Epigenetic Control of Expression Homeostasis during Replication Is Stabilized by the Replication Checkpoint.

DNA replication introduces a dosage imbalance between early and late replicating genes. In budding yeast, buffering gene expression against this imbalance depends on marking replicated DNA by H3K56 acetylation (H3K56ac). Whether additional processes are required for suppressing transcription from H3K56ac-labeled DNA remains unknown. Here, using a database-guided candidate screen, we find that COMPASS, the H3K4 methyltransferase, and its upstream effector, PAF1C, act downstream of H3K56ac to buffer expression. Replicated genes show reduced abundance of the transcription activating mark H3K4me3 and accumulate the transcription inhibitory mark H3K4me2 near transcription start sites. Notably, in hydroxyurea-exposed cells, the S phase checkpoint stabilizes H3K56ac and becomes essential for buffering. We suggest that H3K56ac suppresses transcription of replicated genes by interfering with post-replication recovery of epigenetic marks and assign a new function for the S phase checkpoint in stabilizing this mechanism during persistent dosage imbalance.

LncPRESS1 Is a p53-Regulated LncRNA that Safeguards Pluripotency by Disrupting SIRT6-Mediated De-acetylation of Histone H3K56.

Recent evidence suggests that lncRNAs play an integral regulatory role in numerous functions, including determination of cellular identity. We determined global expression (RNA-seq) and genome-wide profiles (ChIP-seq) of histone post-translational modifications and p53 binding in human embryonic stem cells (hESCs) undergoing differentiation to define a high-confidence set of 40 lncRNAs, which are p53 transcriptional targets. We focused on lncRNAs highly expressed in pluripotent hESCs and repressed by p53 during differentiation to identify lncPRESS1 as a p53-regulated transcript that maintains hESC pluripotency in concert with core pluripotency factors. RNA-seq of hESCs depleted of lncPRESS1 revealed that lncPRESS1 controls a gene network that promotes pluripotency. Further, we found that lncPRESS1 physically interacts with SIRT6 and prevents SIRT6 chromatin localization, which maintains high levels of histone H3K56 and H3K9 acetylation at promoters of pluripotency genes. In summary, we describe a p53-regulated, pluripotency-specific lncRNA that safeguards the hESC state by disrupting SIRT6 activity.

Oxidative replication stress induced by long-term exposure to hydroxyurea in root meristem cells of Vicia faba.

KEY MESSAGE: Low concentrations of hydroxyurea, an inhibitor of DNA replication, induced oxidative and replicative stress in root apical meristem (RAM) cells of Vicia faba. Plant cells are constantly exposed to low-level endogenous stress factors that can affect DNA replication and lead to DNA damage. Long-term treatments of Vicia faba root apical meristems (RAMs) with HU leads to the appearance of atypical cells with intranuclear asynchrony. This rare form of abnormality was manifested by a gradual condensation of chromatin, from interphase to mitosis (so-called IM cells). Moreover, HU-treated root cells revealed abnormal chromosome structure, persisting DNA replication, and elevated levels of intracellular hydrogen peroxide (H2O2) and superoxide anion (O2∙-). Immunocytochemical studies have shown an increased number of fluorescent foci of H3 histones acetylated at lysine 56 (H3K56Ac; canonically connected with the DNA replication process). We show that continuous 3-day exposure to low concentrations (0.75 mM) of hydroxyurea (HU; an inhibitor of DNA replication) induces cellular response to reactive oxygen species and to DNA replication stress conditions.

K-Ras-PI3K regulates H3K56ac through PCAF to elevate the occurrence and growth of liver cancer.

