Epigenetic regulation of nuclear lamina-associated heterochromatin by HAT1 and the acetylation of newly synthesized histones.

A central component of the epigenome is the pattern of histone post-translational modifications that play a critical role in the formation of specific chromatin states. Following DNA replication, nascent chromatin is a 1:1 mixture of parental and newly synthesized histones and the transfer of modification patterns from parental histones to new histones is a fundamental step in epigenetic inheritance. Here we report that loss of HAT1, which acetylates lysines 5 and 12 of newly synthesized histone H4 during replication-coupled chromatin assembly, results in the loss of accessibility of large domains of heterochromatin, termed HAT1-dependent Accessibility Domains (HADs). HADs are mega base-scale domains that comprise ∼10% of the mouse genome. HAT1 globally represses H3 K9 me3 levels and HADs correspond to the regions of the genome that display HAT1-dependent increases in H3 K9me3 peak density. HADs display a high degree of overlap with a subset of Lamin-Associated Domains (LADs). HAT1 is required to maintain nuclear structure and integrity. These results indicate that HAT1 and the acetylation of newly synthesized histones may be critical regulators of the epigenetic inheritance of heterochromatin and suggest a new mechanism for the epigenetic regulation of nuclear lamina-heterochromatin interactions.

Hat2p recognizes the histone H3 tail to specify the acetylation of the newly synthesized H3/H4 heterodimer by the Hat1p/Hat2p complex.

Post-translational modifications of histones are significant regulators of replication, transcription, and DNA repair. Particularly, newly synthesized histone H4 in H3/H4 heterodimers becomes acetylated on N-terminal lysine residues prior to its incorporation into chromatin. Previous studies have established that the histone acetyltransferase (HAT) complex Hat1p/Hat2p medicates this modification. However, the mechanism of how Hat1p/Hat2p recognizes and facilitates the enzymatic activities on the newly assembled H3/H4 heterodimer remains unknown. Furthermore, Hat2p is a WD40 repeat protein, which is found in many histone modifier complexes. However, how the WD40 repeat proteins facilitate enzymatic activities of histone modification enzymes is unclear. In this study, we first solved the high-resolution crystal structure of a Hat1p/Hat2p/CoA/H4 peptide complex and found that the H4 tail interacts with both Hat1p and Hat2p, by which substrate recruitment is facilitated. We further discovered that H3 N-terminal peptides can bind to the Hat2p WD40 domain and solved the structure of the Hat1p/Hat2p/CoA/H4/H3 peptide complex. Moreover, the interaction with Hat2p requires unmodified Arg2/Lys4 and Lys9 on the H3 tail, suggesting a novel model to specify the activity of Hat1p/Hat2p toward newly synthesized H3/H4 heterodimers. Together, our study demonstrated the substrate recognition mechanism by the Hat1p/Hat2p complex, which is critical for DNA replication and other chromatin remodeling processes.

Histone acetyltransferase 1 is required for DNA replication fork function and stability.

The replisome is a protein complex on the DNA replication fork and functions in a dynamic environment at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter DNA duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the activity of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (HAT1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12, which accompanies replication-coupled chromatin assembly. Here, using proximity ligation assay-based chromatin assembly assays and DNA fiber analysis, we analyzed the role of murine HAT1 in replication-coupled chromatin assembly. We demonstrate that HAT1 physically associates with chromatin near DNA replication sites. We found that the association of HAT1 with newly replicated DNA is transient, but can be stabilized by replication fork stalling. The association of HAT1 with nascent chromatin may be functionally relevant, as HAT1 loss decreased replication fork progression and increased replication fork stalling. Moreover, in the absence of HAT1, stalled replication forks were unstable, and newly synthesized DNA became susceptible to MRE11-dependent degradation. These results suggest that HAT1 links replication fork function to the proper processing and assembly of newly synthesized histones.

Hat1-Dependent Lysine Acetylation Targets Diverse Cellular Functions.

