Regulation of Alteration/Deficiency in Activation 3 (ADA3) by Acetylation and its Role in Cell Cycle Regulation and Oncogenesis

The ADA3 (Alteration/Deficiency in Activation 3) protein is a transcriptional adaptor protein that was initially discovered as a component of several HAT (Histone Acetyltransferase) complexes, the enzyme complex responsible for histone acetylation, which is a prerequisite for transcription. Earlier the studies from Dr. Band’s laboratory and that of others’ have deciphered a crucial role of ADA3 in cell cycle regulation (both through G1/S and G2/M phase transitions) and in maintaining the genomic stability. While our laboratory investigated the mechanism behind the role of ADA3 in G1/S transition, the same remained unknown for G2/M phase transition. Based on this prior knowledge about ADA3, I started out my Ph.D. thesis work in Dr. Band’s laboratory directed towards examining the role of ADA3 in mitosis. During my doctoral research, I demonstrated that ADA3 governs the recruitment of a key centromeric protein CENP-B on to the centromeres and regulates the chromosome segregation during mitosis. ADA3 protein has the potential to undergo posttranslational modification, including acetylation, and in the course of my Ph.D. research, I became interested in how these modifications might regulate the function of ADA3. I showed that ADA3 acetylation is regulated by coordinated actions of its associated HATs, GCN5, PCAF and p300, and a new partner I discovered, the deacetylase SIRT1. We used mass-spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300 and found that acetylation defective mutants were capable of interacting with HATs and other components of HAT complexes but deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3 deleted MEFs. A parallel focus of my studies was to define the role of ADA3 in HER2+ breast cancers, which basically emanates from a clinical study from our laboratory that revealed that ADA3 is overexpressed/mislocalized in these types of aggressive tumors. By using cell culture models I have established a link between ADA3 and HER2 signaling pathways. In these cell lines, I found that ADA3 is a downstream target of HER2 and discovered a novel phospho-AKT-phospho-p300-Ac-ADA3 signaling pathway. Importantly, ADA3 knockdown in these cells recapitulates the cell cycle inhibitory effects of a tyrosine kinase inhibitor lapatinib such as accumulation of CDK inhibitor p27 and reduced mitotic index. Taken together these results highlight the importance of ADA3 as a marker for treatment efficacy and a promising therapeutic target. Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including cell cycle, my thesis work provides an insight for the regulation of the function of these complexes through dynamic ADA3 acetylation

Mammalian alteration/deficiency in activation 3 (Ada3) is essential for embryonic development and cell cycle progression.

Ada3 protein is an essential component of histone acetyl transferase containing coactivator complexes conserved from yeast to human. We show here that germline deletion of Ada3 in mouse is embryonic lethal, and adenovirus-Cre mediated conditional deletion of Ada3 in Ada3(FL/FL) mouse embryonic fibroblasts leads to a severe proliferation defect which was rescued by ectopic expression of human Ada3. A delay in G(1) to S phase of cell cycle was also seen that was due to accumulation of Cdk inhibitor p27 which was an indirect effect of c-myc gene transcription control by Ada3. We further showed that this defect could be partially reverted by knocking down p27. Additionally, drastic changes in global histone acetylation and changes in global gene expression were observed in microarray analyses upon loss of Ada3. Lastly, formation of abnormal nuclei, mitotic defects and delay in G(2)/M to G(1) transition was seen in Ada3 deleted cells. Taken together, we provide evidence for a critical role of Ada3 in embryogenesis and cell cycle progression as an essential component of HAT complex

Cytoplasmic localization of alteration/deficiency in activation 3 (ADA3) predicts poor clinical outcome in breast cancer patients.

