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Aberrant glucose metabolism and elevated O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) are hallmarks of hepatocellular carcinoma (HCC). Loss of phosphoenolpyruvate carboxykinase 1 (PCK1), the major rate-limiting enzyme of hepatic gluconeogenesis, increases hexosamine biosynthetic pathway (HBP)-mediated protein O-GlcNAcylation in hepatoma cell and promotes cell growth and proliferation. However, whether PCK1 deficiency and hyper O-GlcNAcylation can induce HCC metastasis is largely unknown. Here, gain- and loss-of-function studies demonstrate that PCK1 suppresses HCC metastasis in vitro and in vivo. Specifically, lysine acetyltransferase 5 (KAT5), belonging to the MYST family of histone acetyltransferases (HAT), is highly modified by O-GlcNAcylation in PCK1 knockout hepatoma cells. Mechanistically, PCK1 depletion suppressed KAT5 ubiquitination by increasing its O-GlcNAcylation, thereby stabilizing KAT5. KAT5 O-GlcNAcylation epigenetically activates TWIST1 expression via histone H4 acetylation, and enhances MMP9 and MMP14 expression via c-Myc acetylation, thus promoting epithelial-mesenchymal transition (EMT) in HCC. In addition, targeting HBP-mediated O-GlcNAcylation of KAT5 inhibits lung metastasis of HCC in hepatospecific Pck1-deletion mice. Collectively, our findings demonstrate that PCK1 depletion increases O-GlcNAcylation of KAT5, epigenetically induces TWIST1 expression and promotes HCC metastasis, and link metabolic enzyme, post-translational modification (PTM) with epigenetic regulation.

Histone acetylation is essential for initiating and maintaining a permissive chromatin conformation and gene transcription. Dysregulation of histone acetylation can contribute to tumorigenesis and metastasis. Using inducible cre-recombinase and CRISPR/Cas9-mediated deletion, we investigated the roles of the histone lysine acetyltransferase TIP60 (KAT5/HTATIP) in human cells, mouse cells, and mouse embryos. We found that loss of TIP60 caused complete cell growth arrest. In the absence of TIP60, chromosomes failed to align in a metaphase plate during mitosis. In some TIP60 deleted cells, endoreplication occurred instead. In contrast, cell survival was not affected. Remarkably, the cell growth arrest caused by loss of TIP60 was independent of the tumor suppressors p53, INK4A and ARF. TIP60 was found to be essential for the acetylation of H2AZ, specifically at lysine 7. The mRNA levels of 6236 human and 8238 mouse genes, including many metabolism genes, were dependent on TIP60. Among the top 50 differentially expressed genes, over 90% were downregulated in cells lacking TIP60, supporting a role for TIP60 as a key co-activator of transcription. We propose a primary role of TIP60 in H2AZ lysine 7 acetylation and transcriptional activation, and that this fundamental role is essential for cell proliferation. Growth arrest independent of major tumor suppressors suggests TIP60 as a potential anti-cancer drug target.

Background Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is upregulated in colon cancer cells and associated with silencing tumor suppressor genes (TSGs) to promote colon cancer cell proliferation. Objective To investigate epigenetic modification of UHRF1 by TIP60. Whether UHRF1 acetylation by TIP60 can induce cell proliferation in colon cancer cells. Methods Acetylation sites of UHRF1 by TIP60 was predicted by ASEB (Acetylation Set Enrichment Based) method and identified by immunoprecipitation assay using anti-pan-acetyl lysine antibody and in vitro acetylation assay. Based on this method, UHRF1 acetylation-deficient mimic 4KR (K644R, K646R, K648R, K650R) mutant was generated to investigate effects of UHRF1 acetylation by TIP60. shRNA system was used to generate stable knockdown cell line of UHRF1. With transient transfection of UHRF1 WT and 4KR, the effects of UHRF1 4KR mutant on Jun dimerization protein 2 (JDP2) gene expression, cell proliferation and cell cycle were investigated by RT-qPCR and FACS analysis in shUHRF1 colon cancer cell line. Results Downregulation of TIP60-mediated UHRF1 acetylation is correlated with suppressed cell cycle progression. Acetylation-deficient mimic of UHRF1 showed poor cell growth through increased expression of JDP2 gene. Conclusions Acetylation of UHRF1 4K residues by TIP60 is important for colon cancer cell growth. Furthermore, upregulated JDP2 expression by acetylation-deficient mutant of UHRF1 might be an important epigenetic target for colon cancer cell proliferation.

The Tip60 lysine acetyltransferase is a tumor suppressor in most cancers but an oncogene in prostate and gastric cancer. Tip60 is commonly found in the nucleus, where it acetylates proteins involved in transcription, DNA repair, and chromatin; however, it has also been shown to acetylate cytoplasmic targets. In this study, we investigated the relationship between Tip60 localization and breast and lung cancer. In cell fractionation experiments, cancer-derived cell lines showed a shift from nuclear to cytoplasmic endogenous Tip60 compared with cell lines derived from normal cells. With immunofluorescence, we observed four different localization patterns of overexpressed Tip60 and found that cancer cells had increased cytoplasmic localization of Tip60 compared with HEK-293 cells. The addition of a nuclear localization signal (NLS) increased the number of cells containing nuclear Tip60, whereas mutation of a putative endogenous NLS increased the number of cells with cytoplasmic Tip60. Overexpression of Tip60 increased cancer cell line sensitivity to paclitaxel regardless of changes in localization. These results suggest that dysregulation of Tip60 in breast and lung cancer is not limited to reduced expression but may also involve subcellular localization.

The DNA sensor cGMP-AMP synthase (cGAS) senses cytosolic microbial or self DNA to initiate a MITA/STING-dependent innate immune response. cGAS is regulated by various posttranslational modifications at its C-terminal catalytic domain. Whether and how its N-terminal unstructured domain is regulated by posttranslational modifications remain unknown. We identified the acetyltransferase KAT5 as a positive regulator of cGAS-mediated innate immune signaling. Overexpression of KAT5 potentiated viral-DNA–triggered transcription of downstream antiviral genes, whereas a KAT5 deficiency had the opposite effects. Mice with inactivated Kat5 exhibited lower levels of serum cytokines in response to DNA virus infection, higher viral titers in the brains, and more susceptibility to DNA-virus–induced death. Mechanistically, KAT5 catalyzed acetylation of cGAS at multiple lysine residues in its N-terminal domain, which promoted its DNA-binding ability. Our findings suggest that KAT5-mediated cGAS acetylation at its N terminus is important for efficient innate immune response to DNA virus.

The XPF-ERCC1 heterodimer is a structure-specific endonuclease that is essential for nucleotide excision repair (NER) and interstrand crosslink (ICL) repair in mammalian cells. However, whether and how XPF binding to ERCC1 is regulated has not yet been established. Here, we show that TIP60, also known as KAT5, a haplo-insufficient tumor suppressor, directly acetylates XPF at Lys911 following UV irradiation or treatment with mitomycin C and that this acetylation is required for XPF-ERCC1 complex assembly and subsequent activation. Mechanistically, acetylation of XPF at Lys911 disrupts the Glu907-Lys911 salt bridge, thereby leading to exposure of a previously unidentified second binding site for ERCC1. Accordingly, loss of XPF acetylation impairs the damage-induced XPF-ERCC1 interaction, resulting in defects in both NER and ICL repair. Our results not only reveal a mechanism that regulates XPF-ERCC1 complex assembly and activation, but also provide important insight into the role of TIP60 in the maintenance of genome stability., The XPF-ERCC1 heterodimer is an endonuclease involved in nucleotide excision (NER) and interstrand crosslink (ICL) repair in mammalian cells. Here, the authors provide insights into its regulation by revealing that TIP60 regulates XPF-ERCC1 complex assembly and activation.

