Histone Acetyltransferases PCAF Research Articles

Single-Cell Analysis of Histone Acetylation Dynamics at Replication Forks Using PLA and SIRF.

Genome integrity is constantly challenged by various processes including DNA damage, structured DNA, transcription, and DNA-protein crosslinks. During DNA replication, active replication forks that encounter these obstacles can result in their stalling and collapse. Accurate DNA replication requires the ability of forks to navigate these threats, which is aided by DNA repair proteins. Histone acetylation participates in this process through an ability to signal and recruit proteins to regions of replicating DNA. For example, the histone acetyltransferase PCAF promotes the recruitment of the DNA repair factors MRE11 and EXO1 to stalled forks by acetylating histone H4 at lysine 8 (H4K8ac). These highly dynamic processes can be detected and analyzed using a modified proximity ligation assay (PLA) method, known as SIRF (in situ protein interactions with nascent DNA replication forks). This single-cell assay combines PLA with EdU-coupled Click-iT chemistry reactions and fluorescence microscopy to detect these interactions at sites of replicating DNA. Here we provide a detailed protocol utilizing SIRF that detects the HAT PCAF and histone acetylation at replication forks. This technique provides a robust methodology to determine protein recruitment and modifications at the replication fork with single-cell resolution.

Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

Oncogenic signaling pathway reprograms cancer cell metabolism to promote aerobic glycolysis in favor of tumor growth. The ability of cancer cells to evade immunosurveillance and the role of metabolic regulators in T-cell functions suggest that oncogene-induced metabolic reprogramming may be linked to immune escape. Notch1 signaling, dysregulated in lung cancer, is correlated with increased glycolysis. Herein, we demonstrate in lung cancer that Notch1 promotes glycolytic gene expression through functional interaction with histone acetyltransferases p300 and pCAF. Notch1 signaling forms a positive feedback loop with TAZ. Notch1 transcriptional activity was increased in the presence of TAZ and the activation was TEAD1 independent. Notably, aerobic glycolysis was critical for Notch1/TAZ axis modulation of lung cancer growth in vitro and in vivo. Increased level of extracellular lactate via Notch1/TAZ axis inhibited cytotoxic T-cell activity, leading to the invasive characteristic of lung cancer cells. Interaction between Notch1 and TAZ promoted aerobic glycolysis and immune escape in lung cancer. Our findings provide potential therapeutic targets against Notch1 and TAZ and would be important for clinical translation in lung cancer.

Carnosol Is a Novel Inhibitor of p300 Acetyltransferase in Breast Cancer.

Carnosol, a natural polyphenol abundant in edible plants such as sage, rosemary, and oregano, has shown promising anticancer activity against various types of cancers. Nonetheless, very little is known about its molecular mechanism of action or its downstream target(s). We have previously shown that carnosol inhibits cellular proliferation, migration, invasion, and metastasis as well as triggers autophagy and apoptosis in the highly invasive MDA-MB-231 breast cancer cells. Here, we report that carnosol induces histone hypoacetylation in MDA-MB-231 and Hs578T breast cancer cells. We show that, while carnosol does not affect HDACs, it promotes a ROS-dependent proteasome degradation of p300 and PCAF histone acetyl transferases (HATs) without affecting other HATs such as GCN5 and hMOF. Carnosol-induced histone hypoacetylation remains persistent even when p300 and PCAF protein levels were rescued from degradation by (i) the inhibition of the proteasome activity by the proteasome inhibitors MG-132 and bortezomib, and (ii) the inhibition of ROS accumulation by the ROS scavenger, N-acetylcysteine. In addition, we report that, in a cell-free system, carnosol efficiently inhibits histone acetyltransferase activity of recombinant p300 but not that of PCAF or GCN5. Molecular docking studies reveal that carnosol inhibits p300 HAT activity by blocking the entry of the acetyl-CoA binding pocket of the catalytic domain. The superimposition of the docked conformation of the p300 HAT domain in complex with carnosol shows a similar orientation as the p300 structure with acetyl-CoA. Carnosol occupies the region where the pantetheine arm of the acetyl-CoA is bound. This study further confirms carnosol as a promising anti-breast cancer therapeutic compound and identifies it as a novel natural p300 inhibitor that could be added to the existing panel of inhibitors.