Transcriptional activation by estrogen receptor (ER) is a key step to breast oncogenesis. Given previous findings that ADA3 is a critical component of HAT complexes that regulate ER function and evidence that overexpression of other ER coactivators such as SRC-3 is associated with clinical outcomes in breast cancer, the current study was designed to assess the potential significance of ADA3 expression/localization in human breast cancer patients. In this study, we analyzed ADA3 expression in breast cancer tissue specimens and assessed the correlation of ADA3 staining with cancer progression and patient outcome. Tissue microarrays prepared from large series of breast cancer patients with long-term follow-ups were stained with anti-ADA3 monoclonal antibody using immunohistochemistry. Samples were analyzed for ADA3 expression followed by correlation with various clinicopathological parameters and patients\u27 outcomes. We report that breast cancer specimens show predominant nuclear, cytoplasmic, or mixed nuclear + cytoplasmic ADA3 staining patterns. Predominant nuclear ADA3 staining correlated with ER+ status. While predominant cytoplasmic ADA3 staining negatively correlated with ER+ status, but positively correlated with ErbB2, EGFR, and Ki67. Furthermore, a positive correlation of cytoplasmic ADA3 was observed with higher histological grade, mitotic counts, Nottingham Prognostic Index, and positive vascular invasion. Patients with nuclear ADA3 and ER positivity have better breast cancer specific survival and distant metastasis free survival. Significantly, cytoplasmic expression of ADA3 showed a strong positive association with reduced BCSS and DMFS in ErbB2+/EGFR+ patients. Although in multivariate analyses ADA3 expression was not an independent marker of survival, predominant nuclear ADA3 staining in breast cancer tissues correlates with ER+ expression and together serves as a marker of good prognosis, whereas predominant cytoplasmic ADA3 expression correlates with ErbB2+/EGFR+ expression and together is a marker of poor prognosis. Thus, ADA3 cytoplasmic localization together with ErbB2+/EGFR+ status may serve as better prognostic marker than individual proteins to predict survival of patients

ADA3 regulates normal and tumor mammary epithelial cell proliferation through c-MYC

Background: We have established the critical role of ADA3 as a coactivator of estrogen receptor (ER), as well as its role in cell cycle progression. Furthermore, we showed that ADA3 is predominantly nuclear in mammary epithelium, and in ER+, but is cytoplasmic in ER- breast cancers, the latter correlating with poor survival. However, the role of nuclear ADA3 in human mammary epithelial cells (hMECs), and in ER+ breast cancer cells, as well as the importance of ADA3 expression in relation to patient prognosis and survival in ER+ breast cancer have remained uncharacterized.Methods: We overexpressed ADA3 in hMECs or in ER+ breast cancer cells and assessed the effect on cell proliferation. The expression of ADA3 was analyzed then correlated with the expression of various prognostic markers, as well as survival of breast cancer patients.Results: Overexpression of ADA3 in ER- hMECs as well as in ER+ breast cancer cell lines enhanced cell proliferation. These cells showed increased cyclin B and c-MYC, decreased p27 and increased SKP2 levels. This was accompanied by increased mRNA levels of early response genes c-FOS, EGR1, and c-MYC. Analysis of breast cancer tissue specimens showed a significant correlation of ADA3 nuclear expression with c-MYC expression. Furthermore, nuclear ADA3 andc-MYC expression together showed significant correlation with tumor grade, mitosis, pleomorphism, NPI, ER/PR status, Ki67 and p27 expression. Importantly, within ER+ cases, expression of nuclear ADA3 and c-MYC also significantly correlated with Ki67 and p27 expression. Univariate Kaplan Meier analysis of four groups in the whole, as well as the ER+ patients showed that c-MYC and ADA3 combinatorial phenotypes showed significantly different breast cancer specific survival with c-MYC-high and ADA3-Low subgroup had the worst outcome. Using multivariate analyses within the whole cohort and the ER+ subgroups, the significant association of ADA3 and c-MYC expression with patients’ outcome was independent of tumor grade, stage and size, and ER status.Conclusion: ADA3 overexpression enhances cell proliferation that is associated with increased expression of c-MYC. Expression patterns with respect to ADA3/c-MYC can divide patients into four significantly different subgroups, with c-MYC High and ADA3 Low status independently predicting poor survival in patients

The Role of ADA3 Overexpression in Proliferation Through Enhancing MYC Expression