H3 modification is related to a wide range of tumors, including liver cancer. The Ras passageway is actuated in human diseases. Thus, we investigated the roles of Ras in liver cancer cells via acetylation of H3K56. Ras-carrying G12V and Y40C site mutation was transfected into liver cancer cell lines SNU-475 and SK-Hep-1. Acetylation of H3K56 and phosphatidylinositol 3-kinase (PI3K), P300/CBP-associated factor (PCAF) and Mouse double minute 2 homolog (MDM2) was tested via western blot. Cell activity, colonies, and migration were tested via Cell Counting Kit-8, soft-agar colony formation, and Transwell experiment, respectively. Sirtuin 6 (SIRT6) and PCAF were tested via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Chromatin immunoprecipitation was employed to test the relationship between Ras and downstream elements. Flow cytometry was employed to test cell cycle series. We found that RasG12V/Y40C transfection reduced the acetylation of H3K56 and activated phosphorylation of protein kinase B. H3K56Q (H3K56ac overexpression) suppressed cell activity, colonies, and migration. H3K56ac changed Ras downstream factors expression. RasG12V/Y40C bound to Ras-PI3K downstream elements' promoters. SIRT6 silencing raised H3K56ac and suppressed cell activity, migration and S phage cell percentage. SIRT6 silence transformed expression of downstream elements. PCAF and H3K56ac demonstrated the close current while MDM2 was conversed. In summary, the Ras-PI3K passageway promoted cell growth and metastasis via decreasing H3K56ac, in which MDM2-mediated PCAF was involved.

Nicotinamide-induced mouse embryo developmental defect rescued by resveratrol and I-CBP112.

Cell cycle of mouse embryo could be delayed by nicotinamide (NAM). Histone H3 lysine 56 (H3K56ac) acetylation plays an important role in mammalian genomic stability and the function of this modification in mouse embryos is not known. Hence, we designed to study the effects of NAM-induced oxidative stress on the developmental ability of mouse embryos, on the acetylation of H3K56ac and the possible functions of this modification related to mouse embryo development. Treatment with NAM (10, 20, or 40 mmol/L for 24 or 48 hr) during in vitro culture significantly decreased developmental rate of blastocyst (24 hr: 90.2 vs. 81.2, 43.2, and 18.2, with p > .05, p < .01, respectively; 48 hr: 89.3 vs. 53.2%, 12.1%, and 0% with p < .05, respectively). NAM treatment (20 mmol/L) for 6 and 31 hr resulted in increased intracellular reactive oxygen species levels in two-cell embryos, and apoptotic cell numbers in blastocysts. Resveratrol (RSV) and I-CBP112 rescued the 20 mmol/L NAM-induced embryo developmental defects. RSV and I-CBP112 increased the level of Sirt1 and decreased the level of H3K56ac induced by NAM in two-cell embryos (p < .05). These data suggest that NAM treatment decreases the expression of Sirt1, which induces high levels of H3K56 acetylation that may be involved in oxidative stress-induced mouse embryo defects, which can be rescued by RSV and I-CBP112.

Retracted: Ras-PI3K-AKT signaling promotes the occurrence and development of uveal melanoma by downregulating H3K56ac expression.

BACKGROUND: Uveal melanoma (UM) is an intraocular malignant tumor characterized by rapid progression and recurrence. The current conventional treatments are unsatisfactory. Histone acetylation at H3 lysine 56 (H3K56ac) has been reported to be a tumor suppressor in breast cancer. However, whether H3K56ac prevents the occurrence and development of UM remains uninvestigated. The study aimed to explore the regulatory effect of H3K56ac on Ras-PI3K-AKT induced UM cells proliferation and migration. METHODS: The vectors of pEGFP-RasWT , pEGFP-K-Ras G12V/Y40C , and pEGFP-N1 were transfected into MP46 cells, and protein levels of phosphorylated AKT Ser473 and H3K56ac were examined using western blot analysis. The effect of H3K56ac on cell proliferation and migration were studied using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, colony formation, and Transwell assays. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and chromatin immunoprecipitation assays were performed to determine the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) downstream genes. Further, the regulatory effects of silent mating type information regulation 2 homolog-1 (SIRT1), general control nonderepressible 5 (GCN5), and mouse double minute 2 homolog (MDM2) on Ras-PI3K-AKT affected H3K56ac expression were also investigated. RESULTS: H3K56ac expression was specifically downregulated by Ras-PI3K-AKT activation pathway. H3K56ac inhibited the tumorigenic effect of Ras-PI3K-AKT on MP46 cells viability, colony formation, and migration, as well as participated in regulating the transcription of PI3K/AKT downstream genes. SIRT1 silence recovered H3K56ac expression, and reversed the tumorigenic effect of Ras-PI3K-AKT activation on MP46 cells. Downregulation of H3K56ac induced by Ras-PI3K-AKT activation was found to be associated with MDM2-mediated the degradation of GCN5. CONCLUSIONS: The results demonstrated that Ras-PI3K-AKT signaling promoted UM cells proliferation and migration via downregulation of H3K56ac expression, which might be related to MDM2-mediated the degradation of GCN5.