PCAF-Mediated Histone Acetylation Promotes Replication Fork Degradation by MRE11 and EXO1 in BRCA-Deficient Cells

Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA-deficient tumors. Epigenetic mechanisms of replication fork stability are emerging but remain poorly understood. Here, we report the histone acetyltransferase PCAF (p300/CBP-associated) as a fork-associated protein that promotes fork degradation in BRCA-deficient cells by acetylating H4K8 at stalled replication forks, which recruits MRE11 and EXO1. A H4K8ac binding domain within MRE11/EXO1 is required for their recruitment to stalled forks. Low PCAF levels, which we identify in a subset of BRCA2-deficient tumors, stabilize stalled forks, resulting in PARPi resistance in BRCA-deficient cells. Furthermore, PCAF activity is tightly regulated by ATR (ataxia telangiectasia and Rad3-related), which phosphorylates PCAF on serine 264 (S264) to limit its association and activity at stalled forks. Our results reveal PCAF and histone acetylation as critical regulators of fork stability and PARPi responses in BRCA-deficient cells, which provides key insights into targeting BRCA-deficient tumors and identifying epigenetic modulators of chemotherapeutic responses.

Abstract LB-184: A lncRNA-histone acetyltransferase complex induces colorectal oncogenesis through regulation of a metabolic kinase network

Abstract Long non-coding RNAs (LncRNAs) through interactions with other cellular macromolecules drive cell processes contributing to important cancer phenotypes. Here we show that HOTAIR, a highly up-regulated lncRNA in human colon cancers, induces transformation of human non-transformed colon epithelial cells. An exon 6 HOTAIR region directly binds the histone acetyltransferase PCAF (KAT2B). The HOTAIR/PCAF complex regulates H3K27 acetylation and transcriptional activation preferentially of genes encoding components of the PI3K/AKT/MEK pathways and induces cell metabolic reprogramming. The transcriptional signature of this HOTAIR/PCAF metabolic circuit is active in colon cancer and correlates with overall patient survival. Topological and mechanistic analyses were employed to characterize the HOTAIR/PCAF interface and design a reversible specific inhibitor (CPDI-1). Selective blocking of the HOTAIR-PCAF interaction, suppresses AKT activation and tumor growth. In conclusion, we demonstrate that disruption of a lncRNA-histone modifier complex interferes with the cancer epigenome and represents a novel approach for cancer therapeutics. Citation Format: Christos Polytarchou, Maria Hatziapostolou, Marina Koutsioumpa, Niki Christodoulou, Tung On Yau, Eleni Birli, Odette Pomenya, Cristina Montiel-Duarte, Swapna Mahurkar Joshi, Daniel W. Hommes, Hein W. Verspaget, Jun Yu, Dimitrios Iliopoulos. A lncRNA-histone acetyltransferase complex induces colorectal oncogenesis through regulation of a metabolic kinase network [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-184.

The Expression and Localization of Histone Acetyltransferases HAT1 and PCAF in Neurons and Astrocytes of the Photothrombotic Stroke-Induced Penumbra in the Rat Brain Cortex.