Background/aim In breast cancer, initiation of carcinogenesis leads to epigenetic dysregulation, which can lead for example to the loss of the heterochromatin skeleton SUV39H1/H3K9me3/HP1 or the supposed secondary skeleton TIP60/P400/H4K12ac/BRD (2/4), which allows the maintenance of chromatin integrity and plasticity. This study investigated the relationship between TIP60, P400 and H4K12ac and their implications in breast tumors. Materials and methods Seventy-seven patients diagnosed with breast cancer were included in this study. Chromatin immunoprecipitation (ChIP) assay was used to identify chromatin modifications. Western blot and reverse transcription and quantitative real-time PCR were used to determine protein and gene expression, respectively. Results We verified the variation in H4K12ac enrichment and the co-localization of H4K12ac and TIP60 on the euchromatin and heterochromatin genes, respectively, by ChIP-qPCR and ChIP-reChIP, which showed an enrichment of H4K12ac on specific genes in tumors compared to the adjacent healthy tissue and a co-localization of H4K12ac with TIP60 in different breast tumor types. Furthermore, RNA and protein expression of TIP60 and P400 was investigated and overexpression of TIP60 and P400 mRNA was associated with tumor aggressiveness. Conclusion There is a potential interaction between H4K12ac and TIP60 in heterochromatin or euchromatin in breast tumors.

Autophagy receptor p62/SQSTM1 promotes the assembly and removal of ubiquitylated proteins by forming p62 bodies and mediating their encapsulation in autophagosomes. Here we show that under nutrient-deficient conditions, cellular p62 specifically undergoes acetylation, which is required for the formation and subsequent autophagic clearance of p62 bodies. We identify K420 and K435 in the UBA domain as the main acetylation sites, and TIP60 and HDAC6 as the acetyltransferase and deacetylase. Mechanically, acetylation at both K420 and K435 sites enhances p62 binding to ubiquitin by disrupting UBA dimerization, while K435 acetylation also directly increases the UBA-ubiquitin affinity. Furthermore, we show that acetylation of p62 facilitates polyubiquitin chain-induced p62 phase separation. Our results suggest an essential role of p62 acetylation in the selective degradation of ubiquitylated proteins in cells under nutrient stress, by specifically regulating the assembly of p62 bodies., The autophagy receptor p62 mediates the assembly and removal of ubiquitylated protein aggregates by forming p62 bodies. Here, the authors identify an acetylation-dependent mechanism that regulates formation and autophagic clearance of p62 bodies under nutrient-deficient conditions.

An estimated 5.4 million cases of nonmelanoma skin cancer are reported in the United States at an associated cost of $4.8 billion. ΔNp63α, a proto-oncogene in the p53 family of transcription factors, is overexpressed in squamous cell carcinoma (SCC) and associated with poor prognosis and survival. ΔNp63α elicits its tumorigenic effects in part by promoting cellular proliferation and cell survival. Despite its importance in SCC, the upstream regulation of ΔNp63α is poorly understood. In this study, we identify TIP60 as a novel upstream regulator of ΔNp63α. Using a combination of overexpression, silencing, stable expression, and pharmacological approaches in multiple cell lines, we showed that TIP60 up-regulates ΔNp63α expression. Utilizing cycloheximide treatment, we showed that TIP60 catalytic activity is required for stabilization of ΔNp63α protein levels. We further showed that TIP60 coexpression inhibits ΔNp63α ubiquitination and proteasomal degradation. Stabilization of ΔNp63α protein was further associated with TIP60-mediated acetylation. Finally, we demonstrated that TIP60-mediated regulation of ΔNp63α increases cellular proliferation by promoting G(2)/M progression through MTS assays and flow cytometry. Taken together, our findings provide evidence that TIP60 may contribute to SCC progression by increasing ΔNp63α protein levels, thereby promoting cellular proliferation.

Wound healing is a complex phenomenon that requires coordination of numerous molecular and cellular changes to facilitate timely and efficient repair of the damaged tissue. Although many of these molecular pathways have been detailed, others remain to be elucidated. In the present work, we show for the first time, roles for the acetyltransferase TIP60 and nuclear receptor transcription factor PXR in this process, participating in wound healing by altering actin dynamics and cellular motility. We found that in response to wound-injury, TIP60 induces rapid formation of filopodia at the wounded cell front, leading to enhanced cell migration and faster closure of the wound. Further, qPCR analysis revealed heightened expression of Cdc42 and ROCK1 genes, key regulators involved in filopodia formation and actin reorganization, exclusively in TIP60-PXR-expressing cells upon wound-induction. We also performed ChIP assays to confirm the context-specific binding of TIP60 on the ROCK1 promoter and demonstrated that the TIP60 chromodomain is essential for loading of the TIP60-PXR complex onto the chromatin. Results from immunoprecipitation assays revealed that during the wounded condition, TIP60 alters the chromatin microenvironment by specifically acetylating histones H2B and H4, thereby modulating the expression of target genes. Overall, findings of this study show that TIP60 is a novel regulator of the wound healing process by regulating the expression of wound repair-related genes.

In eukaryotes, histones and their variants are essential for chromatin structure and function; both play important roles in the regulation of gene transcription, as well as the development of tumors. We aimed to explore the genomics data of hepatocellular carcinoma (HCC), combined with literature analysis, in terms of the histone variant H2A.Z. Cell phenotype assay confirmed the effect of H2A.Z on the proliferation, metastasis, apoptosis, and cell cycle of HCC cells. H2A.Z was shown to function via the tumor dysregulation signaling pathway, with BCL6 as its interacting protein. In addition, the acetylation level of H2A.Z was higher in HCC and was related to tumor formation. We found the acetylation of H2A.Z to be related to and regulated by lincZNF337-AS1. LincZNF337-AS1 was found to bind to H2A.Z and KAT5 at different sites, promoting the acetylation of H2A.Z through KAT5. We concluded that, in HCC, H2A.Z is an oncogene, whose acetylation promotes the transcription of downstream genes, and is regulated by lincZNF331-AS1.

Post-translational modifications of histone variant H2A.Z accompany gene transactivation, but its modifying enzymes still remain elusive. Here, we reveal a hitherto unknown function of human KAT2A (GCN5) as a histone acetyltransferase (HAT) of H2A.Z at the promoters of a set of transactivated genes. Expression of these genes also depends on the DNA repair complex XPC-RAD23-CEN2. We established that XPC-RAD23-CEN2 interacts both with H2A.Z and KAT2A to drive the recruitment of the HAT at promoters and license H2A.Z acetylation. KAT2A selectively acetylates H2A.Z.1 versus H2A.Z.2 in vitro on several well-defined lysines and we unveiled that alanine-14 in H2A.Z.2 is responsible for inhibiting the activity of KAT2A. Notably, the use of a nonacetylable H2A.Z.1 mutant shows that H2A.Z.1ac recruits the epigenetic reader BRD2 to promote RNA polymerase II recruitment. Our studies identify KAT2A as an H2A.Z.1 HAT in mammals and implicate XPC-RAD23-CEN2 as a transcriptional co-activator licensing the reshaping of the promoter epigenetic landscape.