Chromatin signatures at Notch-regulated enhancers reveal large-scale changes in H3K56ac upon activation.

The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. To investigate the influence of chromatin organisation and dynamics on the response to Notch signalling, we partitioned Drosophila chromatin using histone modifications and established the preferred chromatin conditions for binding of Su(H), the Notch pathway transcription factor. By manipulating activity of a co-operating factor, Lozenge/Runx, we showed that it can help facilitate these conditions. While many histone modifications were unchanged by Su(H) binding or Notch activation, we detected rapid changes in acetylation of H3K56 at Notch-regulated enhancers. This modification extended over large regions, required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation appear to be a conserved indicator of enhancer activation as they also occurred at the mammalian Notch-regulated Hey1 gene and at Drosophila ecdysone-regulated genes. This intriguing example of a core histone modification increasing over short timescales may therefore underpin changes in chromatin accessibility needed to promote transcription following signalling activation.

Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases.

During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies.

Both H4K20 mono-methylation and H3K56 acetylation mark transcription-dependent histone turnover in fission yeast.

Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5' promoter and 3' termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNA production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast.

Wu-zhu-yu Decoction reduces early brain injury following subarachnoid hemorrhage in vivo and in vitro by activating the Nrf2 antioxidant system via SIRT6 targeting.

ETHNOPHARMACOLOGICAL RELEVANCE: Early brain damage (EBI) following subarachnoid hemorrhage (SAH) is a long-lasting condition with a high occurrence. However, treatment options are restricted. Wu-zhu-yu Decoction (WZYD) can treat headaches and vomiting, which are similar to the early symptoms of subarachnoid hemorrhage (SAH). However, it is yet unknown if WZYD can reduce EBI following SAH and its underlying mechanisms. AIM OF THE STUDY: This study aimed to investigate whether WZYD protects against EBI following SAH by inhibiting oxidative stress through activating nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling via Sirtuin 6 (SIRT6)-mediated histone H3 lysine 56 (H3K56) deacetylation. MATERIALS AND METHODS: In the current investigation, the principal components of WZYD were identified using high-performance liquid chromatography-diode array detection (HPLC-DAD). The SAH model in rats using the internal carotid artery plug puncture approach and the SAH model in primary neurons using hemoglobin incubation were developed. WZYD with different doses (137 mg kg-1, 274 mg kg-1, 548 mg kg-1) and the positive drug-Nimodipine (40 mg kg-1) were intragastrically administered in SAH model rats, respectively. The PC12 cells were cultured with corresponding medicated for 24h. In our investigation, neurological scores, brain water content, Evans blue leakage, Nissl staining, TUNEL staining, oxidative stress, expression of apoptosis-related proteins, and Nrf2/HO-1 signaling were evaluated. The interaction between SIRT6 and Nrf2 was detected by co-immunoprecipitation. SIRT6 knockdown was used to confirm its role in WZYD's neuroprotection. RESULTS: The WZYD treatment dramatically reduced cerebral hemorrhage and edema, and enhanced neurological results in EBI following SAH rats. WZYD administration inhibited neuronal apoptosis via reducing the expression levels of Cleaved cysteinyl aspartate specific proteinase-3(Cleaved Caspase-3), cysteinyl aspartate specific proteinase-3(caspase-3), and Bcl-2, Associated X Protein (Bax) and increasing the expression of B-cell lymphoma-2(Bal2). It also decreased reactive oxygen species and malondialdehyde levels and increased Nrf2 and HO-1 expression in the rat brain after SAH. In vitro, WZYD attenuated hemoglobin-induced cytotoxicity, oxidative stress and apoptosis in primary neurons. Mechanistically, WZYD enhanced SIRT6 expression and H3K56 deacetylation, activated Nrf2/HO-1 signaling, and promoted the interaction between SIRT6 and Nrf2. Knockdown of SIRT6 abolished WZYD-induced neuroprotection. CONCLUSIONS: WZYD attenuates EBI after SAH by activating Nrf2/HO-1 signaling through SIRT6-mediated H3K56 deacetylation, suggesting its therapeutic potential for SAH treatment.