Stroke is one of the leading reasons of human death. Ischemic penumbra that surrounds the stroke-induced infarction core is potentially salvageable, but molecular mechanisms of its formation are poorly known. Histone acetylation induces chromatin decondensation and stimulates gene expression. We studied the changes in the levels and localization of histone acetyltransferases HAT1 and PCAF in penumbra after photothrombotic stroke (PTS, a stroke model). In PTS, laser irradiation induces local occlusion of cerebral vessels after photosensitization by Rose Bengal. HAT1 and PCAF are poorly expressed in normal cortical neurons and astrocytes, but they are overexpressed 4-24h after PTS. Their predominant localization in neuronal nuclei did not change after PTS, but their levels in the astrocyte nuclei significantly increased. Western blotting showed the increase of HAT1 and PCAF levels in the cytoplasmic fraction of the PTS-induced penumbra. In the nuclear fraction, PCAF level did not change, and HAT1 was overexpressed only at 24h post-PTS. PTS-induced upregulation of HAT1 and PCAF in the penumbra was mainly associated with overexpression in the cytoplasm of neurons and especially astrocytes. HAT1 and PCAF did not co-localize with TUNEL-positive cells that indicated their nonparticipation in PTS-induced apoptosis.

Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation.

Promoter-associated long non-coding RNAs (lncRNAs) regulate the expression of adjacent genes; however, precise roles of these lncRNAs in skeletal muscle remain largely unknown. Here, we characterize a promoter-associated lncRNA, Myoparr, in myogenic differentiation and muscle disorders. Myoparr is expressed from the promoter region of the mouse and human myogenin gene, one of the key myogenic transcription factors. We show that Myoparr is essential both for the specification of myoblasts by activating neighboring myogenin expression and for myoblast cell cycle withdrawal by activating myogenic microRNA expression. Mechanistically, Myoparr interacts with Ddx17, a transcriptional coactivator of MyoD, and regulates the association between Ddx17 and the histone acetyltransferase PCAF Myoparr also promotes skeletal muscle atrophy caused by denervation, and knockdown of Myoparr rescues muscle wasting in mice. Our findings demonstrate that Myoparr is a novel key regulator of muscle development and suggest that Myoparr is a potential therapeutic target for neurogenic atrophy in humans.

PCAF-mediated acetylation of Lin28B increases let-7 biogenesis in lung adenocarcinoma H1299 cells

BackgroundLin28B and its paralog Lin28A are small RNA binding proteins that have similar inhibitory effects, although they target separate steps in the maturation of let-7 miRNAs in mammalian cells. Because Lin28B participates in the promotion and development of tumors mostly by blocking the let-7 tumor suppressor family members, we sought to explore the associated mechanisms to gain insights into how Lin28B might be decreased in human cancer cells to increase let-7 levels and reverse malignancy.ResultsWe demonstrated that the histone acetyltransferase PCAF, via its cold shock domain, directly interacts with and subsequently acetylates Lin28B in lung adenocarcinoma-derived H1299 cells. RT-qPCR assays showed that both let-7a-1 and let-7g were increased in PCAF-transfected H1299 cells. Lin28B is acetylated by ectopic PCAF and translocates from the nucleus to the cytoplasm in H1299 cells.ConclusionsThe effects of acetylated Lin28B on let-7a-1 and let-7g are similar to that of stable knockdown of Lin28B in H1299 cells. The new role of PCAF in mediating Lin28B acetylation and the specific release of its target microRNAs in H1299 cells may shed light on the potential application of let-7 in the clinical treatment of lung cancer patients.

Induction of NKG2D Ligands on Solid Tumors Requires Tumor-Specific CD8+ T Cells and Histone Acetyltransferases.

NKG2D-mediated immune surveillance is crucial for inhibiting tumor growth and metastases. Malignant tumor cells often downregulate NKG2D ligands to escape from immune surveillance. High-profile studies have shown that restoring NKG2D ligand expression via genetic engineering inhibits tumor formation and progression. However, no effective in vivo approaches are available to restore these ligands across different types of solid tumors because the classic stress signal-dependent induction of this ligand in vitro is transient and has rarely been duplicated in solid tumors in vivo We found that coadministration of an immune stimulatory signal (IL12) and chemotherapy (doxorubicin) restored the NKG2D ligand Rae-1 in multiple tumor types, including a human tumor model. The restored expression of NKG2D ligands was associated with tumor cell death and delay of tumor progression in vivo Induction of tumor-specific NKG2D ligands required the engagement of CD8+ T cells and was regulated by the histone acetyltransferases GCN5 and PCAF. The tumor-specific restoration of NKG2D ligands in a variety of tumor models, including a human tumor model, resulted in NKG2D-dependent tumor regression and extended survival time. The elucidation of a CD8+ T cell-dependent mechanism suggests that activated NKG2D+CD8+ T-cell therapy alone may be able to restore the NKG2D ligand in tumors. Cancer Immunol Res; 5(4); 300-11. ©2017 AACR.