Overexpression of KDM2B is frequently occurred in various human solid tumours, and the high levels of KDM2B are associated with tumourigenesis. However, whether and how its activities might be modulated to facilitate tumour progression is still unclear. Immunoprecipitation and immunoblotting were carried out to detect the acetylation of KDM2B. Nucleosomes and mononucleosomes were prepared and the demethylation activity of KDM2B was detected in these two substrates. The effects of KDM2B acetylation on the transcription of target genes, as well as tumour growth and metastasis were then studied. KDM2B was acetylated in osteosarcoma cancer cell lines (MG‐63 and HOS). This modification occurred at lysine 758 and catalysed by Tip60. Acetylation of KDM2B decreased the capacity of KDM2B in binding with nucleosomes. KDM2B acetylation diminished its demethylation activity towards nucleosomal substrates rather than towards bulk histone. Besides, acetylation of KDM2B diminished its ability to bind with the promoters of p21 and puma. Moreover, the promoting effects of KDM2B acetylation on tumour cells' proliferation and metastasis, and in vivo tumour growth were dependent on Tip60. KDM2B is acetylated at lysine 758 by Tip60 in human osteosarcoma cells. Acetylation of KDM2B diminishes its association with nucleosomes, and thus increasing methylation of H3K36 at its target genes as well as enhancing its oncogenic effects.

In rheumatoid arthritis (RA), imbalanced T cells subsets play a critical role in sustaining chronic inflammatory responses in the synovium. Naive T cells in RA patients undergo maldifferentiation, including an increase in the effector Th1/Th17 lineage and a reduction in regulatory T (Treg) cells. Upon stimulation, naive CD4+CD45RO- T cells from RA patients exhibited insufficient expression of Foxp3, which induced a deficiency in Tregs production and an imbalance of Treg/Th17 differentiation. Further mechanistic study indicated that RA T cells failed to produce sufficient levels of the histone acetyltransferase Tip60, leading to reduced acetylation of Foxp3; this, in turn, decreased Foxp3 expression, impaired Treg commitment, and promoted Th17 production. Moreover, in human synovium chimeric mice, suppression of Tip60 activity in healthy T cells promoted tissue infiltration and arthritogenesis, while reconstitution of Tip60 in RA T cells suppressed synovitis and effector T cell infiltration. Our findings link T cell maldifferentiation and tissue infiltration with Tip60-mediated Foxp3 acetylation and identify Tip60 as a potential therapeutic target for suppression of tissue inflammation and autoimmunogenesis in RA.

Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore–microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore–microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80–microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1–80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80–microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore–microtubule attachment in the maintenance of genomic stability during mitosis.

Aim: The acetyltransferase TIP60 is reported to be downregulated in several cancers, in particular breast cancer, but the molecular mechanisms resulting from its alteration are still unclear. Materials & methods: In breast tumors, H3K4ac enrichment and its link with TIP60 were evaluated by chromatin immunoprecipitation-qPCR and re-chromatin immunoprecipitation techniques. To assess the biological roles of TIP60 in breast cancer, two cell lines of breast cancer, MDA-MB-231 (ER-) and MCF-7 (ER+) were transfected with shRNA specifically targeting TIP60 and injected to athymic Balb-c mice. Results: We identified a potential target of TIP60, H3K4. We show that an underexpression of TIP60 could contribute to a reduction of H3K4 acetylation in breast cancer. An increase in tumor development was noted in sh-TIP60 MDA-MB-231 xenografts and a slowdown of tumor growth in sh-TIP60 MCF-7 xenografts. Conclusion: This is evidence that the underexpression of TIP60 observed in breast cancer can promote the tumorigenesis of ER-negative tumors.

Carbonic anhydrases (CAs) play a significant role in maintaining pH balance by catalyzing the conversion of carbon dioxide to bicarbonate. The regulation of pH is critical for all living organisms. Although there are many studies in the literature on the biochemical, functional, and structural features of CAs, there is not sufficient information about the epigenetic regulation of CAs.The lysine acetyltransferase TIP60 (60 kDa Tat-interactive protein) was knocked out specifically in mouse liver using the Cre/loxP system, and knockout rate was shown as 83-88% by Southern blot analysis. The impact of Tip60 on the expression of Ca1, Ca3, and Ca7 was investigated at six Zeitgeber time (ZT) points in the control and liver-specific Tip60 knockout mice (mutant) groups by real-time PCR. In the control group, while Ca1 showed the highest expression at ZT8 and ZT12, the lowest expression profile was observed at ZT0 and ZT20. Hepatic Ca1 displayed robust circadian expression. However, hepatic Ca3 exhibited almost the same level of expression at all ZT points. The highest expression of Ca7 was observed at ZT12, and the lowest expression was determined at ZT4. Furthermore, hepatic Ca7 also showed robust circadian expression. The expression of Ca1 and Ca3 significantly decreased in mutant mice at all time periods, but the expression of Ca7 used as a negative control was not affected.It was suggested for the first time that Tip60 might be considered a candidate protein in the regulation of the Ca1 and Ca3 genes, possibly by acetylation.

A chromosomal translocation found in cannibalistic acute myeloid leukemia (AML) leads to an in-frame fusion of the transcription elongation repressor ZMYND11 to MBTD1, a subunit of the NuA4/TIP60 histone acetyltransferase (HAT) complex. In contrast to the NuA4/TIP60 complex, ZMYND11 is linked to repression of actively transcribed genes through recognition of H3.3K36me3. To understand the abnormal molecular events that expression of this ZMYND11-MBTD1 fusion protein can create, we performed its biochemical and functional characterization in comparison to each individual fusion partner. ZMYND11-MBTD1 is stably incorporated into the endogenous NuA4/TIP60 complex but does not bring any additional interactors as the fusion lacks the MYND domain of ZMYND11. Nevertheless, this truncated ZMYND11 moiety in the fusion leads to mislocalization of the NuA4/TIP60 complex on the body of genes normally bound by ZMYND11 in the genome, in a PWWP-H3.3K36me3 interaction-dependent manner. This can be correlated to increased chromatin acetylation and altered gene transcription, most notably on the MYC oncogene, and alternative splicing. Importantly, expression of ZMYND11-MBTD1, but not the individual fusion partners, during embryonic stem cell differentiation, leads to decreased expression of specific differentiation markers, while favoring Myc-driven pluripotency. It also favors self-renewal of hematopoietic stem/progenitor cells. Altogether, these results indicate that the ZMYND11-MBTD1 fusion protein functions primarily by mistargeting the NuA4/TIP60 complex to the body of genes, altering normal transcription of specific genes, likely driving oncogenesis in part through the Myc regulatory network.Highlights-A recurrent chromosomal translocation detected in cannibalistic acute myeloid leukemia leads to the production of a ZMYND11-MBTD1 fusion protein.-The ZMYND11-MBTD1 fusion protein is stably incorporated into the endogenous NuA4/TIP60 complex.-ZMYND11-MBTD1 leads to mistargeting of NuA4/TIP60 activity to the coding region of ZMYND11-target genes, altering gene expression and splicing.-ZMYND11-MBTD1 binds the MYC gene leading to its upregulation, favoring growth and pluripotency while inhibiting differentiation markers.