Distinct role of histone chaperone Asf1a and Asf1b during fertilization and pre-implantation embryonic development in mice.

BACKGROUND: Asf1 is a well-conserved histone chaperone that regulates multiple cellular processes in different species. Two paralogous genes, Asf1a and Asf1b exist in mammals, but their role during fertilization and early embryogenesis remains to be investigated further. METHODS: We analyzed the dynamics of histone chaperone Asf1a and Asf1b in oocytes and pre-implantation embryos in mice by immunofluorescence and real-time quantitative PCR, and further investigated the role of Asf1a and Asf1b during fertilization and pre-implantation development by specific Morpholino oligos-mediated knock down approach. RESULTS: Immunofluorescence with specific antibodies revealed that both Asf1a and Asf1b were deposited in the nuclei of fully grown oocytes, accumulated abundantly in zygote and 2-cell embryonic nuclei, but turned low at 4-cell stage embryos. In contrast to the weak but definite nuclear deposition of Asf1a, Asf1b disappeared from embryonic nuclei at morula and blastocyst stages. The knockdown of Asf1a and Asf1b by specific Morpholino oligos revealed that Asf1a but not Asf1b was required for the histone H3.3 assembly in paternal pronucleus. However, knockdown of either Asf1a or Asf1b expression decreased developmental potential of pre-implantation embryos. Furthermore, while Asf1a KD severely reduced H3K56 acetylation level and the expression of Oct4 in blastocyst stage embryos, Asf1b KD almost eliminated nuclear accumulation of proliferating cell marker-PCNA in morula stage embryos. These results suggested that histone chaperone Asf1a and Asf1b play distinct roles during fertilization and pre-implantation development in mice. CONCLUSIONS: Our data suggested that both Asf1a and Asf1b are required for pre-implantation embryonic development. Asf1a regulates H3K56ac levels and Oct4 expression, while Asf1b safeguards pre-implantation embryo development by regulating cell proliferation. We also showed that Asf1a, but not Asf1b, was necessary for the assembly of histone H3.3 in paternal pronuclei after fertilization.

DDK/Hsk1 phosphorylates and targets fission yeast histone deacetylase Hst4 for degradation to stabilize stalled DNA replication forks.

