H3 K4 Research Articles

Yng1 PHD Finger Binding to H3 Trimethylated at K4 Promotes NuA3 HAT Activity at K14 of H3 and Transcription at a Subset of Targeted ORFs

Posttranslational histone modifications participate in modulating the structure and function of chromatin. Promoters of transcribed genes are enriched with K4 trimethylation and hyperacetylation on the N-terminal tail of histone H3. Recently, PHD finger proteins, like Yng1 in the NuA3 HAT complex, were shown to interact with H3K4me3, indicating a biochemical link between K4 methylation and hyperacetylation. By using a combination of mass spectrometry, biochemistry, and NMR, we detail the Yng1 PHD-H3K4me3 interaction and the importance of NuA3-dependent acetylation at K14. Furthermore, genome-wide ChIP-Chip analysis demonstrates colocalization of Yng1 and H3K4me3 in vivo. Disrupting the K4me3 binding of Yng1 altered K14ac and transcription at certain genes, thereby demonstrating direct in vivo evidence of sequential trimethyl binding, acetyltransferase activity, and gene regulation by NuA3. Our data support a general mechanism of transcriptional control through which histone acetylation upstream of gene activation is promoted partially through availability of H3K4me3, "read" by binding modules in select subunits.

Proteolysis of MLL Family Proteins Is Essential for Taspase1−Orchestrated Cell Cycle Progression.

Taspase1 was identified as the threonine endopeptidase that cleaves MLL for proper Hox gene expression in vitro. To investigate its functions in vivo, we generated Taspase1−/− mice. Taspase1 deficiency results in non−cleavage (nc) of MLL and MLL2 and homeotic transformations. Remarkably, our in vivo studies uncover an unexpected role of Taspase1 in cell cycle. Taspase1−/− animals are smaller in size. Taspase1−/− MEFs exhibit impaired proliferation and acute deletion of Taspase1 leads to a marked reduction of thymocytes. Taspase1 deficiency incurs down−regulation of Cyclin Es, As, and Bs and up−regulation of p16Ink4a. We show that MLL and MLL2 directly target E2Fs for Cyclin expression. The un−cleaved precursor MLL displays a reduced histone H3 methyl transferase activity in vitro. Accordingly, CHIP assays demonstrate a markedly decreased histone H3 K4 tri−methylation at Cyclin E1 and E2 genes in Taspase1−/− cells. Furthermore, MLLnc/nc;2nc/nc MEFs are also impaired in proliferation. Our data are consistent with a model in which precursor MLLs, activated by Taspase1, target to Cyclins through E2Fs to methylate histone H3 at K4, leading to activation. Lastly, Taspase1−/− cells are resistant to oncogenic transformation and Taspase1 is over−expressed in many cancer cell lines. Thus, Taspase1 may serve as a target for cancer therapeutics.

C-Myb-Interacting Proteins and Their Functions.

