DNA damage in the presence of chemical genotoxic agents induce acetylation of H3K56 and H4K16 but not H3K9 in mammalian cells

Histone covalent modifications play a significant role in the regulation of chromatin structure and function during DNA damage. Hyperacetylation of histones is a DNA damage dependent post translational modification in yeast and mammals. Although acetylation of histones during DNA damage is well established, specific lysine residues that are acetylated is being understood very recently in mammals. Here, in the present study, acetylation of three different lysine residues Histone3Lysine 9 (H3K9), Histone3Lysine 56 (H3K56) and Histone4Lysine 16 (H4K16) were probed with specific antibodies in mammalian cell lines treated with genotoxic agents that induce replication stress or S-phase dependent double strand breaks. Immunoblotting results have shown that DNA damage associated with replication arrest induce acetylation of H3K56 and H4K16 but not H3K9 in mammals. Immunofluorescence experiments further confirmed that acetylated H3K56 and H4K16 form nuclear foci at the site of DNA double strand breaks. Colocalization of H3K56ac with γ H2AX and replication factor PCNA proved the existence of this modification at the site of DNA damage and its probable role in DNA damage repair. Put together, the present data suggests that acetylation of H3K56 and H4K16 are potent DNA damage dependent histone modifications but not H3K9 in mammals.     

Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of γ-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest that Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.     

Chromosome remodeling related to hepatitis B virus replication in HepG2 cells

Hepatitis B Virus (HBV) covalently closed circular DNA (cccDNA) is the main replicative intermediate of HBV and is organized into minichromosomes by the interaction with histone and nonhistone proteins. The remodeling of HBV minichromosomes such as post-translational modifications of histone proteins plays an important role in regulating HBV replication. To determine whether other remodeling occurs in addition to acetylation of cccDNA-bound H3 histones in the presence of HBV replication, a cell culture replication model of HBV was used to assess the dynamic status of acetylation, phosphorylation, and methylation of cccDNA-bound H3 histones at various times after transient transfection of linear HBV DNA into human hepatoma, HepG2 cells. H3 histones bound to cccDNA were found to be phosphorylated, mono-methylated, and acetylated in HepG2 cells containing replicating HBV. The acetylation and methylation status of H3 histones bound to cccDNA paralleled HBV replication. Our results demonstrate that phosphorylation and methylation occur in the remodeling of HBV minichromosomes during HBV replication. The modifications of cccDNA-bound H3 histones were associated with the level of HBV replication. These findings suggest that alterations in the extent of minichromosome remodeling might be a potential target to inhibit HBV replication in the development of effective novel antiviral agents.     

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The coordinated process of DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3-H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three histone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3-H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3-H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.     

Histone H3 lysine 56 acetylation and the response to DNA replication fork damage

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication.     

Chromatin compaction at the mononucleosome level

Using a previously described FRET technique, we measured the distance between the ends of DNA fragments on which nucleosomes were reconstituted from recombinant and native histones. This distance was analyzed in its dependence on the DNA fragment length, concentration of mono- and divalent counterions, presence of linker histone H1, and histone modifications. We found that the linker DNA arms do not cross under all conditions studied but diverge slightly as they leave the histone core surface. Histone H1 leads to a global approach of the linker DNA arms, confirming the notion of a "stem structure". Increasing salt concentration also leads to an approach of the linker DNAs. To study the effect of acetylation, we compared chemically acetylated recombinant histones with histones prepared from HeLa cells, characterizing the sites of acetylation by mass spectroscopy. Nucleosomes from chemically acetylated histones have few modifications in the core domain and form nucleosomes normally. Acetylating all histones or selectively only H3 causes an opening of the nucleosome structure, indicated by the larger distances between the linker DNA ends. Selective acetylation of H4 distances the linker ends for short fragments but causes them to approach each other for fragments longer than 180 bp.     

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The process of coordinated DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3–H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three his-tone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3–H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3–H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.Key words: Gen5, Rtt109, chromatin, nucleosome assembly, genome integrity     

p300-mediated Acetylation of Histone H3 Lysine 56 Functions in DNA Damage Response in Mammals

The packaging of newly replicated and repaired DNA into chromatin is crucial for the maintenance of genomic integrity. Acetylation of histone H3 core domain lysine 56 (H3K56ac) has been shown to play a crucial role in compaction of DNA into chromatin following replication and repair in Saccharomyces cerevisiae. However, the occurrence and function of such acetylation has not been reported in mammals. Here we show that H3K56 is acetylated and that this modification is regulated in a cell cycle-dependent manner in mammalian cells. We also demonstrate that the histone acetyltransferase p300 acetylates H3K56 in vitro and in vivo, whereas hSIRT2 and hSIRT3 deacetylate H3K56ac in vivo. Further we show that following DNA damage H3K56 acetylation levels increased, and acetylated H3K56, which is localized at the sites of DNA repair. It also colocalized with other proteins involved in DNA damage signaling pathways such as phospho-ATM, CHK2, and p53. Interestingly, analysis of occurrence of H3K56 acetylation using ChIP-on-chip revealed its genome-wide spread, affecting genes involved in several pathways that are implicated in tumorigenesis such as cell cycle, DNA damage response, DNA repair, and apoptosis.     