In eukaryotes, paused replication forks are prone to collapse, which leads to genomic instability, a hallmark of cancer. Dbf4-dependent kinase (DDK)/Hsk1Cdc7 is a conserved replication initiator kinase with conflicting roles in replication stress response. Here, we show that fission yeast DDK/Hsk1 phosphorylates sirtuin, Hst4 upon replication stress at C-terminal serine residues. Phosphorylation of Hst4 by DDK marks it for degradation via the ubiquitin ligase SCFpof3. Phosphorylation-defective hst4 mutant (4SA-hst4) displays defective recovery from replication stress, faulty fork restart, slow S-phase progression and decreased viability. The highly conserved fork protection complex (FPC) stabilizes stalled replication forks. We found that the recruitment of FPC components, Swi1 and Mcl1 to the chromatin is compromised in the 4SA-hst4 mutant, although whole cell levels increased. These defects are dependent upon H3K56ac and independent of intra S-phase checkpoint activation. Finally, we show conservation of H3K56ac-dependent regulation of Timeless, Tipin, and And-1 in human cells. We propose that degradation of Hst4 via DDK increases H3K56ac, changing the chromatin state in the vicinity of stalled forks facilitating recruitment and function of FPC. Overall, this study identified a crucial role of DDK and FPC in the regulation of replication stress response with implications in cancer therapeutics.

Transcription levels of a noncoding RNA orchestrate opposing regulatory and cell fate outcomes in yeast.

Transcription through noncoding regions of the genome is pervasive. How these transcription events regulate gene expression remains poorly understood. Here, we report that, in S. cerevisiae, the levels of transcription through a noncoding region, IRT2, located upstream in the promoter of the inducer of meiosis, IME1, regulate opposing chromatin and transcription states. At low levels, the act of IRT2 transcription promotes histone exchange, delivering acetylated histone H3 lysine 56 to chromatin locally. The subsequent open chromatin state directs transcription factor recruitment and induces downstream transcription to repress the IME1 promoter and meiotic entry. Conversely, increasing transcription turns IRT2 into a repressor by promoting transcription-coupled chromatin assembly. The two opposing functions of IRT2 transcription shape a regulatory circuit, which ensures a robust cell-type-specific control of IME1 expression and yeast meiosis. Our data illustrate how intergenic transcription levels are key to controlling local chromatin state, gene expression, and cell fate outcomes.

Changes in Epigenetic Patterns Related to DNA Replication in Vicia faba Root Meristem Cells under Cadmium-Induced Stress Conditions.

Experiments on Vicia faba root meristem cells exposed to 150 µM cadmium chloride (CdCl2) were undertaken to analyse epigenetic changes, mainly with respect to DNA replication stress. Histone modifications examined by means of immunofluorescence labeling included: (1) acetylation of histone H3 on lysine 56 (H3K56Ac), involved in transcription, S phase, and response to DNA damage during DNA biosynthesis; (2) dimethylation of histone H3 on lysine 79 (H3K79Me2), correlated with the replication initiation; (3) phosphorylation of histone H3 on threonine 45 (H3T45Ph), engaged in DNA synthesis and apoptosis. Moreover, immunostaining using specific antibodies against 5-MetC-modified DNA was used to determine the level of DNA methylation. A significant decrease in the level of H3K79Me2, noted in all phases of the CdCl2-treated interphase cell nuclei, was found to correspond with: (1) an increase in the mean number of intranuclear foci of H3K56Ac histones (observed mainly in S-phase), (2) a plethora of nuclear and nucleolar labeling patterns (combined with a general decrease in H3T45Ph), and (3) a decrease in DNA methylation. All these changes correlate well with a general viewpoint that DNA modifications and post-translational histone modifications play an important role in gene expression and plant development under cadmium-induced stress conditions.

SIRT6 as a key event linking P53 and NRF2 counteracts APAP-induced hepatotoxicity through inhibiting oxidative stress and promoting hepatocyte proliferation.

