Component Of Histone Acetyltransferase Complex Research Articles

Loss of OsEAF6, a Subunit of the Histone Acetyltransferase Complex, Causes Hybrid Breakdown in Intersubspecific Rice Crosses.

Gene duplication plays an important role in genetic diversification, adaptive evolution, and speciation. Understanding the mechanisms and effects of postzygotic isolation genes is important for further studies of speciation and crop breeding. The duplicate recessive genes hwe1 and hwe2 cause hybrid breakdown, characterized by poor vegetative growth and reproductive dysgenesis in intersubspecific crosses between Oryza sativa ssp. indica and japonica. Using a map-based cloning strategy, we found that HWE1 and HWE2 encode the Esa1-associated factor 6 (EAF6) protein, a component of histone acetyltransferase complexes. The indica hwe1 and japonica hwe2 alleles lacked functional EAF6, demonstrating that the double recessive homozygote causes hybrid breakdown. Morphological and physiological observations showed that weak plants with double recessive homozygotes had serious morphological defects with a wide range of effects on development and organs, leading to leaves with reduced chlorophyll content, flower and pistil malformation, and anomalies of gametogenesis. These findings suggest that EAF6 plays a pivotal role in the transcriptional regulation of essential genes during the vegetative and reproductive development of rice.

TRRAP stimulates the tumorigenic potential of ovarian cancer stem cells

Ovarian cancer is the most fatal gynecological malignancy in women and identification of new therapeutic targets is essential for the continued development of therapy for ovarian cancer. TRRAP (transformation/transcription domain-associated protein) is an adaptor protein and a component of histone acetyltransferase complex. The present study was undertaken to investigate the roles played by TRRAP in the proliferation and tumorigenicity of ovarian cancer stem cells. TRRAP expression was found to be up-regulated in the sphere cultures of A2780 ovarian cancer cells. Knockdown of TRRAP significantly decreased cell proliferation and the number of A2780 spheroids. In addition, TRRAP knockdown induced cell cycle arrest and increased apoptotic percentages of A2780 sphere cells. Notably, the mRNA levels of stemness-associated markers, that is, OCT4, SOX2, and NANOG, were suppressed in TRRAP-silenced A2780 sphere cells. In addition, TRRAP overexpression increased the mRNA level of NANOG and the transcriptional activity of NANOG promoter in these cells. Furthermore, TRRAP knockdown significantly reduced tumor growth in a murine xenograft transplantation model. Taken together, the findings of the present study suggest that TRRAP plays an important role in the regulation of the proliferation and stemness of ovarian cancer stem cells.

Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation.

Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442-29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation.

The BRPF2/BRD1-MOZ complex is involved in retinoic acid-induced differentiation of embryonic stem cells

The scaffold protein BRPF2 (also called BRD1), a key component of histone acetyltransferase complexes, plays an important role in embryonic development, but its function in the differentiation of embryonic stem cells (ESCs) remains unknown. In the present study, we investigated whether BRPF2 is involved in mouse ESC differentiation. BRPF2 depletion resulted in abnormal formation of embryoid bodies, downregulation of differentiation-associated genes, and persistent maintenance of alkaline phosphatase activity even after retinoic acid-induced differentiation, indicating impaired differentiation of BRPF2-depleted ESCs. We also found reduced global acetylation of histone H3 lysine 14 (H3K14) in BRPF2-depleted ESCs, irrespective of differentiation status. Further, co-immunoprecipitation analysis revealed a physical association between BRPF2 and the histone acetyltransferase MOZ in differentiated ESCs, suggesting the role of BRPF2-MOZ complexes in ESC differentiation. Together, these results suggest that BRPF2-MOZ complexes play an important role in the differentiation of ESCs via H3K14 acetylation.

Identification of transcriptional and phosphatase regulators as interaction partners of human ADA3, a component of histone acetyltransferase complexes

ADA (alteration/deficiency in activation) 3 is a conserved component of several transcriptional adaptor and HAT (histone acetyltransferase) complexes that regulate RNA polymerase II-mediated gene expression. Within the HAT complexes ADA3 is associated with ADA2 and the HAT GCN5 (general control non-repressed 5). ADA3 plays roles in diverse cellular processes and also in malignancies by modulating GCN5 catalytic activity and/or by interactions with other regulators. To gain a better understanding of ADA3 function, we used a yeast two-hybrid approach to screen a human fetal cDNA library for proteins that interacted with hADA3 (human ADA3). We identified three novel hADA3-interacting partners, a transcriptional regulator, AATF (apoptosis-antagonizing transcription factor), and regulatory subunits of the PP1 (protein phosphatase 1) and PP2A (protein phosphatase 2A) [PPP1R7 (PP1 regulatory subunit 7) and PPP2R5D (PP2A 56 kDa regulatory subunit δ isoform) respectively]. Analysis of truncated versions of hADA3 indicated that the C-terminal ADA2-interacting domain was not required for these interactions. Fluorescent microscopy analysis and co-immunoprecipitation provided support for the co-localization and interaction of hADA3 with these proteins in human cells. Expression of the interacting proteins altered expression of an hADA3-regulated reporter gene, suggesting functional consequences for the interactions. The detected interactions of hADA3 might extend the spectrum of mechanisms by which ADA3 can contribute to the regulation of gene expression and shed light on processes mediated by these newly identified ADA3 partners.