An epigenetic code for DNA damage repair pathways?

Exposure of living cells to intracellular or external mutagens results in DNA damage. Accumulation of DNA damage can lead to serious consequences because of the deleterious mutation rate resulting in genomic instability, cellular senescence, and cell death. To counteract genotoxic stress, cells have developed several strategies to detect defects in DNA structure. The eukaryotic genomic DNA is packaged through histone and nonhistone proteins into a highly condensed structure termed chromatin. Therefore the cellular enzymatic machineries responsible for DNA replication, recombination, and repair must circumvent this natural barrier in order to gain access to the DNA. Several studies have demonstrated that histone/chromatin modifications such as acetylation, methylation, and phosphorylation play crucial roles in DNA repair processes. This review will summarize the recent data that suggest a regulatory role of the epigenetic code in DNA repair processes. We will mainly focus on different covalent reversible modifications of histones as an initial step in early response to DNA damage and subsequent DNA repair. Special focus on a potential epigenetic histone code for these processes will be given in the last section. We also discuss new technologies and strategies to elucidate the putative epigenetic code for each of the DNA repair processes discussed.     

Histone H3 K56 acetylation, chromatin assembly, and the DNA damage checkpoint

The role of chromatin and its modulation during DNA repair has become increasingly understood in recent years. A number of histone modifications that contribute towards the cellular response to DNA damage have been identified, including the acetylation of histone H3 at lysine 56. H3 K56 acetylation occurs normally during S phase, but persists in the presence of DNA damage. In the absence of this modification, cellular survival following DNA damage is impaired. Two recent reports provide additional insights into how H3 K56 acetylation functions in DNA damage responses. In particular, this modification appears to be important for both normal replication-coupled nucleosome assembly as well as nucleosome assembly at sites of DNA damage following repair.     

Differential Histone Acetylation in Alfalfa (Medicago sativa) Due to Growth in NaCl : Responses in Salt Stressed and Salt Tolerant Callus Cultures

The steady state distribution of histone variant proteins and their modifications by acetylation were characterized in wild type and salinity stress adapted alfalfa (Medicago sativa). Isotopic labeling detected dynamic acetylation at four sites in the histone H3 variants and five sites in histones H4 and H2B. Histone variant H3.2 was the most highly acetylated histone with 25% higher steady state acetylation and a two- to threefold higher acetylation labeling than histone H3.1. Histone phosphorylation was limited to histone variants H1.A, H1.B, and H1.C and to histone H2A.3, which was also acetylated. Histone variant composition was unaffected by cellular exposure to NaCl. Histone acetylation was qualitatively similar in salt-tolerant and salt-sensitive cells under normal growth conditions. However, short term salt stress in salt sensitive cells or continued growth at 1% NaCl in salt tolerant cells led to major increases in the multiacetylated forms of histone H4 and the two variants of histone H3. These changes were more pronounced in the diploid than in the tetraploid alfalfa strains. The increase in multiacetylation of core histones serves as an in vivo reporter suggesting an altered intranuclear ionic environment in the presence of salt. It may also represent an adaptive response in chromatin structure to permit chromatin function in a more saline intranuclear environment.     

Histone H3 Lysine 56 Acetylation: A New Twist in the Chromosome Cycle

Several recent reports have identified lysine 56 (K56) as a novel site of acetylation in yeast histone H3. K56 acetylation is predicted to disrupt some of the histone-DNA interactions at the entry and exit points of the nucleosome core particle. This modification occurs in virtually all the newly synthesised histones that are deposited into chromatin during S-phase. Cells with mutations that block K56 acetylation show increased genome instability and hypersensitivity to genotoxic agents that interfere with replication. Removal of K56 acetylation takes place in the G2/M phase of the cell cycle and is dependent upon Hst3 and Hst4, two proteins that are related to the NAD+-dependent histone deacetylase Sir2. In response to DNA damage checkpoint activation during S-phase, expression of Hst3/Hst4 is delayed to extend the window of opportunity in which K56 acetylation can act in the DNA damage response. The high abundance of histone H3 K56 acetylation, its regulation and strategic location in the nucleosome core particle raise a number of fascinating issues that we discuss here.