Recurring chromosomal translocation t(10;17)(p15;q21) present in a subset of human acute myeloid leukemia (AML) patients creates an aberrant fusion gene termed ZMYND11-MBTD1 (ZM); however, its function remains undetermined. Here, we show that ZM confers primary murine hematopoietic stem/progenitor cells indefinite self-renewal capability ex vivo and causes AML in vivo. Genomics profilings reveal that ZM directly binds to and maintains high expression of pro-leukemic genes including Hoxa, Meis1, Myb, Myc and Sox4. Mechanistically, ZM recruits the NuA4/Tip60 histone acetyltransferase complex to cis-regulatory elements, sustaining an active chromatin state enriched in histone acetylation and devoid of repressive histone marks. Systematic mutagenesis of ZM demonstrates essential requirements of Tip60 interaction and an H3K36me3-binding PWWP (Pro-Trp-Trp-Pro) domain for oncogenesis. Inhibitor of histone acetylation-‘reading’ bromodomain proteins, which act downstream of ZM, is efficacious in treating ZM-induced AML. Collectively, this study demonstrates AML-causing effects of ZM, examines its gene-regulatory roles, and reports an attractive mechanism-guided therapeutic strategy., The fusion gene ZMYND11-MBTD1 (ZM) is present in a subgroup of patients with acute myeloid leukaemia (AML). Here, the authors show that ZM expression induces AML in a murine model though activating the NuA4/TIP60 histone acetyltransferase complex.

Histone variants differ in amino acid sequence, expression timing and genomic localization sites from canonical histones and convey unique functions to eukaryotic cells. Their tightly controlled spatial and temporal deposition into specific chromatin regions is accomplished by dedicated chaperone and/or remodeling complexes. While quantitatively identifying the chaperone complexes of many human H2A variants by using mass spectrometry, we also found additional members of the known H2A.Z chaperone complexes p400/TIP60/NuA4 and SRCAP. We discovered JAZF1, a nuclear/nucleolar protein, as a member of a p400 sub-complex containing MBTD1 but excluding ANP32E. Depletion of JAZF1 results in transcriptome changes that affect, among other pathways, ribosome biogenesis. To identify the underlying molecular mechanism contributing to JAZF1&rsquo, s function in gene regulation, we performed genome-wide ChIP-seq analyses. Interestingly, depletion of JAZF1 leads to reduced H2A.Z acetylation levels at &gt, 1000 regulatory sites without affecting H2A.Z nucleosome positioning. Since JAZF1 associates with the histone acetyltransferase TIP60, whose depletion causes a correlated H2A.Z deacetylation of several JAZF1-targeted enhancer regions, we speculate that JAZF1 acts as chromatin modulator by recruiting TIP60&rsquo, s enzymatic activity. Altogether, this study uncovers JAZF1 as a member of a TIP60-containing p400 chaperone complex orchestrating H2A.Z acetylation at regulatory regions controlling the expression of genes, many of which are involved in ribosome biogenesis.

Autophagy is a catabolic process through which cytoplasmic components are degraded and recycled in response to various stresses including starvation. Recently, transcriptional and epigenetic regulations of autophagy have emerged as essential mechanisms for maintaining homeostasis. Here, we identify that coactivator-associated arginine methyltransferase 1 (CARM1) methylates Pontin chromatin-remodeling factor under glucose starvation, and methylated Pontin binds Forkhead Box O 3a (FOXO3a). Genome-wide analyses and biochemical studies reveal that methylated Pontin functions as a platform for recruiting Tip60 histone acetyltransferase with increased H4 acetylation and subsequent activation of autophagy genes regulated by FOXO3a. Surprisingly, CARM1-Pontin-FOXO3a signaling axis can work in the distal regions and activate autophagy genes through enhancer activation. Together, our findings provide a signaling axis of CARM1-Pontin-FOXO3a and further expand the role of CARM1 in nuclear regulation of autophagy., Epigenetic regulations of autophagy have emerged as mechanisms for maintaining cellular homeostasis. Here the authors reveal that the CARM1-Pontin-FOXO3a signaling axis can activate autophagy related genes through enhancer activation.

Remyelination of the peripheral and central nervous systems (PNS and CNS, respectively) is a prerequisite for functional recovery after lesion. However, this process is not always optimal and becomes inefficient in the course of multiple sclerosis. Here we show that, when acetylated, eukaryotic elongation factor 1A1 (eEF1A1) negatively regulates PNS and CNS remyelination. Acetylated eEF1A1 (Ac-eEF1A1) translocates into the nucleus of myelinating cells where it binds to Sox10, a key transcription factor for PNS and CNS myelination and remyelination, to drag Sox10 out of the nucleus. We show that the lysine acetyltransferase Tip60 acetylates eEF1A1, whereas the histone deacetylase HDAC2 deacetylates eEF1A1. Promoting eEF1A1 deacetylation maintains the activation of Sox10 target genes and increases PNS and CNS remyelination efficiency. Taken together, these data identify a major mechanism of Sox10 regulation, which appears promising for future translational studies on PNS and CNS remyelination., The molecular mechanisms regulating remyelination are unclear. Here, the authors show that promoting deacetylation of eEF1A1 prevents the translocation of Sox10 outside the nucleus, contributing to maintaining the expression of Sox10 target genes and increasing remyelination efficiency.

Chromosome translocations involving the MLL gene are common rearrangements in leukemia. Such translocations fuse the MLL 5'-region to partner genes in frame, producing MLL-fusions that cause MLL-related leukemia. MLL-fusions activate transcription of target genes such as HoxA cluster and Meis1, but the underlying mechanisms remain to be fully elucidated. In this study, we discovered that Tip60, a MYST-type histone acetyltransferase, was required for the expression of HoxA cluster and Meis1 genes and the development of MLL-fusion leukemia. Tip60 was recruited by MLL-AF10 and MLL-ENL fusions to the Hoxa9 locus, where it acetylated H2A.Z, thereby promoting Hoxa9 gene expression. Conditional deletion of Tip60 prevented the development of MLL-AF10 and MLL-ENL leukemia, indicating that Tip60 is indispensable for the leukemogenic activity of the MLL-AF10 and MLL-ENL-fusions. Our findings provide novel insight about epigenetic regulation in the development of MLL-AF10 and MLL-ENL-fusion leukemia.

