Estrogen regulation of trefoil factor 1 expression by estrogen receptor alpha and Sp proteins

Estrogen-responsive genes in human breast cancer cells often have an estrogen response element (ERE) positioned next to an Sp1 binding site. In chromatin immunoprecipitation (ChIP) assays, we investigated the binding of estrogen receptor alpha (ER), Sp1, and Sp3 to the episomal and native estrogen-responsive trefoil factor 1 (TFF1; formerly pS2) promoter in MCF-7 breast cancer cells. Mutation of the Sp site upstream of the ERE reduced estrogen responsiveness and prevented binding of Sp1 and Sp3, but not ER to the episomal promoter. In the absence of estradiol (E2), Sp1, Sp3, histone deacetylase 1 (HDAC), and HDAC2, and low levels of acetylated H3 and H4 are associated with the native promoter, with the histones being engaged in dynamic reversible acetylation. Following E2 addition, levels of ER and acetylated H3 and H4 bound to the native promoter increases. There is clearance of Sp1, but not of Sp3, from the promoter while HDAC1 and HDAC2 remain bound. These data are consistent with a model in which Sp1 or Sp3 aid in recruitment of HDACs and histone acetyltransferases (HATs) to mediate dynamic acetylation of histones associated with the TFF1 promoter, which is in a state of readiness to respond to events occurring following the addition of estrogen.     

Stimulation of histone deacetylase activity by metabolites of intermediary metabolism

Histone deacetylases (HDACs) function in a wide range of molecular processes, including gene expression, and are of significant interest as therapeutic targets. Although their native complexes, subcellular localization, and recruitment mechanisms to chromatin have been extensively studied, much less is known about whether the enzymatic activity of non-sirtuin HDACs can be regulated by natural metabolites. Here, we show that several coenzyme A (CoA) derivatives, such as acetyl-CoA, butyryl-CoA, HMG-CoA, and malonyl-CoA, as well as NADPH but not NADP(+), NADH, or NAD(+), act as allosteric activators of recombinant HDAC1 and HDAC2 in vitro following a mixed activation kinetic. In contrast, free CoA, like unconjugated butyrate, inhibits HDAC activity in vitro. Analysis of a large number of engineered HDAC1 mutants suggests that the HDAC activity can potentially be decoupled from "activatability" by the CoA derivatives. In vivo, pharmacological inhibition of glucose-6-phosphate dehydrogenase (G6PD) to decrease NADPH levels led to significant increases in global levels of histone H3 and H4 acetylation. The similarity in structures of the identified metabolites and the exquisite selectivity of NADPH over NADP(+), NADH, and NAD(+) as an HDAC activator reveal a previously unrecognized biochemical feature of the HDAC proteins with important consequences for regulation of histone acetylation as well as the development of more specific and potent HDAC inhibitors.     

Histone deacetylase 1-mediated histone modification regulates osteoblast differentiation

Osteogenesis is a complex process associated with dramatic changes in gene expression. To elucidate whether modifications in chromatin structure are involved in osteoblast differentiation, we examined the expression levels of histone deacetylases (HDACs) and the degree of histone acetylation at the promoter regions of osteogenic genes. During osteogenesis, total HDAC enzymatic activity was decreased with significant reduction in HDAC1 expression. Consistently, recruitment of HDAC1 to the promoters of osteoblast marker genes, including osterix and osteocalcin, was down-regulated, whereas histone H3 and H4 were hyperacetylated at those promoters during osteoblast differentiation. Moreover, suppression of HDAC activity with a HDAC inhibitor, sodium butyrate, accelerated osteogenesis by inducing osteoblast marker genes including osteopontin and alkaline phosphatase. Consistently, knockdown of HDAC1 by the short interference RNA system stimulated osteoblast differentiation. Taken together, these data propose that down-regulation of HDAC1 is an important process for osteogenesis.     

Role of histone deacetylation in cell-specific expression of endothelial nitric-oxide synthase

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of endothelial nitric-oxide synthase (eNOS) are not fully understood. In this study we investigated whether histone deacetylation was involved in repression of eNOS expression in non-endothelial cells. Induction of eNOS expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate was observed in all four different types of non-endothelial cells examined. Chromatin immunoprecipitation assays showed that the induction of eNOS expression by TSA was accompanied by a remarkable increase of acetylation of histone H3 associated with the eNOS 5'-flanking region in the non-endothelial cells. Moreover, DNA methylation-mediated repression of eNOS promoter activity was partially reversed by TSA treatment, and combined treatment of TSA and 5-aza-2'-deoxycytidine (AzadC) synergistically induced eNOS expression in non-endothelial cells. The proximal Sp1 site is critical for basal activity of eNOS promoter. The induction of eNOS by inhibition of HDACs in non-endothelial cells, however, appeared not mediated by the changes in Sp1 DNA binding activity. We further showed that Sp1 bound to the endogenous eNOS promoter and associated with HDAC1 in non-endothelial HeLa cells. Combined TSA and AzadC treatment increased Sp1 binding to the endogenous eNOS promoter but decreased the association between HDAC1 and Sp1 in HeLa cells. Our data suggest that HDAC1 plays a critical role in eNOS repression, and the proximal Sp1 site may serve a key target for HDCA1-mediated eNOS repression in non-endothelial cells.     

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.