Lysine acetylation has emerged as one of the most important post-translational modifications, regulating different biological processes. However, its regulation by lysine acetyltransferases is still unclear in most cases. Hat1 is a lysine acetyltransferase originally identified based on its ability to acetylate histones. Using an unbiased proteomics approach, we have determined how loss of Hat1 affects the mammalian acetylome. Hat1+/+ and Hat1-/- mouse embryonic fibroblast cell lines were grown in both glucose- and galactose-containing media, as Hat1 is required for growth on galactose, and Hat1-/- cells exhibit defects in mitochondrial function. Following trypsin digestion of whole cell extracts, acetylated peptides were enriched by acetyllysine affinity purification, and acetylated peptides were identified and analyzed by label-free quantitation. Comparison of the acetylome from Hat1+/+ cells grown on galactose and glucose demonstrated that there are large carbon source-dependent changes in the mammalian acetylome where the acetylation of enzymes involved in glycolysis were the most affected. Comparisons of the acetylomes from Hat1+/+ and Hat1-/- cells identified 65 proteins whose acetylation decreased by at least 2.5-fold in cells lacking Hat1. In Hat1-/- cells, acetylation of the autoregulatory loop of CBP (CREB-binding protein) was the most highly affected, decreasing by up to 20-fold. In addition to the proteins involved in chromatin structure, Hat1-dependent acetylation was also found in a number of transcriptional regulators, including p53 and mitochondrial proteins. Hat1 mitochondrial localization suggests that it may be directly involved in the acetylation of mitochondrial proteins. Data are available via ProteomeXchange with identifier PXD017362.

Replication-dependent histone isoforms: a new source of complexity in chromatin structure and function.

Replication-dependent histones are expressed in a cell cycle regulated manner and supply the histones necessary to support DNA replication. In mammals, the replication-dependent histones are encoded by a family of genes that are located in several clusters. In humans, these include 16 genes for histone H2A, 22 genes for histone H2B, 14 genes for histone H3, 14 genes for histone H4 and 6 genes for histone H1. While the proteins encoded by these genes are highly similar, they are not identical. For many years, these genes were thought to encode functionally equivalent histone proteins. However, several lines of evidence have emerged that suggest that the replication-dependent histone genes can have specific functions and may constitute a novel layer of chromatin regulation. This Survey and Summary reviews the literature on replication-dependent histone isoforms and discusses potential mechanisms by which the small variations in primary sequence between the isoforms can alter chromatin function. In addition, we summarize the wealth of data implicating altered regulation of histone isoform expression in cancer.

Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly.

Histone acetyltransferase 1 (Hat1) catalyzes the acetylation of newly synthesized histone H4 at lysines 5 and 12 that accompanies replication-coupled chromatin assembly. The acetylation of newly synthesized H4 occurs in the cytoplasm and the function of this acetylation is typically ascribed to roles in either histone nuclear import or deposition. Using cell lines from Hat1+/+ and Hat1-/- mouse embryos, we demonstrate that Hat1 is not required for either histone nuclear import or deposition. We employed quantitative proteomics to characterize Hat1-dependent changes in the composition of nascent chromatin structure. Among the proteins depleted from nascent chromatin isolated from Hat1-/- cells are several bromodomain-containing proteins, including Brg1, Baz1A and Brd3. Analysis of the binding specificity of their bromodomains suggests that Hat1-dependent acetylation of H4 is directly involved in their recruitment. Hat1-/- nascent chromatin is enriched for topoisomerase 2α and 2ß. The enrichment of topoisomerase 2 is functionally relevant as Hat1-/- cells are hyper-sensitive to topoisomerase 2 inhibition suggesting that Hat1 is required for proper chromatin topology. In addition, our results indicate that Hat1 is transiently recruited to sites of chromatin assembly, dissociating prior to the maturation of chromatin structure.

Identification of replication-dependent and replication-independent linker histone complexes: Tpr specifically promotes replication-dependent linker histone stability.

BACKGROUND: There are 11 variants of linker histone H1 in mammalian cells. Beyond their shared abilities to stabilize and condense chromatin, the H1 variants have been found to have non-redundant functions, the mechanisms of which are not fully understood. Like core histones, there are both replication-dependent and replication-independent linker histone variants. The histone chaperones and other factors that regulate linker histone dynamics in the cell are largely unknown. In particular, it is not known whether replication-dependent and replication-independent linker histones interact with distinct or common sets of proteins. To better understand linker histone dynamics and assembly, we used chromatography and mass spectrometry approaches to identify proteins that are associated with replication-dependent and replication-independent H1 variants. We then used a variety of in vivo analyses to validate the functional relevance of identified interactions. RESULTS: We identified proteins that bind to all linker histone variants and proteins that are specific for only one class of variant. The factors identified include histone chaperones, transcriptional regulators, RNA binding proteins and ribosomal proteins. The nuclear pore complex protein Tpr, which was found to associate with only replication-dependent linker histones, specifically promoted their stability. CONCLUSION: Replication-dependent and replication-independent linker histone variants can interact with both common and distinct sets of proteins. Some of these factors are likely to function as histone chaperones while others may suggest novel links between linker histones and RNA metabolism. The nuclear pore complex protein Tpr specifically interacts with histone H1.1 and H1.2 but not H1x and can regulate the stability of these replication-dependent linker histones.