Post-translational modifications of chromatin have fundamental roles in many biological processes including transcriptional regulation, heterochromatin organization, and x-chromosome inactivation. One of these modifications, histone methylation, is recognized as an important component of an epigenetic indexing system demarcating transcriptionally active and inactive domains. It is possible that methylation induces alterations in chromatin architecture, either condensing or relaxing its structure. These alterations not only regulate accessibility for DNA binding proteins, but also create binding sites for regulatory proteins that contain specialized binding domains. The c-Myb gene is the normal cellular counterpart of an oncogene transduced by the avian myeloblastosis virus. It is a nuclear protein that functions as a DNA binding transcription factor and a number of complementary gene silencing strategies have demonstrated that it plays a pivotal role during hematopoietic cell development and is an obligate requirement for adult hematopoiesis. c-Myb is highly expressed in immature hematopoietic cells and down-regulated upon differentiation. It has also been shown that c-Myb expression is important in promoting the proliferation of leukemic cells.However, the biologically relevant molecular mechanisms that regulate c-Myb activity during normal and leukemic hematopoiesis remain unclear. In order to investigate c-Myb function in leukemic vs non-leukemic cells, and ultimately in normal cells, more mechanistically, we expressed epitope-tagged c-Myb in transiently transfected 293T cells, and both transiently and stably transfected K562 human leukemia cells. We then carried out a series of immunoprecipitation experiments to examine proteins that c-Myb might interact with in both environments. In accord with prior reports, we found that c-Myb is associated with the tumor suppressor protein menin, a product of the MEN1 gene that is mutated in familial multiple endocrine neoplasia type 1. Menin has been reported to associate with a Trithorax family histone methyltransferase complex, and to be an essential oncogenic cofactor for MLL-associated leukemogenesis. Stable interaction between c-Myb and menin was confirmed by reciprocal immunoprecipitation in co-transfected 293T cells and retrovirus infected K562 human leukemia cells. We also examined whether c-Myb is associated with the histone methyltransferase complex in transfected 293T cells. Again, using an immunoprecipitation strategy we found that c-Myb is associated with both WDR5 and RbBp5, which are common components of multiple H3 K4 methyltransferase complexes. A Luciferase reporter assay revealed that transfection of 100 ng of WDR5 with 200 ng of c-Myb resulted in 2-fold activation of the reporter, as compared to signal obtained from cells transfected with 200 ng of c-Myb cDNA alone. Furthermore, c-Myb-associated methyltransferase complexes, isolated from c-Myb transfected 293T cells, K562 human leukemia cells expressing epitope-tagged c-Myb, as well as untransfected K562 cells, were able to methylate histone H3. In aggregate, these results suggest that in addition to regulating gene transcription by binding to DNA, c-Myb may also regulate transcription via a new paradigm, that of covalent histone modification.

A Highly Specific Mechanism of Histone H3-K4 Recognition by Histone Demethylase LSD1

Human lysine-specific demethylase (LSD1) is a chromatin-modifying enzyme that specifically removes methyl groups from mono- and dimethylated Lys4 of histone H3 (H3-K4). We used a combination of in vivo and in vitro experiments to characterize the substrate specificity and recognition by LSD1. Biochemical assays on histone peptides show that essentially all epigenetic modifications on the 21 N-terminal amino acids of histone H3 cause a significant reduction in enzymatic activity. Replacement of Lys4 with Arg greatly enhances binding affinity, and a histone peptide incorporating this mutation has a strong inhibitory power. Conversely, a peptide bearing a trimethylated Lys4 is only a weak inhibitor of the enzyme. Rapid kinetics measurements evidence that the enzyme is efficiently reoxidized by molecular oxygen with a second-order rate constant of 9.6x10(3) M-1 s-1, and that the presence of the reaction product does not greatly influence the rate of flavin reoxidation. In vivo experiments provide a correlation between the in vitro inhibitory properties of the tested peptides and their ability of affecting endogenous LSD1 activity. Our results show that epigenetic modifications on histone H3 need to be removed before Lys4 demethylation can efficiently occur. The complex formed by LSD1 with histone deacetylases 1/2 may function as a "double-blade razor" that first eliminates the acetyl groups from acetylated Lys residues and then removes the methyl group from Lys4. We suggest that after H3-K4 demethylation, LSD1 recruits the forthcoming chromatin remodelers leading to the introduction of gene repression marks.

The genomic landscape of histone modifications in human T cells

To understand the molecular basis that supports the dynamic gene expression programs unique to T cells, we investigated the genomic landscape of activating histone modifications, including histone H3 K9/K14 diacetylation (H3K9acK14ac), H3 K4 trimethylation (H3K4me3), and the repressive histone modification H3 K27 trimethylation (H3K27me3) in primary human T cells. We show that H3K9acK14ac and H3K4me3 are associated with active genes required for T cell function and development, whereas H3K27me3 is associated with silent genes that are involved in development in other cell types. Unexpectedly, we find that 3,330 gene promoters are associated with all of these histone modifications. The gene expression levels are correlated with both the absolute and relative levels of the activating H3K4me3 and the repressive H3K27me3 modifications. Our data reveal that rapidly inducible genes are associated with the H3 acetylation and H3K4me3 modifications, suggesting they assume a chromatin structure poised for activation. In addition, we identified a subpopulation of chromatin regions that are associated with high levels of H3K4me3 and H3K27me3 but low levels of H3K9acK14ac. Therefore, these regions have a distinctive chromatin modification pattern and thus may represent a distinct class of chromatin domains.

