell embryo nuclei, but intense signals were detected in nuclei from 4-cell embryo to blastula. The H2A.Z signal was absent from pronucleus to morula chromatin, whereas a fluorescent signal was detected in blastula nuclei. Our results suggest that histone H2A variants are probably involved in reprogramming of genomes during oocyte meiosis or after fertilization.

Purine receptor P2Y&lt;sub&gt;6&lt;/sub&gt; mediates cellular response to γ-ray-induced DNA damage

We previously showed that nucleotide P2 receptor agonists such as ATP and UTP amplify γ-ray-induced focus formation of phosphorylated histone H2A variant H2AX (γH2AX), which is considered to be an indicator of DNA damage so far, by activating purine P2Y6 and P2Y12 receptors. Therefore, we hypothesized that these P2 receptors play a role in inducing the repair response to γ-ray-induced DNA damage. In the present study, we tested this idea by using human lung cancer A549 cells. First, reverse-transcription polymerase chain reaction (RT-PCR) showed that P2Y6 receptor is highly expressed in A549 cells, but P2Y12 receptor is only weakly expressed. Next, colony formation assay revealed that P2Y6 receptor antagonist MRS2578 markedly reduced the survival rate of γ-ray-exposed A549 cells. The survival rate was also significantly reduced in P2Y6-knock-down cells, compared with scramble siRNA-transfected cells. Since it has reported that phosphorylation of ERK1/2 after activation of EGFR via P2Y6 and P2Y12 receptors is involved in the repair response to γ-ray-induced DNA damage, we next examined whether γ-ray-induced phosphorylation of ERK1/2 was also inhibited by MRS2578 in A549 cells. We found that it was. Taken together, these findings indicate that purinergic signaling through P2Y6 receptor, followed by ERK1/2 activation, promotes the cellular repair response to γ-ray-induced DNA damage.

Trivalent methylated arsenic metabolites induce apoptosis in human myeloid leukemic HL-60 cells through generation of reactive oxygen species.

Arsenic trioxide (As2O3) has remarkable therapeutic efficacy against leukemia. However, after As2O3 biotransformation, the role of arsenic metabolites in the clinical efficacy against leukemia still needs to be elucidated. Therefore, to explore the contribution of trivalent methylated arsenicals in the therapeutic effects, we investigated and compared the effects of arsenite (iAs(III)), monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) on HL-60 cells. Methylated arsenic species MMA(III) and DMA(III) showed potentially reduced cell survival with IC50 values of 3 and 2 μM, respectively. We found that methylated metabolites caused apoptosis through oxidative stress and loss of mitochondrial membrane potential. Furthermore, we found that the caspase-9 and -3 were markedly activated by exposure to methylated metabolites, with cleavage of poly-ADP ribose polymerase (PARP). Conversely, cellular apoptosis, generation of ROS, activation of caspase-3, -9 as well as PARP cleavage were significantly attenuated by pretreatment with an antioxidant, N-acetylcysteine (NAC). DNA damage was also markedly observed in HL-60 cells exposed to either MMA(III) or DMA(III), while iAs(III) did not show any relevant effects in HL-60 cells. Likewise, phosphorylation of the histone H2A variant (γ-H2AX), a biomarker of DNA damage, significantly occurred in cellular nuclei following exposure to two methylated species, which was reduced in the presence of NAC, suggesting that the induction of DNA damage was predominantly caused by the two metabolites via oxidative stress. In conclusion, we suggest that arsenic intermediate metabolites; MMA(III) and DMA(III) might prove to be of clinical relevance in future as such approaches may help in the treatment of leukemia and other types of cancers.