Histone acetyl transferase 1 is essential for mammalian development, genome stability, and the processing of newly synthesized histones H3 and H4.

Histone acetyltransferase 1 is an evolutionarily conserved type B histone acetyltransferase that is thought to be responsible for the diacetylation of newly synthesized histone H4 on lysines 5 and 12 during chromatin assembly. To understand the function of this enzyme in a complex organism, we have constructed a conditional mouse knockout model of Hat1. Murine Hat1 is essential for viability, as homozygous deletion of Hat1 results in neonatal lethality. The lungs of embryos and pups genetically deficient in Hat1 were much less mature upon histological evaluation. The neonatal lethality is due to severe defects in lung development that result in less aeration and respiratory distress. Many of the Hat1(-/-) neonates also display significant craniofacial defects with abnormalities in the bones of the skull and jaw. Hat1(-/-) mouse embryonic fibroblasts (MEFs) are defective in cell proliferation and are sensitive to DNA damaging agents. In addition, the Hat1(-/-) MEFs display a marked increase in genome instability. Analysis of histone dynamics at sites of replication-coupled chromatin assembly demonstrates that Hat1 is not only responsible for the acetylation of newly synthesized histone H4 but is also required to maintain the acetylation of histone H3 on lysines 9, 18, and 27 during replication-coupled chromatin assembly.

Proteomic profiling identifies specific histone species associated with leukemic and cancer cells.

BACKGROUND: Chromatin is an extraordinarily complex structure. Much of this complexity results from the presence of numerous histone post-translational modifications and histone variants. Alterations in the patterns of histone post-translational modifications are emerging as a feature of many types of cancer and have been shown to have prognostic value. RESULTS: We have applied a liquid chromatography/mass spectrometry-based approach to comprehensively characterize the histone proteome in primary samples from chronic lymphocytic leukemia (CLL) patients, as well as bladder and breast cancer cell culture models. When compared to non-malignant CD19+ B cells from healthy donors, the CLL histone proteome showed a distinct signature of differentially expressed species, spanning all the histones studied and including both post-translationally modified species and unmodified, non-allelic replication-dependent histone isoforms. However, the large changes in histone H3 and H4 that are characteristic of many cancer types were not observed. One of species of H2A (mass = 14,063 Da) was the most strongly associated with time to treatment in CLL patients. CLL patient samples also demonstrated histone profiles that were distinct from those of the bladder and breast cancer cells. CONCLUSIONS: Signatures of histone profiles are complex and can distinguish between healthy individuals and CLL patients and may provide prognostic markers. In addition, histone profiles may define tissue specific malignancies.

Discovery of histone modification crosstalk networks by stable isotope labeling of amino acids in cell culture mass spectrometry (SILAC MS).

In this paper we describe an approach that combines stable isotope labeling of amino acids in cells culture, high mass accuracy liquid chromatography tandem mass spectrometry and a novel data analysis approach to accurately determine relative peptide post-translational modification levels. This paper describes the application of this approach to the discovery of novel histone modification crosstalk networks in Saccharomyces cerevisiae. Yeast histone mutants were generated to mimic the presence/absence of 44 well-known modifications on core histones H2A, H2B, H3, and H4. In each mutant strain the relative change in H3 K79 methylation and H3 K56 acetylation were determined using stable isotope labeling of amino acids in cells culture. This approach showed relative changes in H3 K79 methylation and H3 K56 acetylation that are consistent with known histone crosstalk networks. More importantly, this study revealed additional histone modification sites that affect H3 K79 methylation and H3 K56 acetylation.

H1 histones: current perspectives and challenges.