Opposing roles for Set2 and yFACT in regulating TBP binding at promoters

Previous work links histone methylation by Set2 with transcriptional elongation. yFACT (Spt16-Pob3 and Nhp6) reorganizes nucleosomes and functions in both transcriptional initiation and elongation. We show that growth defects caused by spt16 or pob3 mutations can be suppressed by deleting SET2, suggesting that Set2 and yFACT have opposing roles. Set2 methylates K36 of histone H3, and K36 substitutions also suppress yFACT mutations. In contrast, set1 enhances yFACT mutations. Methylation at H3 K4 by Set1 is required for set2 to suppress yFACT defects. We did not detect an elongation defect at an 8 kb ORF in yFACT mutants. Instead, pob3 mutants displayed reduced binding of both pol II and TBP to the GAL1 promoter. Importantly, both GAL1 transcription and promoter binding of pol II and TBP are significantly restored in the pob3 set2 double mutant. Defects caused by an spt16 mutation are enhanced by either TBP or TFIIA mutants. These synthetic defects are suppressed by set2, demonstrating that yFACT and Set2 oppose one another during transcriptional initiation at a step involving DNA binding by TBP and TFIIA.

Proline Isomerization of Histone H3 Regulates Lysine Methylation and Gene Expression

The cis-trans isomerization of proline serves as a regulatory switch in signaling pathways. We identify the proline isomerase Fpr4, a member of the FK506 binding protein family in Saccharomyces cerevisiae, as an enzyme which binds the amino-terminal tail of histones H3 and H4 and catalyses the isomerization of H3 proline P30 and P38 in vitro. We show that P38 is necessary for methylation of K36 and that isomerization by Fpr4 inhibits the ability of Set2 to methylate H3 K36 in vitro. These results suggest that the conformational state of P38, controlled by Fpr4, is important for methylation of H3K36 by Set2. Consistent with such an antagonistic role, abrogation of Fpr4 catalytic activity in vivo results in increased levels of H3K36 methylation and delayed transcriptional induction kinetics of specific genes in yeast. These results identify proline isomerization as a novel noncovalent histone modification that regulates transcription and provides evidence for crosstalk between histone lysine methylation and proline isomerization.

Histone H3 Acetylation and H3 K4 Methylation Define Distinct Chromatin Regions Permissive for Transgene Expression

Histone modifications are associated with distinct transcription states and serve as heritable epigenetic markers for chromatin structure and function. While H3 K9 methylation defines condensed heterochromatin that is able to silence a nearby gene, how gene silencing within euchromatin regions is achieved remains elusive. We report here that histone H3 K4 methylation or K9/K14 acetylation defines distinct chromatin regions permissive or nonpermissive for transgene expression. A permissive chromatin region is enriched in H3 K4 methylation and H3 acetylation, while a nonpermissive region is poor in or depleted of these two histone modifications. The histone modification states of the permissive chromatin can spread to transgenic promoters. However, de novo histone H3 acetylation and H3 K4 methylation at a transgenic promoter in a nonpermissive chromatin region are stochastic, leading to variegated transgene expression. Moreover, nonpermissive chromatin progressively silences a transgene, an event that is accompanied by the reduction of H3 K4 methylation and H3 acetylation levels at the transgenic promoter. These repressive effects of nonpermissive chromatin cannot be completely countered by strong transcription activators, indicating the dominance of the chromatin effects. We therefore propose a model in which histone H3 acetylation and H3 K4 methylation localized to discrete sites in the mammalian genome mark distinct chromatin functions that dictate transgene expression or silencing.

Proteolysis of MLL family proteins is essential for Taspase1-orchestrated cell cycle progression