Abstract A21: The role of SOX4 in epithelial to mesenchymal transition in prostate cancer

Abstract Prostate cancer remains the most commonly diagnosed cancer in U.S. males, and ranks second in mortality with over 28,000 deaths per year. Prostate cancer mortality is typically due to metastases, necessitating the understanding of how metastatic disease develops. One major step for cancer cells to metastasize and acquire migratory and invasive capabilities is the epithelial to mesenchymal transition (EMT). EMT encompasses vast molecular changes in gene expression, mediated by aberrant developmental signaling pathway activation, leading to the loss of cell-cell junctions and planar and apical-basal polarity, increased motility, and resistance to apoptosis and anoikis (cell death due to the detachment from the extracellular matrix). The sex determining region Y-box 4 (SOX4) gene is a developmental transcription factor that is over expressed in prostate cancer and plays a critical role in many developmental pathways inappropriately activated during EMT, such as the transforming growth factor (TGF-beta) and epidermal growth factor receptor (EGFR) signaling pathways. Our data suggest that concomitant treatment of TGF-beta and EGF induces SOX4, and initiates the EMT program in prostate epithelial cell lines. We also demonstrate that SOX4 over expression is sufficient to induce EMT in prostate epithelial cell lines. Currently, the molecular mechanisms that govern how SOX4 initiates EMT via transcriptional activation of target genes are poorly understood. We identified candidate SOX4 targets using the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE) on 9755 Pan Cancer samples from The Cancer Genome Atlas (TCGA). Gene ontology analysis of the top 1000 candidate SOX4 targets revealed genes that were enriched for epigenetic processes such as chromatin modification and DNA methylation. One putative target gene, PCAF, is a subunit of a large histone acetyltransferase (HAT) complex. Interestingly, our preliminary data also suggest that SOX4 interacts not only with PCAF, but also with TRRAP, another subunit of the PCAF HAT complex. These data suggest that SOX4 may induce and recruit the PCAF/TRRAP HAT complex in order to transcriptionally activate downstream targets to initiate the EMT program. Citation Format: Nitya V. Sharma, Soma Sannigrahi, YuHeng Lai, Carlos Moreno. The role of SOX4 in epithelial to mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A21.

Nuclear myosin 1 contributes to a chromatin landscape compatible with RNA polymerase II transcription activation.

BackgroundNuclear myosin 1c (NM1) is emerging as a regulator of transcription and chromatin organization.ResultsUsing chromatin immunoprecipitation and deep sequencing (ChIP-Seq) in combination with molecular analyses, we investigated the global association of NM1 with the mammalian genome. Analysis of the ChIP-Seq data demonstrates that NM1 binds across the entire mammalian genome with occupancy peaks correlating with distributions of RNA Polymerase II (Pol II) and active epigenetic marks at class II gene promoters. In mouse embryonic fibroblasts subjected to RNAi mediated NM1 gene silencing, we show that NM1 synergizes with polymerase-associated actin to maintain active Pol II at the promoter. NM1 also co-localizes with the nucleosome remodeler SNF2h at class II promoters where they assemble together with WSTF as part of the B-WICH complex. A high resolution micrococcal nuclease (MNase) assay and quantitative real time PCR shows that this mechanism is required for local chromatin remodeling. Following B-WICH assembly, NM1 mediates physical recruitment of the histone acetyl transferase PCAF and the histone methyl transferase Set1/Ash2 to maintain and preserve H3K9acetylation and H3K4trimethylation for active transcription.ConclusionsWe propose a novel genome-wide mechanism where myosin synergizes with Pol II-associated actin to link the polymerase machinery with permissive chromatin for transcription activation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0147-z) contains supplementary material, which is available to authorized users.