HTZ-1/H2A.z and MYS-1/MYST HAT act redundantly to maintain cell fates in somatic gonadal cells through repression of ceh-22 in C. elegans

The stable maintenance of acquired cell fates is important during development and for maintaining tissue homeostasis. Although histone modification is one of the major strategies used by cells to maintain their fates, the mechanisms by which histone variants maintain cell fates are not well understood. In C. elegans, the acetylated-histone-H4 (AcH4)-binding protein BET-1 acts downstream of the MYST family histone acetyltransferases MYS-1 and MYS-2 to establish and maintain cell fates in multiple cell lineages. Here we show that, in the bet-1 pathway, the histone H2A variant HTZ-1/H2A.z and MYS-1 are required for the maintenance of cell fates in a redundant manner. BET-1 controlled the subnuclear localization of HTZ-1. HTZ-1 and MYS-1 maintained the fates of the somatic gonadal cells (SGCs) through the repression of a target, ceh-22/Nkx2.5, which induced the formation of the leader cells of the gonad. H3K27 demethylase, UTX-1, had an antagonistic effect relative to HTZ-1 in the regulation of ceh-22. Nuclear spot assay revealed that HTZ-1 localized to the ceh-22 locus in SGCs in an utx-1-dependent manner. We propose that HTZ-1 and MYS-1 repress ceh-22 when UTX-1 removes its silencing mark, H3K27 methylation on the ceh-22 locus, thereby maintaining the fates of SGCs.

Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatin.

Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to canonical H2A, and preferentially associates with actively transcribed genes. We found that H2A.B transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemical function of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of DNA, and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/exit sites from the histone surface. These results suggested that H2A.B rapidly and transiently forms nucleosomes with short DNA segments during chromatin reorganization.

Epigenetically induced paucity of histone H2A.Z stabilizes fission-yeast ectopic centromeres

In most eukaryotes, centromeres are epigenetically defined by nucleosomes that contain the histone H3 variant centromere protein A (CENP-A). Specific targeting of the CENP-A-loading chaperone to the centromere is vital for stable centromere propagation; however, the existence of ectopic centromeres (neocentromeres) indicates that this chaperone can function in different chromatin environments. The mechanism responsible for accommodating the CENP-A chaperone at noncentromeric regions is poorly understood. Here, we report the identification of transient, immature neocentromeres in Schizosaccharomyces pombe that show reduced association with the CENP-A chaperone Scm3, owing to persistence of the histone H2A variant H2A.Z. After the acquisition of adjacent heterochromatin or relocation of the immature neocentromeres to subtelomeric regions, H2A.Z was depleted and Scm3 was replenished, thus leading to subsequent stabilization of the neocentromeres. These findings provide new insights into histone variant-mediated epigenetic control of neocentromere establishment.

H2AX a Promising Biomarker for Lung Cancer: A Review

Histone's H2A variant (H2AX) phosphorylation is an early indicator of DNA double-strand breaks formation and DNA damage response. Thus, it may act as a novel biomarker to monitor genotoxic events that can drive cancer development and tumor progression. This review will focus on the possible applications of H2AX as a key regulator of DNA damage response in lung cancer and as a biomarker of: sensitivity of lung tumors to chemotherapy and radiotherapy, treatment with PARP inhibitors, bystander effect, multistep lung carcinogenesis, environmental smoking, and chemical genotoxicity, chemoprevention, prognosis, and also as therapeutic targets in lung cancers.