Summary: Phosphoglycerate dehydrogenase (PHGDH) is the first enzyme in the serine synthesis pathway in which it is also the rate-limiting enzyme. It is significantly upregulated in many cancers, especially breast cancer. However, the posttranslational mechanism of PHGDH upregulation in breast cancer is unknown. In this study, we find that RNF5, an E3 ubiquitin ligase, is essential for the degradation of PHGDH protein. PHGDH is degraded by RNF5 to prevent the proliferation of breast cancer cells. The acetylation of PHGDH at K58 is able to disrupt the interaction of RNF5-PHGDH and promote the proliferation of breast cancer cells. Tip60 and SIRT2 regulate the reversible acetylation modification of PHGDH in response to glucose alteration. Moreover, PHGDH is significantly upregulated in samples of human breast cancer and is associated with decreased RNF5 expression. This implies a potential therapeutic target through the interference interaction of PHGDH-RNF5 to degrade PHGDH in breast cancer.

Inflammation supports the progression of alcohol-related organ injury. Recent research findings have linked ethanol exposure to changes in histone acetylation and deacetylation in the brain and in peripheral tissues, leading to ethanol-dependence related damage. One of the mechanisms that has been shown to play a major role during inflammation is the cannabinoid system. Previous research has demonstrated that ethanol can modulate cannabinoid receptors' functions. Our lab has shown that the G protein-coupled receptor (GPR55), a novel cannabinoid receptor, is upregulated in binge drinkers and in cells treated acutely with ethanol. Additionally, our group has also uncovered that chronic ethanol exposure leads to an increase in histone modifications, such as acetylation. However, the regulatory mechanism of GPR55 within the immune system under the influence of ethanol is poorly understood. Since changes in histone modifications might lead to changes in gene expression, we hypothesize that the mechanism of ethanol-induced upregulation of GPR55 is linked to epigenetic changes on histone proteins. Taking into account previous findings from our lab, the goal of the present study was to determine whether there is any relevant association between histone hyperacetylation and the regulation of the novel cannabinoid receptor GPR55 in monocyte-derived dendritic cells (MDDCs) of human origin treated acutely with ethanol. Therefore, monocytes were isolated from buffy coats and allowed to differentiate into MDDCs. The cells were treated with ethanol for 24 h, harvested, fixed, and stained with antibodies against GPR55. As expected, based on previous findings, confocal microscopy showed that ethanol exposure increases GPR55 expression. In order to demonstrate the correlation between histone acetylation and GPR55 expression regulation, the cells were treated with ethanol, harvested, and then the chromatin was extracted and fractionated for chromatin immunoprecipitation (ChIP) assay, followed by real-time qPCR for the analysis of DNA fragments. The results showed an enrichment of the histone modification H4K12ac in the GPR55 gene of MDDCs treated with ethanol. Furthermore, siRNA against the histone acetyltransferase Tip60 (responsible for the acetylation of H4K12) resulted in a downregulation of GPR55. In conjunction, these results indicate that in the presence of ethanol, the upregulation of GPR55 expression is accompanied by H4K12 acetylation, which might have a significant effect in the ability of this innate immune system's cells to cope with cellular stress induced by ethanol. However, the causality of ethanol regulation of H4K12ac in GPR55 expression changes still lacks further elucidation; therefore, additional experimental approaches to confirm a significant causality between H4K12 acetylation and ethanol regulation of GPR55 are currently undergoing in our lab.

Acute lung injury (ALI) is often associated with inflammation. Increasing evidence has identified the role for ubiquitin-specific protease 7 (USP7) in activating the expression of inflammatory factors in macrophages. The present study evaluated whether USP7 also mediates histone acetyltransferase Tat-interactive protein 60 (Tip60) in the development of ALI inflammation. An ALI mouse model was induced by intratracheal lipopolysaccharide (LPS) administration. Next, lung myeloperoxidase (MPO) activity and the ratio of dry weight/wet weight of lung were examined to evaluate tissue damage. In addition, RAW 264.7 cells were treated with LPS to induce an in vitro LPS-induced inflammatory cell model. ELISA was performed to measure expression of IL-1β, TNF-α, IL-6, and IL-8 in cells and tissues. TUNEL was used to detect LPS-induced cell apoptosis. Furthermore, the interaction between USP7 and Tip60 was identified by IP, Western blot analysis, and cycloheximide (CHX) treatment. The enrichment of Tip60 and H3K27ac in the promoter region of IL-6 and IL-8 was assessed by ChIP. USP7 was highly expressed in LPS-endotoxin-induced ALI mouse models and silencing of USP7 delayed the progression of ALI in mice. Silencing of USP7 protected RAW 264.7 cells against LPS-induced inflammation and apoptosis by downregulating IL-1β, TNF-α, IL-6, and IL-8. USP7 enhanced Tip60 protein stability, and Tip60 increased the enrichment of H3K27ac on IL-6 and IL-8 promoter region and activated NF-κB p65 to increase IL-6 and IL-8 expression. These findings reveal a new post-transcriptional role for USP7 in inflammation by stabilizing Tip60 and increasing the release of the pro-inflammatory cytokines, and implicate USP7 inhibitors as potential therapeutic agents for ALI. KEY MESSAGES: USP7 expresses highly in an acute lung injury (ALI) mouse models. Silencing of USP7 inhibits inflammation and cell apoptosis in ALI mouse. USP7 stabilizes Tip60 to boost the release of IL-6 and IL-8. Tip60 increases IL-6 and IL-8 expression by acetylating NF-κB p65. Silencing of USP7 alleviates ALI by repressing NF-κB p65 and Tip60.

Aberrant histone acetylation contributes to age-dependent cognitive decline and neurodegenerative diseases. We analyze the function of lysine acetyltransferase TIP60/KAT5 in neurons of the hippocampus using an inducible mouse model. TIP60-deficiency in the adult forebrain leads within days to extensive transcriptional dysfunction characterized by the presence of a neurodegeneration-related signature in CA1. Cell cycle- and immunity-related genes are upregulated while learning- and neuronal plasticity-related genes are downregulated. The dysregulated genes seen under TIP60-deficiency overlap with those in the well-characterized CK-p25 neurodegeneration model. We found that H4K12 is hypoacetylated at the transcriptional start sites of those genes whose expression is dampened in TIP60-deficient mice. Transcriptional dysregulation is followed over a period of weeks by activation of Caspase 3 and fragmentation of β-actin in CA1 neurites, eventually leading to severe neuronal loss. TIP60-deficient mice also develop mild memory impairment. These phenotypes point to a central role of TIP60 in transcriptional networks that are critical for neuronal viability.

The Aurora kinases represent a group of serine/threonine kinases which are crucial regulators of mitosis. Dysregulated Aurora kinase B (AurkB) expression, stemming from genomic amplification, increased gene transcription or overexpression of its allosteric activators, is capable of initiating and sustaining malignant phenotypes. Although AurkB level in cells is well-orchestrated, studies that relate to its stability or activity, independent of mitosis, are lacking. We report that AurkB undergoes acetylation in vitro by lysine acetyltransferases (KATs) belonging to different families, namely by p300 and Tip60. The haploinsufficient tumor suppressor Tip60 acetylates two highly conserved lysine residues within the kinase domain of AurkB which not only impinges the protein stability but also its kinase activity. These results signify a probable outcome on the increase in “overall activity” of AurkB upon Tip60 downregulation, as observed under cancerous conditions. The present work, therefore, uncovers an important functional interplay between AurkB and Tip60, frailty of which may be an initial event in carcinogenesis.