Posttranslational Modification Cross Talk Regulates the Critical Adaptive Immune Molecule, The Class II Transactivator

Major histocompatibility class II (MHC II) molecules present pathogenic and tumorigenic antigens to CD4+T cells and play vital roles in regulating adaptive immune responses. MHC II is controlled at the level of transcription by the Class II Transactivator (CIITA), which in turn is regulated through a series of posttranslational modifications (PTMs). Acetylation of CIITA by the histone acetyltransferase (HAT) pCAF, results in CIITA nuclear localization. CIITA is also phosphorylated by Extracellular Signal Regulated Kinase (ERK1/2) at S280, a critical site for regulating additional PTMs such as mono‐ubiquitination. Both phosphorylation and mono‐ubiquitination is key in CIITA's stability and increased transactivation. We have identified that phosphorylation of CIITA by ERK1/2 enhances K63 linked ubiquitination leading to CIITA's ability to bind to the MHC II promoter. pCAF was recently identified as an E3 ligase for the nuclear protein Mdm2. We show here pCAF increases both CIITA transactivation and ubiquitination; suggesting pCAF also plays a role in regulating CIITA ubiquitination whether through its HAT or E3 ligase capability has yet to be elucidated. These data provide novel links between acetylation, phosphorylation and ubiquitination of this critical inflammatory protein. Our data suggests particular posttranslational modifications of CIITA result in very different functional outcomes, and may enable the identification of biomarkers for immune diseases associated with dysregulated antigen presentation. Support for research provided by Molecular Basis of Disease area of Focus, Georgia State University

Histone acetyltransferase PCAF accelerates apoptosis by repressing a GLI1/BCL2/BAX axis in hepatocellular carcinoma.

P300/CBP-associated factor (PCAF), a histone acetyltransferase (HAT), has been found to regulate numerous cell signaling pathways controlling cell fate by acetylating both histone and non-histone proteins. We previously reported that PCAF upregulates cell apoptosis by inactivating Serine/Threonine Protein Kinase 1 (AKT1) signaling and consequently inhibits hepatocellular carcinoma (HCC) cell growth. Here, we show that PCAF can directly acetylate cytoplasmic GLI1 protein at lysine 518, preventing its nuclear translocation and promoter occupancy, and consequently suppressing Hedgehog (Hh) signaling in HCC. Further, our results show that GLI1 can increase Bcl-2 expression and downregulate BAX. Interestingly, forced expression of PCAF reduced Bcl-2 expression, upregulated BAX and repressed cell apoptosis. Further, we provide evidence that knockdown of GLI1 abrogates the inhibitory effect of PCAF on the growth of HCC in vitro. PCAF was also found to sensitize HCC cells to 5-fluorouracil (5-FU) treatment by regulating GLI1/Bcl-2/BAX axis-dependent apoptosis. In vivo experiments also confirmed the regulatory effect of PCAF on the GLI1/Bcl-2/BAX axis and its synergistic antitumor effects with 5-FU. Gene expression microarray studies showed that PCAF was downregulated in HCC tissues compared with adjacent liver tissues and that PCAF expression was significantly associated with longer overall survival and recurrence-free survival after surgery. Together, these results show that PCAF can induce cell apoptosis by modulating a GLI1/Bcl-2/BAX axis that in turn suppresses HCC progression, and suggest that 5-FU may exert a stronger anti-tumor effect in patients with PCAF expression in HCC tumors.

Targeting colon cancer metabolism through a long noncoding RNA.