HAda3 Degradation by Papillomavirus Type 16 E6 Correlates with Abrogation of the p14ARF-p53 Pathway and Efficient Immortalization of Human Mammary Epithelial Cells

Two activities of human papillomavirus type 16 E6 (HPV16 E6) are proposed to contribute to the efficient immortalization of human epithelial cells: the degradation of p53 protein and the induction of telomerase. However, the requirement for p53 inactivation has been debated. Another E6 target is the hAda3 protein, a p53 coactivator and a component of histone acetyltransferase complexes. We have previously described the role of hAda3 and p53 acetylation in p14ARF-induced human mammary epithelial cell (MEC) senescence (P. Sekaric, V. A. Shamanin, J. Luo, and E. J. Androphy, Oncogene 26:6261-6268, 2007). In this study, we analyzed a set of HPV16 E6 mutants for the ability to induce hAda3 degradation. E6 mutants that degrade hAda3 but not p53 could abrogate p14ARF-induced growth arrest despite the presence of normal levels of p53 and efficiently immortalized MECs. However, two E6 mutants that previously were reported to immortalize MECs with low efficiency were found to be defective for both p53 and hAda3 degradation. We found that these immortal MECs select for reduced p53 protein levels through a proteasome-dependent mechanism. The findings strongly imply that the inactivation of the p14ARF-p53 pathway, either by the E6-mediated degradation of p53 or hAda3 or by cellular adaptation, is required for MEC immortalization.

Analyses of Heme-Regulated Genes during Erythoid Differentiation.

(Introduction) During erythroid differentiation, the level of erythroid-specific genes increases synchronizing with the intracellular heme content. In addition, heme has been shown to play a role in transcription and protein synthesis. Based on these evidences, it is possible that heme widely regulates the expression of erythroid specific genes. With this hypothesis, we compared the gene expression profile between wild-type and heme-deficient erythroblasts generated from wild-type and ALAS2 (−) ES cells in in vitro, and identified 4 heme-regulated erythroid-specific genes (UCP2, CNBP, NuSAP and unknown EST1), (BBRC 2006; 340:105–110). Among them, unknown EST1 is consisted of 110 a.a. with a conserved acetyl-CoA binding domain, which was characteristic of GNAT (GCN5-related N-acetyltransferase) superfamily. Thus, it is likely that EST1 is a novel acetyltransferase. In the present study, we focused two genes, EST1 and NuSAP, and investigated their function during erythoid differentiation.(Methods) First, the expression and regulation of NuSAP gene during erythroid differentiation was examined. For the expression analysis, in vivo erythroblasts were fractionated according to the surface expression of TER119/CD71, and the level of expression of NuSAP mRNA was examined by quantitative RT-PCR. For the promoter analysis, the promoter region of mouse NuSAP gene was cloned, and the regulatory cis-element was determined by luciferase assay and EMSA. Next, for defining the properties of EST1, EST1 was constitutively expressed using Flag/HA tagged retroviral vector into mouse erythroleukemia (MEL) cell line, and nuclear extract of EST1-expessed MEL cells was purified by affinity chromatography, which was loaded on an SDS/PAGE gel and subjected to electrophoresis. In addition, for in vitro histone acetyltransferase (HAT) assay, free histone and purified EST1 protein were incubated with [3H]acetyl-CoA, and acetyltransferase activity was measured by scintigraphy.(Results) (1) NuSAP mRNA was more significantly abundant in the subset corresponding to immature erythroblasts (TER119+CD71high) than mature erythroblasts (TER119+CD71low), and it was significantly increased in TER119+ cells from in vivo phlebotomized mice compared with control mice. Furthermore, during erythroid maturation of MEL cells by dimethylsulfoxide, NuSAP mRNA was increased at 24–72 hrs. Promoter analyses of NuSAP gene demonstrated that duplicated CCAAT boxes located at −81/−85 and −30/−34 were essential for promoter activity, which was trans-activated by NF-YA. These results suggested that NuSAP might contribute to the expansion of immature erythroblast pool under the control of NF-Y. (2) By the gel electrophoresis, it was revealed that EST1 protein forms a multimeric protein complex. Furthermore, whereas recombinant EST1 protein did not show HAT activity, EST1 complex could acetylate free histones in vitro, suggesting that EST1 might be a component of HAT complex.(Conclusion) The novel functions of EST1 and NuSAP suggest that heme regulates erythroid differentiation by controlling the expression of variety of genes.