Metformin has beneficial effects of preventing and treating cancers on type 2 diabetic patients. However, the role of metformin in non-diabetic cancer patients and the precise molecular mechanisms against cancer have not yet been sufficiently elucidated. We recently reported that the pseudokinase protein TRIB3 acts as a stress sensor linking metabolic stressors to cancer promotion by inhibiting autophagy and ubiquitin-proteasomal degradation systems; genetically abrogating of TRIB3 expression reduces tumourigenesis and cancer progression. Thus, TRIB3 is a potential therapeutic target for diverse cancers. In this study, we found that metformin attenuates melanoma growth and metastasis by reducing TRIB3 expression in non-diabetic C57BL/6 mice and diabetic KK-Ay mice; overexpression of TRIB3 protects metformin from the activation of autophagic flux, the clearance of accumulated tumour-promoting factors and the attenuation of tumour progression. We further elucidated that TRIB3 acts as an adaptor to recruit lysine acetyltransferase 5 (KAT5) to SMAD3 and induce a phosphorylation-dependent K333 acetylation of SMAD3, which sustains transcriptional activity of SMAD3 and subsequently enhances TRIB3 transcription. Metformin suppresses SMAD3 phosphorylation and decreases the KAT5/SMAD3 interaction, to attenuate the KAT5-mediated K333 acetylation of SMAD3, reduce the SMAD3 transcriptional activity and subsequent TRIB3 expression, thereby antagonizes melanoma progression. Together, our study not only defines a molecular mechanism by which metformin protects against melanoma progression by disturbing the KAT5/TRIB3/SMAD3 positive feedback loop in diabetes and non-diabetes mice, but also suggests a candidate diverse utility of metformin in tumour prevention and therapy because of suppressing stress protein TRIB3 expression.

Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation. The Ran GTPase plays a key role in mitotic spindle assembly. However, how the generation of a chemical gradient of Ran-GTP at the spindle is coupled to mitotic post-translational modifications has never been characterized. Here, we solved the complex structure of Ran with the nucleotide release factor Mog1 and delineated a novel mitosis-specific acetylation-regulated Ran–Mog1 interaction during chromosome segregation. Our structure-guided functional analyses revealed that Mog1 competes with RCC1 for Ran binding in a GTP/GDP-dependent manner. Biochemical characterization demonstrated that Mog1-bound Ran prevents RCC1 binding and subsequent GTP loading. Surprisingly, Ran is a bona fide substrate of TIP60, and the acetylation of Lys134 by TIP60 liberates Mog1 from Ran binding during mitosis. Importantly, this acetylation-elicited switch of Ran binding to RCC1 promotes high level of Ran-GTP, which is essential for chromosome alignment. These results establish a previously uncharacterized regulatory mechanism in which TIP60 provides a homeostatic control of Ran-GTP level by tuning Ran effector binding for chromosome segregation in mitosis.

Arsenic is a ubiquitous environmental contaminant with widespread public health concern. Epidemiological studies have revealed that chronic human exposure to arsenic in drinking water is associated with the prevalence of skin, lung, and bladder cancers. Aberrant histone modifications (e.g., methylation, acetylation, and ubiquitination) were previously found to be accompanied by arsenic exposure; thus, perturbation of epigenetic pathways is thought to contribute to arsenic carcinogenesis. Arsenite is known to interact with zinc finger motifs of proteins, and zinc finger motif is present in and indispensable for the enzymatic activities of crucial histone-modifying enzymes especially the MYST family of histone acetyltransferases (e.g., TIP60). Hence, we reasoned that trivalent arsenic may target the zinc finger motif of these enzymes, disturb their enzymatic activities, and alter histone acetylation. Herein, we found that As3+ could bind directly to the zinc-finger motif of TIP60 in vitro and in cells. In addition, exposure to As3+ could lead to a dose-dependent decrease in TIP60 protein level via the ubiquitin-proteasome pathway. Thus, the results from the present study revealed, for the first time, that arsenite may target cysteine residues in the zinc-finger motif of the TIP60 histone acetyltransferase, thereby altering the H4K16Ac histone epigenetic mark. Our results also shed some new light on the mechanisms underlying the arsenic-induced epigenotoxicity and carcinogenesis in humans.

Post-translational modifications of histone proteins regulate numerous cellular processes. Among these modifications, trimethylation of lysine 36 in histone H3 (H3K36me3) and acetylation of lysine 16 in histone H4 (H4K16ac) have important roles in transcriptional regulation and DNA damage response signaling. However, whether these two epigenetic histone marks are mechanistically linked remains unclear. Here we discovered a new pathway through which H3K36me3 stimulates H4K16ac upon DNA double-strand break (DSB) induction in human cells. In particular, we examined, using Western blot analysis, the levels of H3K36me3 and H4K16ac in cells after exposure to various DSB-inducing agents, including neocarzinostatin, γ rays, and etoposide, and found that H3K36me3 and H4K16ac were both elevated in cells upon these treatments. We also observed that DSB-induced H4K16 acetylation was abolished in cells upon depletion of the histone methyltransferase gene SET-domain containing 2 (SETD2) and the ensuing loss of H3K36me3. Furthermore, the H3K36me3-mediated increase in H4K16ac necessitated lens epithelium–derived growth factor p75 splicing variant (LEDGF), which is a reader protein of H3K36me3, and the KAT5 (TIP60) histone acetyltransferase. Mechanistically, the chromatin-bound LEDGF, through its interaction with KAT5, promoted chromatin localization of KAT5, thereby stimulating H4K16 acetylation. In this study, we unveiled cross-talk between two important histone epigenetic marks and defined the function of this cross-talk in DNA DSB repair.

PXR is a member of nuclear receptor superfamily and a well-characterized mediator of xenobiotic metabolism. The classical mode of PXR activation involves its binding to appropriate ligand and subsequent heterodimerization with its partner RXR. However, various factors such as post-translational modifications and crosstalk with different cellular factors may also regulate the functional dynamics and behavior of PXR. In the present study, we have identified that TIP60, an essential lysine acetyltransferase protein interacts with unliganded PXR and together this complex promotes cell migration & adhesion. TIP60 utilizes its NR Box to interact with LBD region of PXR and acetylates PXR at lysine 170 to induce its intranuclear reorganization. Also, RXR is not required for TIP60-PXR complex formation and this complex does not induce ligand-dependent PXR target gene transactivation. Interestingly, we observed that PXR augments the catalytic activity of TIP60 for histones. This is the first report demonstrating the exclusive interaction of TIP60 with PXR and uncovers a potential role for the TIP60-PXR complex in cell migration and adhesion.