604 Background: Long non-coding RNAs (lncRNAs) have gained massive attention in recent years as a potentially new and crucial layer of gene regulation. We studied the function of the lncRNA HOTAIR in colorectal cancer, one of the most common causes of cancer mortality worldwide. Methods: Molecular profiling was performed in colonic normal (40) and cancer (160) tissues. Gene expression was evaluated by qRT-PCR and gene expression arrays. LncRNA-protein interactions were investigated by RNA-immunoprecipitation, mass spectrometry and mutational analyses. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) was employed for the histone acetylome analysis. Subcutaneous xenograft mouse models were used to evaluate tumor growth in vivo. Results: HOTAIR is significantly upregulated (>30 fold) in colon cancer and inversely correlates with patient survival. Molecular profiling revealed a gene signature downstream of HOTAIR that links metabolic pathways to colon cancer cell growth and invasiveness. RNA immunoprecipitation/mass spectrometry analysis revealed the interaction of HOTAIR with the histone acetyltransferase PCAF. Mutational analyses identified the domains of both the lincRNA and acetyltransferase responsible for their interaction and function. Histone acetylome analysis demonstrated that HOTAIR is required for the recruitment and activity of PCAF towards a set of genes involved in the mTOR/insulin receptor/PI3K-Akt pathways. In specific, 44 genes involved in these pathways are regulated by Lysine 27 histone H3 acetylation, catalyzed by PCAF only when recruited by HOTAIR. Genome-wide profiling of colon cancer and normal colonic tissues revealed that the expression of this gene set correlates positively with HOTAIR levels and inversely with patient survival. Knockdown of either HOTAIR or PCAF sensitized colon cancer cells to dietary restriction and inhibited tumor growth in subcutaneous xenograft mouse models. Conclusions: Our results provide the mechanistic link between an lncRNA, a transcription activating chromatin mark and metabolic signaling in colorectal cancer. In conclusion, we identified a novel RNA-protein complex that affects growth factor/metabolic pathways essential for colon oncogenesis.

Histone Acetyltransferase Pcaf Is Required for ATRA-Induced Granulocytic Differentiation in Acute Promyelocytic Leukemia Cells

Differentiation therapy with All-trans retinoic acid (ATRA) improves the treatment outcome of acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML); however, its molecular mechanism remains elusive. We have previously reported that inhibition of NAD-dependent histone deacetylase SIRT2 induces granulocytic differentiation in APL cells (PLOS ONE. 2013; 8(2): e57633), suggesting a possible involvement of protein acetylation in the differentiation of APL cells by ATRA.To assess this possibility, in the present study, we examined expression of major histone-acetyltrasnferases such as GCN5, PCAF, and CBP/p300 upon ATRA-treatment, and found that PCAF-expression was dramatically increased at mRNA (1.8 to 7 fold, Figure 1A) as well as protein levels in NB4, HL-60, and APL primary cells. Consistent with this, PCAF mRNA expression was greatly induced (10 to 40 fold) in the bone marrow of APL patients who received ATRA-containing treatment (Figure 1B). A search in the public expression database revealed that PCAF-expression in bone marrow was generally reduced in not only APL but also other acute hematologic malignancies such as AML and acute lymphoblastic leukemia (ALL) (healthy donor; n = 74, APL; n=37, AML; n=505, ALL; n=750, healthy donor vs. AML, p < 0.001, healthy donor vs. APL, p < 0.001, healthy donor vs. ALL, p < 0.001, adopted from oncomine database). Interestingly, the reduction of PCAF-expression was much more evident in acute than chronic hematologic malignancies such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS) (CML; n = 76, MDS; n=206), suggesting that PCAF reduction is related to a blockade of proper differentiation in malignant cells. Based on these findings we hypothesized that PCAF plays an important role in ATRA-induced APL cell differentiation.To prove this, we performed a loss-of-function assay using lentivirus vectors expressing 3 independent shRNA against PCAF or non-targeting shRNA (control) (Figure 1C). When PCAF was knocked down in HL-60 cells, these cells failed to differentiate into granulocytes with ATRA-treatment (31.9±11.9% vs. 4.9±2.2%, 5.2±0.7%, 8.6±3.7%, CD11b positive cells, control vs. 3 independent PCAF shRNA) (Figure 1C). Moreover, transduction of PCAF shRNA or non-target shRNA to primary APL cells made cells resistant to ATRA-induced granulocytic differentiation (data not shown), suggesting that PCAF is required for the ATRA-induced granulocytic differentiation in APL cells. To further investigate how PCAF promotes APL cell differentiation upon ATRA-treatment, we performed an acetylome analysis to identify a downstream molecule whose activity is regulated by PCAF through acetylation. We pulled-down acetylated proteins using anti-acetylated lysine antibody from cell extracts prepared 3 or 24 hours after either mock- or ATRA-treatment, and identified 5 proteins preferentially acetylated by ATRA-treatment including histone H3, which is a known acetylation target of PCAF. These results strongly support our hypothesis that upon ATRA-treatment, PCAF induction and subsequent acetylation of PCAF substrates promotes APL cell differentiation. More detailed understanding of PCAF-dependent APL cell differentiation may lead to the development of differentiation therapy against not only APL but also other AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Redox-sensitive gene-regulatory events controlling aberrant matrix metalloproteinase-1 expression