Taspase1 was identified as the threonine endopeptidase that cleaves mixed-lineage leukemia (MLL) for proper Hox gene expression in vitro. To investigate its functions in vivo, we generated Taspase1(-/-) mice. Taspase1 deficiency results in noncleavage (nc) of MLL and MLL2 and homeotic transformations. Remarkably, our in vivo studies uncover an unexpected role of Taspase1 in the cell cycle. Taspase1(-/-) animals are smaller in size. Taspase1(-/-) mouse embryonic fibroblasts (MEFs) exhibit impaired proliferation, and acute deletion of Taspase1 leads to a marked reduction of thymocytes. Taspase1 deficiency incurs down-regulation of Cyclin Es, As, and Bs and up-regulation of p16(Ink4a) . We show that MLL and MLL2 directly target E2Fs for Cyclin expression. The uncleaved precursor MLL displays a reduced histone H3 methyl transferase activity in vitro. Accordingly, chromatin immunoprecipitation assays demonstrate a markedly decreased histone H3 K4 trimethylation at Cyclin E1 and E2 genes in Taspase1(-/-) cells. Furthermore, MLL(nc/nc;2nc/nc) MEFs are also impaired in proliferation. Our data are consistent with a model in which precursor MLLs, activated by Taspase1, target to Cyclins through E2Fs to methylate histone H3 at K4, leading to activation. Lastly, Taspase1(-/-) cells are resistant to oncogenic transformation, and Taspase1 is overexpressed in many cancer cell lines. Thus, Taspase1 may serve as a target for cancer therapeutics.

Different roles of histone H3 lysine 4 methylation in chromatin maintenance

Histone H3 methyltransferases are involved in the epigenetic control of transcription and heterochromatin maintenance. In Saccharomyces cerevisiae, deletion of a histone H3 methyltransferase SET1 leads to the induction of a subset of stress responsive genes in a Rad53 dependent manner. This type of induction was observed only in the absence of SET1 and not in the absence of other histone methyltransferases, SET2 or DOT1. We show that the increased expression of the stress responsive genes results from a lack of histone H3 lysine (K) 4 methylation. The loss of mono-methylation on H3 K4 is necessary to increase the expression of the stress responsive genes, while the loss of di- or tri-methylation induced by deletion of either RRM domain of Set1 or the upstream effector molecules hardly affected their expression. These results suggest that mono- and multiple methylation of H3 K4 have different roles. The mono-methylation of H3 K4 might be required for the global integrity of chromatin structure, which is normally monitored by the Rad53 dependent chromatin surveillance system.

The RAD6/BRE1 Histone Modification Pathway in Saccharomyces Confers Radiation Resistance Through a RAD51-Dependent Process That Is Independent of RAD18

We examine ionizing radiation (IR) sensitivity and epistasis relationships of several Saccharomyces mutants affecting post-translational modifications of histones H2B and H3. Mutants bre1Delta, lge1Delta, and rtf1Delta, defective in histone H2B lysine 123 ubiquitination, show IR sensitivity equivalent to that of the dot1Delta mutant that we reported on earlier, consistent with published findings that Dot1p requires H2B K123 ubiquitination to fully methylate histone H3 K79. This implicates progressive K79 methylation rather than mono-methylation in IR resistance. The set2Delta mutant, defective in H3 K36 methylation, shows mild IR sensitivity whereas mutants that abolish H3 K4 methylation resemble wild type. The dot1Delta, bre1Delta, and lge1Delta mutants show epistasis for IR sensitivity. The paf1Delta mutant, also reportedly defective in H2B K123 ubiquitination, confers no sensitivity. The rad6Delta, rad51null, rad50Delta, and rad9Delta mutations are epistatic to bre1Delta and dot1Delta, but rad18Delta and rad5Delta show additivity with bre1Delta, dot1Delta, and each other. The bre1Delta rad18Delta double mutant resembles rad6Delta in sensitivity; thus the role of Rad6p in ubiquitinating H2B accounts for its extra sensitivity compared to rad18Delta. We conclude that IR resistance conferred by BRE1 and DOT1 is mediated through homologous recombinational repair, not postreplication repair, and confirm findings of a G1 checkpoint role for the RAD6/BRE1/DOT1 pathway.

