The yeast transcriptional adaptor, Gcn5p, is a catalytic subunit of a nuclear (type A) histone acetyltransferase linking histone acetylation to gene activation. Here we report that Gcn5p acetylates histones H3 and H4 non-randomly at specific lysines in the amino-terminal domains. Lysine 14 of H3 and lysines 8 and 16 of H4 are highly preferred acetylation sites for Gcn5p. We also demonstrate that lysine 9 is the preferred position of acetylation in newly synthesized yeast H3 in vivo. This finding, along with the fact that lysines 5 and 12 in H4 are predominant acetylation sites during chromatin assembly of many organisms, indicates that Gcn5p acetylates a distinct set of lysines that do not overlap with those sites characteristically used by type B histone acetyltransferases for histone deposition and chromatin assembly.

mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) signaling pathways and plays an important role in the viability response of these cytokines. In this study, we demonstrated that cytokine stimulation of mcl-1 mRNA and protein expression were attenuated by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-K) inhibitors. Reporter gene assays further showed that the PI3-K/Akt signaling pathway was involved in IL-3 activation of mcl-1 gene transcription. Analysis of the mcl-1 promoter revealed that both promoter elements, SIE at position ؊87 and CRE-2 at ؊70, contribute to IL-3 stimulation of mcl-1 gene expression. Although either the SIE site or the CRE-2 site alone was sufficient to confer IL-3 inducibility on a heterologous promoter, only IL-3 activation of the CRE-2 reporter was mediated via the PI3-K/Akt pathway. The SIE binding activity was constitutively high in cells deprived of or stimulated by IL-3. In contrast, the CRE-2 binding activity was low in cytokine-starved cells and was strongly induced within 1 h following cytokine treatment of cells. In addition, cytokine induction of the CRE-2 but not of the SIE binding activity was dependent on activation of the PI3-K/Akt signaling pathway. Lastly, we showed that CREB was one component of the CRE-2 binding complex and played a role in IL-3 activation of the mcl-1 reporter gene. Taken together, our results suggest that both PI3-K/Aktdependent and -independent pathways contribute to the IL-3 activation of mcl-1 gene expression. Activation of mcl-1 by the PI3-K/Akt-dependent pathway is through a transcription factor complex containing CREB.

Within chromatin, reversible acetylation of core histones is critical for transcriptional activation of eukaryotic target genes. The recent identification of intrinsic histone acetyltransferase (HAT) catalytic activity from a number of transcriptional co-activators (including yeast GCN5, p300/CBP, P/CAF, and TAFII250), has underscored the importance of protein acetylation in transcriptional control. The GCN5 family is the prototype for a diverse group of at least four distinct human HATs families. Although there is now a clear link between in vivo HAT catalytic activity and gene activation, little is known about the molecular mechanisms of histone acetylation. Herein, we report the first detailed biochemical study that probes the catalytic mechanism and the function of invariant glutamic acid 173 within the GCN5 family of HATs. Our results suggest that the HAT reaction involves the formation of a ternary complex (histones, acetyl-CoA, and enzyme) where the ⑀-amino group of histone lysine residues directly attacks the bound acetyl-CoA. The acetylation reaction requires deprotonation of the ⑀-amino group prior to nucleophilic attack. Employing site-directed mutagenesis, chemical modification, steady-state, and pH-dependent rate analysis, it is demonstrated that glutamic acid 173 is an essential catalytic residue, acting as a general base catalyst by deprotonating the histone substrate.

No abstract

BackgroundOvarian cancer (OCa) peritoneal metastasis is the leading cause of cancer–related deaths in women with limited therapeutic options available for treating it and poor prognosis, as the underlying mechanism is not fully understood.MethodThe clinicopathological correlation of G9a expression was assessed in tumor specimens of ovarian cancer patients. Knockdown or overexpression of G9a in ovarian cancer cell lines was analysed with regard to its effect on adhesion, migration, invasion and anoikis-resistance. In vivo biological functions of G9a were tested by i.p. xenograft ovarian cancer models. Microarray and quantitative RT-PCR were used to analyze G9a-regulated downstream target genes.ResultsWe found that the expression of histone methyltransferase G9a was highly correlated with late stage, high grade, and serous-type OCa. Higher G9a expression predicted a shorter survival in ovarian cancer patients. Furthermore, G9a expression was higher in metastatic lesions compared with their corresponding ovarian primary tumors. Knockdown of G9a expression suppressed prometastatic cellular activities including adhesion, migration, invasion and anoikis-resistance of ovarian cancer cell lines, while G9a over-expression promoted these cellular properties. G9a depletion significantly attenuated the development of ascites and tumor nodules in a peritoneal dissemination model. Importantly, microarray and quantitative RT-PCR analysis revealed that G9a regulates a cohort of tumor suppressor genes including CDH1, DUSP5, SPRY4, and PPP1R15A in ovarian cancer. Expression of these genes was also inversely correlated with G9a expression in OCa specimens.ConclusionWe propose that G9a contributes to multiple steps of ovarian cancer metastasis and represents a novel target to combat this deadly disease.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-189) contains supplementary material, which is available to authorized users.