Previously we demonstrated that fluoride increased acetylated-p53 (Ac-p53) in LS8 cells that are derived from mouse enamel organ epithelia and in rodent ameloblasts. However, how p53 is acetylated by fluoride and how the p53 upstream molecular pathway responds to fluoride is not well characterized. Here we demonstrate that fluoride activates histone acetyltransferases (HATs) including CBP, p300, PCAF and Tip60 to acetylate p53. HAT activity is regulated by post-translational modifications such as acetylation and phosphorylation. HAT proteins and their post-translational modifications (p300, Acetyl-p300, CBP, Acetyl-CBP, Tip60 and phospho-Tip60) were analyzed by Western blots. p53-HAT binding was detected by co-immunoprecipitation (co-IP). Cell growth inhibition was analyzed by MTT assays. LS8 cells were treated with NaF with/without HAT inhibitors MG149 (Tip60 inhibitor) and Anacardic Acid (AA; inhibits p300/CBP and PCAF). MG149 or AA was added 1 h prior to NaF treatment. Co-IP results showed that NaF increased p53-CBP binding and p53-PCAF binding. NaF increased active Acetyl-p300, Acetyl-CBP and phospho-Tip60 levels, suggesting that fluoride activates these HATs. Fluoride-induced phospho-Tip60 was decreased by MG149. MG149 or AA treatment reversed fluoride-induced cell growth inhibition at 24 h. MG149 or AA treatment decreased fluoride-induced p53 acetylation to inhibit caspase-3 cleavage, DNA damage marker γH2AX expression and cytochrome-c release into the cytosol. These results suggest that acetylation of p53 by HATs contributes, at least in part, to fluoride-induced toxicity in LS8 cells via cell growth inhibition, apoptosis, DNA damage and mitochondrial damage. Modulation of HAT activity may, therefore, be a potential therapeutic target to mitigate fluoride toxicity in ameloblasts.

Many physiological processes exhibit circadian rhythms driven by cellular clocks composed of interlinked activating and repressing elements. To investigate temporal regulation in this molecular oscillator, we combined mouse genetic approaches and analyses of interactions of key circadian proteins with each other and with clock gene promoters. We show that transcriptional activators control BRD4-PTEFb recruitment to E-box-containing circadian promoters. During the activating phase of the circadian cycle, the lysine acetyltransferase TIP60 acetylates the transcriptional activator BMAL1 leading to recruitment of BRD4 and the pause release factor P-TEFb, followed by productive elongation of circadian transcripts. We propose that the control of BRD4-P-TEFb recruitment is a novel temporal checkpoint in the circadian clock cycle.

E2F1 and retinoblastoma (RB) tumor-suppressor protein not only regulate the periodic expression of genes important for cell proliferation, but also localize to DNA double-strand breaks (DSBs) to promote repair. E2F1 is acetylated in response to DNA damage but the role this plays in DNA repair is unknown. Here we demonstrate that E2F1 acetylation creates a binding motif for the bromodomains of the p300/KAT3B and CBP/KAT3A acetyltransferases and that this interaction is required for the recruitment of p300 and CBP to DSBs and the induction of histone acetylation at sites of damage. A knock-in mutation that blocks E2F1 acetylation abolishes the recruitment of p300 and CBP to DSBs and also the accumulation of other chromatin modifying activities and repair factors, including Tip60, BRG1 and NBS1, and renders mice hypersensitive to ionizing radiation (IR). These findings reveal an important role for E2F1 acetylation in orchestrating the remodeling of chromatin structure at DSBs to facilitate repair., E2F1, which localises to DNA double-strand breaks (DSBs) to promote repair, is acetylated in response to DNA damage but the role this plays in DNA repair is unknown. Here the authors show that E2F1 acetylation creates a binding motif for the bromodomains of the p300/KAT3B and CBP/KAT3A acetyltransferases, which is required for recruitment of p300 and CBP to DSBs, to facilate repair.

UHRF2 is a ubiquitin-protein ligase E3 that regulates cell cycle, genomic stability and epigenetics. We conducted a co-immunoprecipitation assay and found that TIP60 and HDAC1 interact with UHRF2. We previously demonstrated that UHRF2 regulated H3K9ac and H3K14ac differentially in normal and cancer cells. However, the accurate signal transduction mechanisms were not clear. In this study, we found that TIP60 acted downstream of UHRF2 to regulate H3K9ac and H3K14ac expression. TIP60 is stabilized in normal cells by UHRF2 ubiquitination. However, TIP60 is destabilized in cancer cells. Depletion or inhibition of TIP60 disrupts the regulatory relationship between UHRF2, H3K9ac and H3K14ac. In summary, the findings suggest that UHRF2 mediated the post-translational modification of histones and the initiation and progression of cancer. Electronic supplementary material The online version of this article (doi:10.1007/s13238-016-0324-z) contains supplementary material, which is available to authorized users.

Chemotherapy-induced emergence of drug resistant cells is frequently observed and is exemplified by the expression of family of drug resistance proteins including, multidrug resistance protein 1 (MDR1). However, a concise mechanism for chemotherapy-induced MDR1 expression is unclear. Mechanistically, mutational selection, epigenetic alteration, activation of the Wnt pathway or impaired p53 function have been implicated. The present study describes that the surviving fraction of cisplatin resistant cells co- upregulate MDR1, BMI1 and acetyl transferase activity of TIP60. Using complementary gain and loss of function approaches, we demonstrate that the expression of MDR1 is positively regulated by BMI1, a stem-cell factor classically known as a transcriptional repressor. Our study establishes a functional interaction between TIP60 and BMI-1 resulting in upregulation of MDR1 expression. Chromatin immunoprecipitation (ChIP) assays further establish that the proximal MDR1 promoter responds to cisplatin in a BMI1 dependent manner. BMI1 interacts with a cluster of E-box elements on the MDR1 promoter and recruits TIP60 resulting in acetylation of histone H2A and H3. Collectively, our data establish a hitherto unknown liaison among MDR1, BMI1 and TIP60 and provide mechanistic insights into cisplatin-induced MDR1 expression resulting in acquired cross-resistance against paclitaxel, doxorubicin and likely other drugs. In conclusion, our results advocate utilizing anti-BMI1 strategies to alleviate acquired resistance to chemotherapy.

The dynamic exchange of histones alleviates the nucleosome barrier and simultaneously facilitates various aspects of cellular DNA metabolism, such as DNA repair and transcription. In response to DNA damage, the acetylation of Lys5 in the histone variant H2AX, catalyzed by TIP60, plays a key role in promoting histone exchange; however, the detailed molecular mechanism still is unclear. Here, we show that the TIP60 complex includes poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 is required for the rapid exchange of H2AX on chromatin at DNA damage sites. It is known that PARP-1 binds dynamically to damaged chromatin and is crucial for the subsequent recruitment of other repair factors, and its auto-poly(ADP-ribosyl)ation is required for the dynamics. We also show that the acetylation of histone H2AX at Lys5 by TIP60, but not the phosphorylation of H2AX, is required for the ADP-ribosylation activity of PARP-1 and its dynamic binding to damaged chromatin. Our results indicate the reciprocal regulation of K5 acetylation of H2AX and PARP-1, which could modulate the chromatin structure to facilitate DNA metabolism at damage sites. This could explain the rather undefined roles of PARP-1 in various DNA damage responses.