Breast cancer is a heterogeneous disease that is the second leading cause of cancer related deaths in women. Cancer is defined as abnormally heightened proliferation. In order for gene transcription and eventual translation to occur to drive the cell cycle to generate more cells, DNA must be uncoiled from nucleosomes by histone acetylation complexes. One of the key evolutionarily conserved components of these HAT complexes is alteration/deficiency in activation 3 (ADA3). In addition to the role in histone acetylation, this protein also functions as a coactivator for nuclear hormone receptors. Recent findings indicated that nuclear Ada3 correlates with ER+ breast cancers and a favorable survival, while cytoplasmic (overexpression) Ada3 correlates with ErbB2+ and EGFR+ breast cancers and a poor survival. My thesis work focused on the role of Ada3 knockdown and overexpression on cellular proliferation in human derived cell lines. We showed knockdown of ADA3 in immortal hMECs led to inhibition of cell cycle progression, decrease in cyclin B, c-myc or H3K56 levels, and increase in p27 protein. In contrast, ADA3 overexpression in two immortal hMECs and two ER+ breast cancer cell lines enhanced proliferation. Cell cycle analyses showed increased cyclin B and decreased p27 upon ADA3 overexpression. Decreased p27 levels were due to increased turnover of p27 protein. Furthermore, ADA3 overexpression led to increased mRNA levels of early response genes c-fos, EGR1, and c-myc. Analysis of a large cohort of ER+ breast cancer tissue specimens showed a subset of ER+ tumors express higher levels of ADA3 and these tumors also express higher levels of c-myc (

Human ADA3 regulates RARα transcriptional activity through direct contact between LxxLL motifs and the receptor coactivator pocket

The alternation/deficiency in activation-3 (ADA3) is an essential component of the human p300/CBP-associated factor (PCAF) and yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) histone acetyltransferase complexes. These complexes facilitate transactivation of target genes by association with transcription factors and modification of local chromatin structure. It is known that the yeast ADA3 is required for nuclear receptor (NR)-mediated transactivation in yeast cells; however, the role of mammalian ADA3 in NR signaling remains elusive. In this study, we have investigated how the human (h) ADA3 regulates retinoic acid receptor (RAR) α-mediated transactivation. We show that hADA3 interacts directly with RARα in a hormone-dependent manner and this interaction contributes to RARα transactivation. Intriguingly, this interaction involves classical LxxLL motifs in hADA3, as demonstrated by both ‘loss’ and ‘gain’ of function mutations, as well as a functional coactivator pocket of the receptor. Additionally, we show that hADA3 associates with RARα target gene promoter in a hormone-dependent manner and ADA3 knockdown impairs RARβ2 expression. Furthermore, a structural model was established to illustrate an interaction network within the ADA3/RARα complex. These results suggest that hADA3 is a bona fide transcriptional coactivator for RARα, acting through a conserved mechanism involving direct contacts between NR boxes and the receptor’s co-activator pocket

Interaction between the SAGA complex and chromatin regulates nucleosome acetylation and inducible gene transcription

The SAGA family of coactivators is a group of highly conserved histone acetyltransferases that is essential for transcription of genes participating in stress adaptation and developemental processes. Misregulation of SAGA function can lead to developmental diseases and cancer. One important function of SAGA in regulating gene expression is to acetylate nucleosomes at both gene promoters and coding regions. To understand the molecular mechanism of nucleosome acetylation, much work has been done using recombinant Gcn5, the catalytic subunit of SAGA, and histone proteins or peptides. However, little is known about how histones are acetylated by full SAGA complex in the context of chromatin and how this relates to expression of SAGA-dependent genes. In this study, we performed histone acetyltransferase (HAT) kinetic assay using purified SAGA complex and reconstituted nucleosomal arrays. We found that SAGA complex acetylates nucleosomes in a highly cooperative manner with a cooperativity coefficiency of 1.97 y 0.15, suggesting that its maximal acetylation activity can be augmented by binding to multiple nucleosomes. Next, we explored the requirement for cooperative nucleosome acetylation with respect to substrates and SAGA enzyme complex, respectively. To determine the substrate requirement for cooperativity, various novel nucleosomes were generated and investigated through HAT kinetic assay. We found that SAGA-mediated cooperativity occurs only when both histone H3 tails are present and properly oriented in the nucleosomes. Furthermore, the four acetylation lysine residues on both H3 tails must be present and unacetylated. To determine factors in SAGA complex required for cooperative nucleosome acetylation, we performed HAT kinetic assay using a SAGA trimeric subcomplex composed of Ada2, Ada3, and Gcn5 and our studies showed that these three subunits are the minimal requirement to recapitulate SAGA-mediated cooperativity. Additionally, we discovered a novel regulatory mechanism of SAGA that enhances its activity when cells were grown under stressful conditions. Our results showed that SAGA acetylates one of its subunit Ada3 and undergoes acetylation-dependent dimerization when cells sense stress signals in the environment. Most importantly, this acetylation-dependent dimerization of SAGA contributes to cooperative nucleosome acetylation and facilitates cell growth under stress conditions. To probe which protein domains within these three subunits are required for cooperativity, we generated a series of subcomplex mutations and discovered that Gcn5 bromodomain is necessary for cooperativity. To further study the functions of Gcn5 bromodomain in SAGA-mediated nucleosome acetylation, we performed HAT kinetic assay on nucleosomes containing either pre-acetylated single H3 tail or pre-acetylated histone H4K16. We found that the Gcn5 bromodomain facilitates cross-tail H3 acetylation in pre-acetylated nucleosomes. Collectively, our studies revealed a novel mechanism which SAGA modulates its activity and nucleosome acetylation. Furthermore, this regulatory mechanism has important implications in inducible gene transctiption and co-regulation of functionally related genes