Gene dysregulation by histone variant H2A.Z in bladder cancer

BackgroundThe incorporation of histone variants into nucleosomes is one of the main strategies that the cell uses to regulate the structure and function of chromatin. Histone H2A.Z is an evolutionarily conserved histone H2A variant that is preferentially localized within nucleosomes at the transcriptional start site (TSS). H2A.Z reorganizes the local chromatin structure and recruits the transcriptional machinery for gene activation. High expression of H2A.Z has been reported in several types of cancers and is causally linked to genomic instability and tumorigenesis. However, it is not entirely clear how H2A.Z overexpression in cancer cells establishes aberrant chromatin states and promotes gene expression.ResultsThrough integration of genome-wide H2A.Z ChIP-seq data with microarray data, we demonstrate that H2A.Z is enriched around the TSS of cell cycle regulatory genes in bladder cancer cells, and this enrichment is correlated with the elevated expression of cancer-promoting genes. RNAi-mediated knockdown of H2A.Z in the cancer cells causes transcriptional suppression of multiple cell cycle regulatory genes with a distinct decrease in cell proliferation. H2A.Z nucleosomes around the TSS have higher levels of H3K4me2/me3, which coincides with the recruitment of two chromatin factors, WDR5 and BPTF. The observed recruitment is functional, as the active states of H2A.Z target genes are largely erased by suppressing the expression of WDR5 or BPTF, effects resembling H2A.Z knockdown.ConclusionsWe conclude that H2A.Z is overexpressed in bladder cancer cells and contributes to cancer-related transcription pathways. We also provide evidence in support of the engagement of H3K4me2/me3 and WDR5/BPTF in H2A.Z-induced cancer pathogenesis. Further studies are warranted to understand how H2A.Z overexpression contributes to the recruitment of the full repertoire of transcription machinery to target genes in bladder cancer cells.

Oocyte-Specific H2A Variant H2af1o Is Required for Cell Synchrony Before Midblastula Transition in Early Zebrafish Embryos1

Oocyte-specific histone variants have been expected to play significant roles in early embryonic development, but the exact evidence and the biological function have remained unclear. Here, we present evidence that H2af1o, an oocyte-specific H2A variant, is required for cell synchrony before midblastula transition in early zebrafish embryos. The H2A variant is oocyte specific, peaks in mature eggs, and is supplied to early embryos. We constructed a series of deletion plasmids of the zebrafish h2af1o tagged with EGFP and determined the main key function regions including nuclear localization signal of N-terminal 25 amino acids and nucleosome binding region of 110-122 amino acid sequence in the C-terminus by microinjecting them into one-cell-stage zebrafish embryos. In comparison with ubiquitous H2A.X, the H2af1o was revealed to confer a more open structure than canonical H2A in the nucleosomes. Furthermore, we conducted the h2af1o-specific morpholino knockdown analysis in early embryos of zebrafish and revealed its biological function for maintaining cell synchrony division because the H2af1o deficiency disturbed cell synchrony in early cleavages before midblastula transition. Therefore, our current findings provided the first case to understand the biological function of maternal oocyte-specific histone variants in vertebrates.

Mutations in Non-Acid Patch Residues Disrupt H2A.Z’s Association with Chromatin through Multiple Mechanisms

The incorporation of histone variants into nucleosomes is a critical mechanism for regulating essential DNA-templated processes and for establishing distinct chromatin architectures with specialised functions. H2A.Z is an evolutionarily conserved H2A variant that has diverse roles in transcriptional regulation, heterochromatin boundary definition, chromosome stability and DNA repair. The H2A.Z C-terminus diverges in sequence from canonical H2A and imparts unique functions to H2A.Z in the yeast S. cerevisiae. Although mediated in part through the acid patch-containing M6 region, many molecular determinants of this divergent structure-function relationship remain unclear. Here, by using an unbiased random mutagenesis screen of H2A.Z alleles, we identify point mutations in the C-terminus outside of the M6 region that disrupt the normal function of H2A.Z in response to cytotoxic stress. These functional defects correlate with reduced chromatin association, which we attribute to reduced physical stability within chromatin, but also to altered interactions with the SWR and INO80 chromatin remodeling complexes. Together with experimental data, computational modelling of these residue changes in the context of protein structure suggests the importance of C-terminal domain integrity and configuration for maintaining the level of H2A.Z in nucleosomes.