A new compaction pathway of mammalian pericentric heterochromatin is identified, which relies on H4K12ac by Tip60, probably followed by recruitment of BRD2, and therefore chromatin compaction, which can contribute to genetic stability., Pericentric heterochromatin is a highly compacted structure required for accurate chromosome segregation in mitosis. In mammals, it relies on methylation of histone H3K9 by Suv39H enzymes, which provides a docking site for HP1 proteins, therefore mediating heterochromatin compaction. Here we show that, when this normal compaction pathway is defective, the histone acetyltransferase Tip60 is recruited to pericentric heterochromatin, where it mediates acetylation of histone H4K12. Furthermore, in such a context, depletion of Tip60 leads to derepression of satellite transcription, decompaction of pericentric heterochromatin, and defects in chromosome segregation in mitosis. Finally, we show that depletion of BRD2, a double bromodomain–containing protein that binds H4K12ac, phenocopies the Tip60 depletion with respect to heterochromatin decompaction and defects in chromosome segregation. Taking the results together, we identify a new compaction pathway of mammalian pericentric heterochromatin relying on Tip60 that might be dependent on BRD2 recruitment by H4K12 acetylation. We propose that the underexpression of Tip60 observed in many human tumors can promote genetic instability via defective pericentric heterochromatin.

Faithful chromosome segregation in mammalian cells requires the bi-orientation of sister chromatids which relies on sensing correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation that triggers mitotic entry is extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via acetyltransferase TIP60 (Tat-interactive protein 60 kDa) in human cell division. CDK1-cyclin B phosphorylated Ser90 of TIP60, which elicited TIP60-dependent acetylation of Aurora B and promoted accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protected the phosphorylation of its activation loop from PP2A reactivation-elicited dephosphorylation to ensure a robust, error-free metaphase-anaphase transition. These findings delineated a conserved signaling cascade that integrates protein phosphorylation and acetylation to cell cycle progression for maintenance of genomic stability.

Summary: MBTD1, a H4K20me reader, has recently been identified as a component of the NuA4/TIP60 acetyltransferase complex, regulating gene expression and DNA repair. NuA4/TIP60 inhibits 53BP1 binding to chromatin through recognition of the H4K20me mark by MBTD1 and acetylation of H2AK15, blocking the ubiquitination mark required for 53BP1 localization at DNA breaks. The NuA4/TIP60 non-catalytic subunit EPC1 enlists MBTD1 into the complex, but the detailed molecular mechanism remains incompletely explored. Here, we present the crystal structure of the MBTD1-EPC1 complex, revealing a hydrophobic C-terminal fragment of EPC1 engaging the MBT repeats of MBTD1 in a site distinct from the H4K20me binding site. Different cellular assays validate the physiological significance of the key residues involved in the MBTD1-EPC1 interaction. Our study provides a structural framework for understanding the mechanism by which MBTD1 recruits the NuA4/TIP60 acetyltransferase complex to influence transcription and DNA repair pathway choice. : EPC1 is a critical component of the NuA4/TIP60 acetyltransferase complex involved in gene expression and genome maintenance. Zhang et al. illuminate the structural and molecular basis for EPC1 bound to the H4K20me reader MBTD1, highlighting the important role of their association in gene transcription and DNA repair. Keywords: MBTD1, EPC1, NuA4, TIP60, H4K20me, DNA repair, crystal structure

Chromatin remodelers regulate the nucleosome barrier during transcription, DNA replication, and DNA repair. The chromatin remodeler RSF1 is enriched at mitotic centromeres, but the functional consequences of this enrichment are not completely understood. Shugoshin (Sgo1) protects centromeric cohesion during mitosis and requires BuB1-dependent histone H2A phosphorylation (H2A-pT120) for localization. Loss of Sgo1 at centromeres causes chromosome missegregation. Here, we show that RSF1 regulates Sgo1 localization to centromeres through coordinating a crosstalk between histone acetylation and phosphorylation. RSF1 interacts with and recruits HDAC1 to centromeres, where it counteracts TIP60-mediated acetylation of H2A at K118. This deacetylation is required for the accumulation of H2A-pT120 and Sgo1 deposition, as H2A-K118 acetylation suppresses H2A-T120 phosphorylation by Bub1. Centromeric tethering of HDAC1 prevents premature chromatid separation in RSF1 knockout cells. Our results indicate that RSF1 regulates the dynamics of H2A histone modifications at mitotic centromeres and contributes to the maintenance of chromosome stability.

Anaplastic thyroid cancer (ATC) is an aggressive cancer with poor clinical prognosis. However, mechanisms driving ATC aggressiveness is not well known. Components of the DNA damage response (DDR) are frequently found mutated or aberrantly expressed in ATC. The goal of this study is to establish the functional link between histone acetyltransferase lysine (K) acetyltransferase 5 (KAT5, a critical DDR protein) and ATC invasiveness using clinical, in vitro and in vivo models. We analyzed the expression of KAT5 by immunohistochemistry and assessed its relationship with metastasis and overall survival in 82 ATC patients. Using cellular models, we established functional connection of KAT5 expression and C-MYC stabilization. We then studied the impact of genetically modified KAT5 expression on ATC metastasis in nude mice. In clinical samples, there is a strong correlation of KAT5 expression with ATC metastasis (P = 0.0009) and overall survival (P = 0.0017). At the cellular level, upregulation of KAT5 significantly promotes thyroid cancer cell proliferation and invasion. We also find that KAT5 enhances the C-MYC protein level by inhibiting ubiquitin-mediated degradation. Further evidence reveals that KAT5 acetylates and stabilizes C-MYC. Finally, we prove that altered KAT5 expression influences ATC lung metastases in vivo. KAT5 promotes ATC invasion and metastases through stabilization of C-MYC, demonstrating it as a new biomarker and therapeutic target for ATC.

Most tumor cells take up more glucose than normal cells. Splicing dysregulation is one of the molecular hallmarks of cancer. However, the role of splicing factor in glucose metabolism and tumor development remains poorly defined. Here, we show that upon glucose intake, the splicing factor SRSF5 is specifically induced through Tip60-mediated acetylation on K125, which antagonizes Smurf1-mediated ubiquitylation. SRSF5 promotes the alternative splicing of CCAR1 to produce CCAR1S proteins, which promote tumor growth by enhancing glucose consumption and acetyl-CoA production. Conversely, upon glucose starvation, SRSF5 is deacetylated by HDAC1, and ubiquitylated by Smurf1 on the same lysine, resulting in proteasomal degradation of SRSF5. The CCAR1L proteins accumulate to promote apoptosis. Importantly, SRSF5 is hyperacetylated and upregulated in human lung cancers, which correlates with increased CCAR1S expression and tumor progression. Thus, SRSF5 responds to high glucose to promote cancer development, and SRSF5–CCAR1 axis may be valuable targets for cancer therapeutics., Changes in glucose metabolism can lead to tumor development, but the involvement of splicing factors is unclear. Here, the authors screened for SR proteins and identified SRSF5 stability is enhanced in response to glucose elevation to promote alternative splicing of CCAR1 which facilitates tumor growth.

The acetylation of p53 plays an essential role in regulating its transcriptional activity. Nevertheless, how p53 acetylation is modulated in cell fate decision is less understood. We developed a network model to reveal how Tip60-dependent p53 acetylation is regulated to modulate cellular outcome. We proposed that p53 is progressively activated and exhibits distinct dynamics depending on the severity of DNA damage. For mild damage, p53 is primarily activated to trigger cell cycle arrest by transactivating p21, with its concentration showing pulses. For severe damage, p53 is acetylated at Lysine 120 (K120) by Tip60 to trigger apoptosis by inducing PUMA, and its concentration increases to high levels. Several p53-centered feedback loops coordinate to regulate its acetylation status, ensuring a robust decision on cell fate.