H1 and related linker histones are important both for maintenance of higher-order chromatin structure and for the regulation of gene expression. The biology of the linker histones is complex, as they are evolutionarily variable, exist in multiple isoforms and undergo a large variety of posttranslational modifications in their long, unstructured, NH2- and COOH-terminal tails. We review recent progress in understanding the structure, genetics and posttranslational modifications of linker histones, with an emphasis on the dynamic interactions of these proteins with DNA and transcriptional regulators. We also discuss various experimental challenges to the study of H1 and related proteins, including limitations of immunological reagents and practical difficulties in the analysis of posttranslational modifications by mass spectrometry.

Increasing the complexity of chromatin: functionally distinct roles for replication-dependent histone H2A isoforms in cell proliferation and carcinogenesis.

Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant. However, the abundance of specific replication-dependent isoforms of histone H2A is altered in patients with chronic lymphocytic leukemia. Similar changes in the abundance of H2A isoforms are also associated with the proliferation and tumorigenicity of bladder cancer cells. To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity. We also observe that regulation of replication-dependent histone H2A expression can occur on a gene-specific level. Specific replication-dependent histone H2A genes are either up- or downregulated in chronic lymphocytic leukemia tumor tissue samples. In addition, discreet elements are identified in the 5' untranslated region of the HIST1H2AC locus that confer translational repression. Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.

miR-155 targets histone deacetylase 4 (HDAC4) and impairs transcriptional activity of B-cell lymphoma 6 (BCL6) in the Eµ-miR-155 transgenic mouse model.

Multiple studies have established that microRNAs (miRNAs) are involved in the initiation and progression of cancer. Notably, miR-155 is one of the most overexpressed miRNAs in several solid and hematological malignancies. Ectopic miR-155 expression in mice B cells (Eµ-miR-155 transgenic mice) has been shown to induce pre-B-cell proliferation followed by high-grade lymphoma/leukemia. Loss of miR-155 in mice resulted in impaired immunity due to defective T-cell-mediated immune response. Here we provide a mechanistic insight into miR-155-induced leukemogenesis in the Eµ-miR-155 mouse model through genome-wide transcriptome analysis of naïve B cells and target studies. We found that a key transcriptional repressor and proto-oncogene, Bcl6 is significantly down-regulated in Eµ-miR-155 mice. The reduction of Bcl6 subsequently leads to de-repression of some of the known Bcl6 targets like inhibitor of differentiation (Id2), interleukin-6 (IL6), cMyc, Cyclin D1, and Mip1α/ccl3, all of which promote cell survival and proliferation. We show that Bcl6 is indirectly regulated by miR-155 through Mxd1/Mad1 up-regulation. Interestingly, we found that miR-155 directly targets HDAC4, a corepressor partner of BCL6. Furthermore, ectopic expression of HDAC4 in human-activated B-cell-type diffuse large B-cell lymphoma (DLBCL) cells results in reduced miR-155-induced proliferation, clonogenic potential, and increased apoptosis. Meta-analysis of the diffuse large B-cell lymphoma patient microarray data showed that miR-155 expression is inversely correlated with Bcl6 and Hdac4. Hence this study provides a better understanding of how miR-155 causes disruption of the BCL6 transcriptional machinery that leads to up-regulation of the survival and proliferation genes in miR-155-induced leukemias.

SILAC peptide ratio calculator: a tool for SILAC quantitation of peptides and post-translational modifications.

This paper describes an algorithm to assist in relative quantitation of peptide post-translational modifications using stable isotope labeling by amino acids in cell culture (SILAC). The described algorithm first determines the normalization factor and then calculates SILAC ratios for a list of target peptide masses using precursor ion abundances. Four yeast histone mutants were used to demonstrate the effectiveness of this approach for quantitation of peptide post-translational modifications changes. The details of the algorithm's approach for normalization and peptide ratio calculation are described. The examples demonstrate the robustness of the approach as well as its utility to rapidly determine changes in peptide post-translational modifications within a protein.

The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms.

Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones.

Histone acetyltransferase 1: more than just an enzyme?

Histone acetyltransferase 1 (HAT1) is an enzyme that is likely to be responsible for the acetylation that occurs on lysines 5 and 12 of the NH2-terminal tail of newly synthesized histone H4. Initial studies suggested that, despite its evolutionary conservation, this modification of new histone H4 played only a minor role in chromatin assembly. However, a number of recent studies have brought into focus the important role of both this modification and HAT1 in histone dynamics. Surprisingly, the function of HAT1 in chromatin assembly may extend beyond just its catalytic activity to include its role as a major histone binding protein. These results are incorporated into a model for the function of HAT1 in histone deposition and chromatin assembly. This article is part of a Special issue entitled: Histone chaperones and Chromatin assembly.