Aberrant matrix metalloproteinase-1 (MMP-1) expression contributes to the pathogenesis of many degenerative disease processes that are associated with increased oxidative damage or stress. We and others have established that shifts in steady-state H2O2 production resulting from enforced antioxidant gene expression, senescence, or UV irradiation control MMP-1 expression. Here we establish that histone deacetylase-2 (HDAC2) protein levels and its occupancy of the MMP-1 promoter are decreased in response to enforced manganese superoxide dismutase (Sod2) expression. Inhibition of HDAC activity further accentuates the redox-dependent expression of MMP-1. Sod2-dependent decreases in HDAC2 are associated with increases in a proteasome-sensitive pool of ubiquitinylated HDAC2 and MMP-1-specific histone H3 acetylation. Sod2 overexpression also enhanced recruitment of Ets-1, c-Jun, c-Fos, and the histone acetyltransferase PCAF to the distal and proximal regions of the MMP-1 promoter. Furthermore, the Sod2-dependent expression of MMP-1 can be reversed by silencing the transcriptional activator c-Jun. All of the above Sod2-dependent alterations are largely reversed by catalase coexpression, indicating that the redox control of MMP-1 is H2O2-dependent. These findings identify a novel redox regulation of MMP-1 transcription that involves site-specific promoter recruitment of both activating factors and chromatin-modifying enzymes, which converge to maximally drive MMP-1 gene expression.

Combinatorial action of transcription factors orchestrates cell cycle‐dependent expression of the ribosomal protein genes and ribosome biogenesis

Nucleolar assembly begins at the early G1 phase of the cell cycle and is a hub of ribosomal DNA transcription and rRNA biosynthesis. The newly-formed rRNAs together with ribosomal proteins (RPs) constitute the building block of the ribosomal machinery. Although RPs play a major role in protein biosynthesis, their own regulation and expression is rather poorly understood. In the present study, we investigated the regulation of RP genes RPS27a, RPS24, RPS6, RPL9 and RPL4 in synchronized mammalian cell culture. Quantitative RT-PCR analysis indicated their expression during the mid to late G1 phase, whereas the rRNA genes were expressed during the early G1 phase of the cell cycle. The promoter reporter analysis of the RPS27a gene revealed that it could be synergistically stimulated by the transcription factors specificity protein 1 (Sp1) and cAMP response element-binding protein (CREB). However, E2F transcription factor 1 (E2F1) appeared to negatively regulate gene expression. Chromatin immunoprecipitation studies confirmed the promoter occupancy of Sp1, CREB and E2F1. Although Sp1 and CREB binding enhanced the promoter occupancy of histone acetyltransferases PCAF, p300 and CREB binding protein, E2F1 facilitated the recruitment of histone deacetylases. Both acetylation (histone H4 pan-acetyl, histone H3 acetyl Lys 14) and methylation (histone H3 trimethyl Lys 9) marks were observed in the RPS27a promoter region, suggesting their important regulatory role in gene expression. Because the promoter regions of most RP genes are well conserved, we propose that their orchestrated regulation and synthesis during the cell cycle facilitates ribosome biogenesis.