Acetaminophen (APAP) overdose is the leading cause of drug-induced liver injury, and its prognosis depends on the balance between hepatocyte death and regeneration. Sirtuin 6 (SIRT6) has been reported to protect against oxidative stress-associated DNA damage. But whether SIRT6 regulates APAP-induced hepatotoxicity remains unclear. In this study, the protein expression of nuclear and total SIRT6 was up-regulated in mice liver at 6 and 48 h following APAP treatment, respectively. Sirt6 knockdown in AML12 cells aggravated APAP-induced hepatocyte death and oxidative stress, inhibited cell viability and proliferation, and downregulated CCNA1, CCND1 and CKD4 protein levels. Sirt6 knockdown significantly prevented APAP-induced NRF2 activation, reduced the transcriptional activities of GSTµ and NQO1 and the mRNA levels of Nrf2, Ho-1, Gstα and Gstµ. Furthermore, SIRT6 showed potential protein interaction with NRF2 as evidenced by co-immunoprecipitation (Co-IP) assay. Additionally, the protective effect of P53 against APAP-induced hepatocytes injury was Sirt6-dependent. The Sirt6 mRNA was significantly down-regulated in P53 -/- mice. P53 activated the transcriptional activity of SIRT6 and exerted interaction with SIRT6. Our results demonstrate that SIRT6 protects against APAP hepatotoxicity through alleviating oxidative stress and promoting hepatocyte proliferation, and provide new insights in the function of SIRT6 as a crucial docking molecule linking P53 and NRF2.

Human CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1.

Acetylated histone H3 lysine 56 (H3K56Ac) diminishes in response to DNA damage but is restored following DNA repair. Here, we report that CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1. We show that H3K56Ac accumulates at DNA damage sites. The restoration of H3K56Ac but not H3K27Ac, H3K18Ac and H3K14Ac depends on CAF-1 function, whereas all these acetylations are mediated by CBP/p300. The CRL4DDB2 components, DDB1, DDB2 and CUL4A, are also required for maintaining the H3K56Ac and H3K9Ac level in chromatin, and for restoring H3K56Ac following induction of DNA photolesions and strand breaks. Depletion of CUL4A decreases the recruitment of CAF-1 p60 and p150 to ultraviolet radiation- and phleomycin-induced DNA damage. Neddylation inhibition renders CRL4DDB2 inactive, decreases H3K56Ac level, diminishes CAF-1 recruitment and prevents H3K56Ac restoration. Mutation in the PIP box of DDB2 compromises its capability to elevate the H3K56Ac level but does not affect XPC ubiquitination. These results demonstrated a function of CRL4DDB2 in differential regulation of histone acetylation in response to DNA damage, suggesting a novel role of CRL4DDB2 in repair-driven chromatin assembly.

A role for Rtt109 in buffering gene-dosage imbalance during DNA replication.

Chromatin can function as an integrator of DNA-related processes, allowing communication, for example, between DNA replication and gene transcription. Such communication is needed to overcome the gene-dosage imbalance introduced during DNA replication, when certain genes are replicated prior to others. Increased transcription of early replicating genes could alter regulatory balances. This does not occur, suggesting a mechanism that suppresses expression from newly replicated DNA. Critical to this buffering is Rtt109, which acetylates the internal K56 residue of newly synthesized histone H3 prior to incorporation onto DNA. H3K56ac distinguishes replicated from non-replicated DNA, communicating this information to the transcription machinery to ensure expression homeostasis during S phase.

Cell cycle-dependent changes in H3K56ac in human cells.

The incorporation of histone H3 with an acetylated lysine 56 (H3K56ac) into the nucleosome is important for chromatin remodeling and serves as a marker of new nucleosomes during DNA replication and repair in yeast. However, in human cells, the level of H3K56ac is greatly reduced, and its role during the cell cycle is controversial. Our aim was to determine the potential of H3K56ac to regulate cell cycle progression in different human cell lines. A significant increase in the number of H3K56ac foci, but not in H3K56ac protein levels, was observed during the S and G2 phases in cancer cell lines, but was not observed in embryonic stem cell lines. Despite this increase, the H3K56ac signal was not present in late replication chromatin, and H3K56ac protein levels did not decrease after the inhibition of DNA replication. H3K56ac was not tightly associated with the chromatin and was primarily localized to active chromatin regions. Our results support the role of H3K56ac in transcriptionally active chromatin areas but do not confirm H3K56ac as a marker of newly synthetized nucleosomes in DNA replication.