Characterization of novel genetic interactions between the Ipl1 Aurora kinase, the Set1 methyltransferase, and the Rad6‐Bre1 E2–E3 ubiquitin ligases

Proper chromosome segregation during mitosis is crucial for the faithful inheritance of genetic material. Ipl1 is the founding member of the Aurora kinases, a fundamental class of regulators of chromosome segregation and cytokinesis from yeast to humans. In the yeast S. cerevisiae, Ipl1 phosphorylates histone H3 and several kinetochore proteins including Dam1. Another histone modifying enzyme is the methyltransferase Set1. Both the Paf1 complex and ubiquitination of H2B by Rad6/Bre1 are necessary for Set1 methylation of H3. Our lab has shown loss of SET1 suppresses the temperature sensitive (ts) and chromosome segregation defects of the ipl1-2 mutant allele. Further experiments indicate that this suppression is through Set1 dimethylation of Dam1 at lysine 233, and that a balance of dimethylation and phosphorylation of flanking serines are important for proper Dam1 function. Here I show that Set1 is functioning within the COMPASS complex at the kinetochore. I also show that suppression of ipl1-2 ts phenotype by loss of SET1 is not through methylation of H3 K4 as the loss of genes necessary for H3 K4 dimethylation does not suppress the ipl1-2 ts phenotype. Surprisingly, I found that loss of RAD6, BRE1, and the Paf1 complex member RTF1 suppress the ipl1-2 ts phenotype, whereas loss of PAF1 does not. Therefore, some but not all of the factors required for H3 K4 dimethylation may also be required for Dam1 K233 dimethylation by Set1. This work supported by NIH T32 HD07325 & Robert A. Welch Foundation (G1371)

Resetting the Epigenetic Histone Code in the MRL-lpr/lpr Mouse Model of Lupus by Histone Deacetylase Inhibition

The baseline level of gene expression varies between healthy controls and systemic lupus erythematosus (SLE) patients, and among SLE patients themselves. These variations may explain the different clinical manifestations and severity of disease observed in SLE. Epigenetic mechanisms, which involve DNA and histone modifications, are predictably associated with distinct transcriptional states. To understand the interplay between various histone modifications, including acetylation and methylation, and lupus disease, we performed differential expression histone modification analysis in splenocytes from the MRL-lpr/lpr mouse model of lupus. Using stable isotope labeling in combination with mass spectrometry, we found global site-specific hypermethylation (except H3 K4 methylation) and hypoacetylation in histone H3 and H4 MRL-lpr/lpr mice compared to control MRL/MPJ mice. Moreover, we have identified novel histone modifications such as H3 K18 methylation, H4 K31 methylation, and H4 K31 acetylation that are differentially expressed in MRL-lpr/lpr mice compared to controls. Finally, in vivo administration of the histone deacetylase inhibitor trichostatin A (TSA) corrected the site-specific hypoacetylation states on H3 and H4 in MRL-lpr/lpr mice with improvement of disease phenotype. Thus, this study is the first to establish the association between aberrant histone codes and pathogenesis of autoimmune disease SLE. These aberrant post-translational histone modifications can therefore be reset with histone deacetylase inhibition in vivo.

BUR Kinase Selectively Regulates H3 K4 Trimethylation and H2B Ubiquitylation through Recruitment of the PAF Elongation Complex

Histone-lysine methylation is linked to transcriptional regulation and the control of epigenetic inheritance. Lysine residues can be mono-, di-, or trimethylated, and it has been suggested that each methylation state of a given lysine may impart a unique biological function. In yeast, histone H3 lysine 4 (K4) is mono-, di-, and trimethylated by the Set1 histone methyltransferase. Previous studies show that Set1 associates with RNA polymerase II and demarcates transcriptionally active genes with K4 trimethylation. To determine whether K4 trimethylation might be selectively regulated, we screened a library of yeast deletion mutants associated with transcriptional regulation and chromatin function. We identified BUR2, a cyclin for the Bur1/2 (BUR) cyclin-dependent protein kinase, as a specific regulator of K4 trimethylation. Surprisingly, BUR also regulated H2B monoubiquitylation, whereas other K4 methylation states and H3 lysine 79 (K79) methylation were unaffected. Synthetic genetic array (SGA) and transcription microarray analyses of a BUR2 mutant revealed that BUR is functionally similar to the PAF, Rad6, and Set1 complexes. These data suggest that BUR acts upstream of these factors to control their function. In support, we show that recruitment of the PAF elongation complex to genes is significantly impaired in a BUR2 deletion. Our data reveal a novel function for the BUR kinase in transcriptional regulation through the selective control of histone modifications.