Pulling a ligase out of a “HAT” (555.13)

Major histocompatibility class II (MHC II) molecules present pathogenic and tumorigenic antigens to CD4+T cells and play vital roles in regulating adaptive immune responses. MHC II is controlled at the level of transcription by the Class II Transactivator (CIITA) which is in turn regulated through a series of sequential posttranslational modification. Acetylation of CIITA by the histone acetyltransferase (HAT) pCAF, results in CIITA nuclear localization. Once nuclear, CIITA is ubiquitinated and binds the MHC II promoter. In addition to its HAT activity, pCAF was recently identified as an E3 ligase for the nuclear phospho protein Mdm2. We show here pCAF increases CIITA transactivation and ubiquitination; suggesting pCAF also drives CIITA ubiquitination. We have previously linked CIITA phosphorylation with subsequent mono‐ubiquitination and increased CIITA transactivation. We now further identify K63 linked ubiquitination of CIITA and provide novel links between acetylation, phosphorylation and ubiquitination of this critical inflammatory protein. Our data suggests particular posttranslational modifications of CIITA result in very different functional outcomes, and may enable the identification of biomarkers for immune diseases associated with dysregulated antigen presentation.

Azide–alkyne cycloaddition affording enzymatically tunable bisubstrate based inhibitors of histone acetyltransferase PCAF

A novel strategy to prepare bisubstrate based inhibitors for histone acetyltransferases is presented. To obtain these, azido peptides derived from histone H3 incorporating either a serine or a phosphoserine residue were connected to a propargyl coenzyme A derivative through copper catalyzed click chemistry. The resulting inhibitors were tested with therapeutically relevant acetyltransferase PCAF. Increased potency of the phosphoserine containing inhibitor was observed. The synthetic strategy presented may be used for developing bisubstrate based inhibitors against any acetyltransferase.

Combinatorial H3K9acS10ph Histone Modification in IgH Locus S Regions Targets 14-3-3 Adaptors and AID to Specify Antibody Class-Switch DNA Recombination

Class-switch DNA recombination (CSR) is central to the antibody response, in that it changes the immunoglobulin heavy chain (IgH) constant region, thereby diversifying biological effector functions of antibodies. The activation-induced cytidine deaminase (AID)-centered CSR machinery excises and rejoins DNA between an upstream (donor) and a downstream (acceptor) S region, which precede the respective constant region DNA. AID is stabilized on S regions by 14-3-3 adaptors. These adaptors display a high affinity for 5'-AGCT-3' repeats, which recur in all S regions. However, how 14-3-3, AID, and the CSR machinery target exclusively the donor and acceptor S regions is poorly understood. Here, we show that histone methyltransferases and acetyltransferases are induced by CD40 or Toll-like receptor signaling and catalyze H3K4me3 and H3K9ac/K14ac histone modifications, which are enriched in S regions but do not specify the S region targets of CSR. By contrast, the combinatorial H3K9acS10ph modification specifically marks the S regions set to recombine and directly recruits 14-3-3 adaptors for AID stabilization there. Inhibition of the enzymatic activity of GCN5 and PCAF histone acetyltransferases reduces H3K9acS10ph in S regions, 14-3-3 and AID stabilization, and CSR. Thus, H3K9acS10ph is a histone code that is "written" specifically in S regions and is "read" by 14-3-3 adaptors to target AID for CSR as an important biological outcome.