HAda3 regulates p14ARF-induced p53 acetylation and senescence

Acetylation is thought to be a key event for p53 activation. We demonstrate that p14ARF-induced senescence of human mammary epithelial cells (MEC) is associated with p53 acetylation and requires hAda3, a component of histone acetyltransferase complexes and a p53 transcriptional coactivator. Expression of the N-terminal domain of hAda3 that binds p53 but not p300 blocked p14ARF-induced p53 acetylation and protected MECs from senescence. Consistent with these findings, the human papillomavirus 16 E6 mutant Y54D, which selectively targets hAda3 but not p53 for degradation and protects MECs from p14ARF-induced senescence, inhibited p53 acetylation. In H1299 cells, hAda3 overexpression increased p300-mediated p53 acetylation, which conversely decreased following small interfering RNA (siRNA) knockdown of hAda3. Moreover, depletion of hAda3 by siRNA inhibited endogenous p53 acetylation and accumulation of p21cip1 in response to ectopic p14ARF. These studies reveal that, in addition to its known ability to inhibit Mdm2-mediated p53 degradation, p14ARF signals through hAda3 to stimulate p53 acetylation and the induction of cell senescence.

The Epc-N domain: a predicted protein-protein interaction domain found in select chromatin associated proteins

BackgroundAn underlying tenet of the epigenetic code hypothesis is the existence of protein domains that can recognize various chromatin structures. To date, two major candidates have emerged: (i) the bromodomain, which can recognize certain acetylation marks and (ii) the chromodomain, which can recognize certain methylation marks.ResultsThe Epc-N (Enhancer of Polycomb-N-terminus) domain is formally defined herein. This domain is conserved across eukaryotes and is predicted to form a right-handed orthogonal four-helix bundle with extended strands at both termini. The types of amino acid residues that define the Epc-N domain suggest a role in mediating protein-protein interactions, possibly specifically in the context of chromatin binding, and the types of proteins in which it is found (known components of histone acetyltransferase complexes) strongly suggest a role in epigenetic structure formation and/or recognition. There appear to be two major Epc-N protein families that can be divided into four unique protein subfamilies. Two of these subfamilies (I and II) may be related to one another in that subfamily I can be viewed as a plant-specific expansion of subfamily II. The other two subfamilies (III and IV) appear to be related to one another by duplication events in a primordial fungal-metazoan-mycetozoan ancestor. Subfamilies III and IV are further defined by the presence of an evolutionarily conserved five-center-zinc-binding motif in the loop connecting the second and third helices of the four-helix bundle. This motif appears to consist of a PHD followed by a mononuclear Zn knuckle, followed by a PHD-like derivative, and will thus be referred to as the PZPM. All non-Epc-N proteins studied thus far that contain the PZPM have been implicated in histone methylation and/or gene silencing. In addition, an unusual phyletic distribution of Epc-N-containing proteins is observed.ConclusionThe data suggest that the Epc-N domain is a protein-protein interaction module found in chromatin associated proteins. It is possible that the Epc-N domain serves as a direct link between histone acetylation and methylation statuses. The unusual phyletic distribution of Epc-N-containing proteins may provide a conduit for future insight into how different organisms form, perceive and respond to epigenetic information.

Adenovirus E1A requires the yeast SAGA histone acetyltransferase complex and associates with SAGA components Gcn5 and Tra1

The budding yeast Saccharomyces cerevisiae was used as a model system to study the function of the adenovirus E1A oncoprotein. Previously we demonstrated that expression of the N-terminal 82 amino acids of E1A in yeast causes pronounced growth inhibition and specifically interferes with SWI/SNF-dependent transcriptional activation. Further genetic analysis identified the yeast transcription factor Adr1 as a high copy suppressor of E1A function. Transcriptional activation by Adr1 requires interaction with co-activator proteins Ada2 and Gcn5, components of histone acetyltransferase complexes including ADA and SAGA. Analysis of mutant alleles revealed that several components of the SAGA complex, including proteins from the Ada, Spt, and Taf classes were required for E1A-induced growth inhibition. Growth inhibition also depended on the Gcn5 histone acetyltransferase, and point mutations within the Gcn5 HAT domain rendered cells E1A-resistant. Also required was SAGA component Tra1, a homologue of the mammalian TRRAP protein which is required for c-myc and E1A induced cellular transformation. Additionally, Gcn5 protein could associate with E1A in vitro in a manner that depended on the N-terminal domain of E1A, and Tra1 protein was co-immunoprecipitated with E1A in vivo. These results indicate a strong requirement for intact SAGA complex for E1A to function in yeast, and suggest a role for SAGA-like complexes in mammalian cell transformation.