ADA1, a novel component of the ADA/GCN5 complex, has broader effects than GCN5, ADA2, or ADA3

9 pages, 5 figures, 3 tables.-- PMID: 9154821 [PubMed].-- PMCID: PMC232175.The ADA genes encode factors which are proposed to function as transcriptional coactivators. Here we describe the cloning, sequencing, and initial characterization of a novel ADA gene, ADA1. Similar to the previously isolated ada mutants, ada1 mutants display decreases in transcription from various reporters. Furthermore, ADA1 interacts with the other ADAs in the ADA/GCN5 complex as demonstrated by partial purification of the complex and immunoprecipitation experiments. We estimate that the complex has a molecular mass of approximately 2 MDa. Previously, it had been demonstrated that ada5 mutants displayed more severe phenotypic defects than the other ada mutants (G. A. Marcus, J. Horiuchi, N. Silverman, and L. Guarente, Mol. Cell. Biol. 16:3197-3205, 1996; S. M. Roberts and F. Winston, Mol. Cell. Biol. 16:3206-3213, 1996). ada1 mutants display defects similar to those of ada5 mutants and different from those of the other mutants with respect to promoters affected, inositol auxotrophy, and Spt- phenotypes. Thus, the ADAs can be separated into two classes, suggesting that the ADA/GCN5 complex may have two separate functions. We present a speculative model on the possible roles of the ADA/GCN5 complex.We thank A. Koleske, D. Chao, C. Wilson, C. Hengartner, and R. Young for sharing antibodies and for technical assistance. We also thank A. Greenleaf and C. Peterson for sharing antisera and K. Kaplan for assistance with the Sprint BIOCAD apparatus. We are grateful to S. Roberts and F. Winston for strains, T. D. Fox and D. Allis for sharing unpublished results, and D. McNabb for critical comments on the manuscript. G.M. thanks C. Armstrong and S. Treadway for help with the GCN5 antibody. J.H. was an HHMI predoctoral fellow. This work was supported by grant GM50207 from NIH to L.G.Peer reviewe

Characterization of yeast histone H3-specific type B histone acetyltransferases identifies an ADA2-independent Gcn5p activity

BACKGROUND: The acetylation of the core histone NH(2)-terminal tails is catalyzed by histone acetyltransferases. Histone acetyltransferases can be classified into two distinct groups (type A and B) on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4. RESULTS: To better understand the role of histone acetylation in the assembly of chromatin structure, we have identified additional type B histone acetyltransferase activities specific for histone H3. One such activity, termed HatB3.1, acetylated histone H3 with a strong preference for free histones relative to chromatin substrates. Deletion of the GCN5 and ADA3 genes resulted in the loss of HatB3.1 activity while deletion of ADA2 had no effect. In addition, Gcn5p and Ada3p co-fractionated with partially purified HatB3.1 activity while Ada2p did not. CONCLUSIONS: Yeast extracts contain several histone acetyltransferase activities that show a strong preference for free histone H3. One such activity, termed HatB3.1, appears to be a novel Gcn5p-containing complex which does not depend on the presence of Ada2p