Dynamic Reassociation of the Nuclear Lamina with Newly Replicated DNA.

The physical association of specific regions of chromatin with components of the nuclear lamina provides the framework for the 3-dimensionl architecture of the genome. The regulation of these interactions plays a critical role in the maintenance of gene expression patterns and cell identity. The breakdown and reassembly of the nuclear membrane as cells transit mitosis plays a central role in the regulation of the interactions between the genome and the nuclear lamina. However, other nuclear processes, such as transcription, have emerged as regulators of the association of DNA with the nuclear lamina. To determine whether DNA replication also has the potential to regulate DNA-nuclear lamina interactions, we adapted proximity ligation-based chromatin assembly assays to analyze the dynamics of nuclear lamina association with newly replicated DNA. We observe that lamin A/C and lamin B, as well as inner nuclear membrane proteins LBR and emerin, are found in proximity to newly replicated DNA. While core histones rapidly reassociate with DNA following passage of the replication fork, the complete reassociation of nuclear lamina components with newly replicated DNA occurs over a period of approximately 30 minutes. We propose models to describe the disassembly and reassembly of nascent chromatin with the nuclear lamina.

Nuclear Hat1p complex (NuB4) components participate in DNA repair-linked chromatin reassembly.

Chromatin is disassembled and reassembled during DNA repair. To assay chromatin reassembly accompanying DNA double strand break repair, ChIP analysis can be used to monitor the presence of histone H3 near the lesion. The chromatin assembly factor Asf1p, as well as the acetylation of histone H3 lysine 56, have been shown to promote chromatin reassembly when DNA double strand break repair is complete. Using Gal-HO-mediated double strand break repair, we have tested each of the components of the nuclear Hat1p-containing type B histone acetyltransferase complex (NuB4) and have found that they can affect repair-linked chromatin reassembly but that their contributions are not equivalent. In particular, deletion of the catalytic subunit, Hat1p, caused a significant defect in chromatin reassembly. In addition, loss of the histone chaperone Hif1p, when combined with an allele of H3 that mutates lysines 14 and 23 to arginine, has a pronounced effect on chromatin reassembly that is similar to that observed in an asf1Δ. The role of Hat1p and Hif1p is at least partially redundant with the role of Asf1p. Consistent with a more prominent role for Hif1p in chromatin reassembly than either Hat1p or Hat2p, Hif1p exists in complex(es) independent of Hat1p and Hat2p and influences the activity of an H3-specific histone acetyltransferase activity. Our data directly demonstrate the role of the nuclear HAT1 complex (NuB4) components in DNA repair-linked chromatin reassembly.

Genetic interactions between POB3 and the acetylation of newly synthesized histones.

Pob3p is an essential component of the S. cerevisiae FACT complex (yFACT). Several lines of evidence indicate that the yFACT complex plays an important role in chromatin assembly including the observation that the pob3 Q308K allele is synthetically lethal with an allele of histone H4 that prevents the diacetylation of newly synthesized molecules. We have analyzed the genetic interactions between the Q308K allele of POB3 and mutations in all of the sites of acetylation that have been identified on newly synthesized histones. Genetic interactions were observed between POB3 and sites of acetylation on the NH(2)-terminal tails of H3 and H4. For histone H3, lysine residues 14 and 23 were particularly important when POB3 activity is compromised. Surprisingly, synthetic defects observed when the pob3 Q308K allele was combined with mutations of H4 lysines 5 and 12, were not phenocopied by deletion of HAT1, which encodes the enzyme that is thought to generate this pattern of acetylation on H4. Genetic interactions were also observed between POB3 and sites of acetylation found in the core domain of newly synthesized histones H3 and H4. These include synthetic lethality with an allele of H4 lysine 91 that mimics constitutive acetylation. While the mutations that alter H4 lysines 5, 12 and 91 do not affect binding to Pob3p, mutation of histone H3 lysine 56 decreases the association of histones with Pob3p. These results support the model that the yFACT complex plays a central role in chromatin assembly pathways regulated by acetylation of newly synthesized histones.