Histone Variant H2A.Z Functions in Sister Chromatid Cohesion inSaccharomyces cerevisiae

H2A.Z is a highly conserved variant of histone H2A with well-characterized roles in transcriptional regulation. We previously reported that H2A.Z and Mcd1, a subunit of the cohesin complex, regulate the establishment of transcriptional silencing at telomeres in Saccharomyces cerevisiae and that H2A.Z broadly dissociated from chromatin during the anaphase-to-telophase transition, coincident with the dissociation of Mcd1 from chromosomes and dissolution of cohesion. In this study, we show that depletion of H2A.Z causes precocious loss of sister chromatid cohesion in yeast without loss of Mcd1 from chromosomes. H2A.Z is deposited into chromatin by the SWR1 complex and is subject to acetylation of its four N-terminal tail lysine residues by the NuA4 and SAGA histone acetyltransferase complexes. We found that cells compromised for function of the SWR1 complex were defective in cohesion, as were cells expressing a form of H2A.Z not subject to acetylation. Finally, inactivation of H2A.Z in metaphase-blocked cells led immediately to cohesion defects, suggesting a direct role for H2A.Z in the maintenance of sister chromatid cohesion.

Structure and Subunit Topology of the INO80 Chromatin Remodeler and Its Nucleosome Complex

INO80/SWR1 family chromatin remodelers are complexes composed of >15 subunits and molecular masses exceeding 1 MDa. Their important role in transcription and genome maintenance is exchanging the histone variants H2A and H2A.Z. We report the architecture of S. cerevisiae INO80 using an integrative approach of electron microscopy, crosslinking and mass spectrometry. INO80 has an embryo-shaped head-neck-body-foot architecture and shows dynamic open and closed conformations. We can assign an Rvb1/Rvb2 heterododecamer to the head in close contact with the Ino80 Snf2 domain, Ies2, and the Arp5 module at the neck. The high-affinity nucleosome-binding Nhp10 module localizes to the body, whereas the module that contains actin, Arp4, and Arp8 maps to the foot. Structural and biochemical analyses indicate that the nucleosome is bound at the concave surface near the neck, flanked by the Rvb1/2 and Arp8 modules. Our analysis establishes a structural and functional framework for this family of large remodelers.

H2A.Z Acidic Patch Couples Chromatin Dynamics to Regulation of Gene Expression Programs during ESC Differentiation

The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.ZAP3) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.ZAP3 interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.ZAP3 was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.ZAP3 ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.ZAP3 ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.ZAP3 displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.ZAP3 mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment.

Histone variant Htz1 promotes histone H3 acetylation to enhance nucleotide excision repair in Htz1 nucleosomes

Nucleotide excision repair (NER) is critical for maintaining genome integrity. How chromatin dynamics are regulated to facilitate this process in chromatin is still under exploration. We show here that a histone H2A variant, Htz1 (H2A.Z), in nucleosomes has a positive function in promoting efficient NER in yeast. Htz1 inherently enhances the occupancy of the histone acetyltransferase Gcn5 on chromatin to promote histone H3 acetylation after UV irradiation. Consequently, this results in an increased binding of a NER protein, Rad14, to damaged DNA. Cells without Htz1 show increased UV sensitivity and defective removal of UV-induced DNA damage in the Htz1-bearing nucleosomes at the repressed MFA2 promoter, but not in the HMRa locus where Htz1 is normally absent. Thus, the effect of Htz1 on NER is specifically relevant to its presence in chromatin within a damaged region. The chromatin accessibility to micrococcal nuclease in the MFA2 promoter is unaffected by HTZ1 deletion. Acetylation on previously identified lysines of Htz1 plays little role in NER or cell survival after UV. In summary, we have identified a novel aspect of chromatin that regulates efficient NER, and we provide a model for how Htz1 influences NER in Htz1 nucleosomes.

The ATP-dependent Chromatin Remodeling Enzyme Fun30 Represses Transcription by Sliding Promoter-proximal Nucleosomes

The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5' and 3' ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30 target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, a genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5' end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2, and +3 nucleosomes at the 5' end of target genes were shifted significantly, whereas the position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity-purified, single-component Fun30 exhibited a nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in the regulation of transcription and indicate that Fun30 remodels chromatin at the 5' end of genes by sliding promoter-proximal nucleosomes.