A conserved, extended chromatin opening within α-globin locus during development

Histone modifications play an important role in eukaryotic gene regulation. However, the dynamic alteration of histone modification during development is poorly understood. In addition, the relationship between histone modification and globin gene switching remains unclear. Here, we assessed the dynamic pattern of histone modification (H3 acetylation, H4 acetylation, H3 K4 methylation, and H3 K79 methylation) along the murine α-globin locus, as well as along the human α-globin locus in transgenic mice, during globin gene switching in vivo. During the switching, histone modification at embryonic ζ-gene and fetal/adult α-genes displayed different developmental patterns. The level of histone modification at ζ-gene was developmentally regulated, in accordance with the level of ζ-gene expression, whereas the α-genes kept high level of histone modification at both developmental stages, regardless of their expression levels. Histone deacetylase inhibition selectively increased acetylation at the inactive ζ-gene in fetal livers, although it did not reactivate the gene expression. More importantly, an obvious increasing of histone modification level at major regulatory elements and fetal/adult α-genes was observed during the switching, suggesting that a conserved, extended chromatin opening within the locus occurs during globin gene switching.

WDR5 Associates with Histone H3 Methylated at K4 and Is Essential for H3 K4 Methylation and Vertebrate Development

Histone H3 lysine 4 (K4) methylation has been linked to the transcriptional activation in a variety of eukaryotic species. Here we show that a common component of MLL1, MLL2, and hSet1 H3 K4 methyltransferase complexes, the WD40-repeat protein WDR5, directly associates with histone H3 di- and trimethylated at K4 and with H3-K4-dimethylated nucleosomes. WDR5 is required for binding of the methyltransferase complex to the K4-dimethylated H3 tail as well as for global H3 K4 trimethylation and HOX gene activation in human cells. WDR5 is essential for vertebrate development, in that WDR5-depleted X. laevis tadpoles exhibit a variety of developmental defects and abnormal spatial Hox gene expression. Our results are the first demonstration that a WD40-repeat protein acts as a module for recognition of a specific histone modification and suggest a mechanism for reading and writing an epigenetic mark for gene activation.

Physical Association and Coordinate Function of the H3 K4 Methyltransferase MLL1 and the H4 K16 Acetyltransferase MOF

A stable complex containing MLL1 and MOF has been immunoaffinity purified from a human cell line that stably expresses an epitope-tagged WDR5 subunit. Stable interactions between MLL1 and MOF were confirmed by reciprocal immunoprecipitation, cosedimentation, and cotransfection analyses, and interaction sites were mapped to MLL1 C-terminal and MOF zinc finger domains. The purified complex has a robust MLL1-mediated histone methyltransferase activity that can effect mono-, di-, and trimethylation of H3 K4 and a MOF-mediated histone acetyltransferase activity that is specific for H4 K16. Importantly, both activities are required for optimal transcription activation on a chromatin template in vitro and on an endogenous MLL1 target gene, Hox a9, in vivo. These results indicate an activator-based mechanism for joint MLL1 and MOF recruitment and targeted methylation and acetylation and provide a molecular explanation for the closely correlated distribution of H3 K4 methylation and H4 K16 acetylation on active genes.

Epigenetic Silencing Mechanisms in Budding Yeast and Fruit Fly: Different Paths, Same Destinations

Transcriptional silencing in budding yeast and fruit fly is mediated by fundamentally unrelated proteins that assemble very different chromatin structures. Surprisingly, the repressive mechanisms evolved from these very different materials have similar features, including an epigenetic mode of inheritance and a block to transcription based on interference with the assembly or function of the promoter complex rather than with the binding of gene-specific activators.

Variant histone H3.3 marks promoters of transcriptionally active genes during mammalian cell division

Variant histone H3.3 is incorporated into nucleosomes by a mechanism that does not require DNA replication and has also been implicated as a potential mediator of epigenetic memory of active transcriptional states. In this study, we have used chromatin immunoprecipitation analysis to show that H3.3 is found mainly at the promoters of transcriptionally active genes. We also show that H3.3 combines with H3 acetylation and K4 methylation to form a stable mark that persists during mitosis. Our results suggest that H3.3 is deposited principally through the action of chromatin-remodelling complexes associated with transcriptional initiation, with deposition mediated by RNA polymerase II elongation having only a minor role.

Cell cycle regulation of chromatin at an origin of DNA replication

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.