Histone deacetylase inhibitors decrease reelin promoter methylation in vitro

We investigated the effects of agents that induce reelin mRNA expression in vitro on the methylation status of the human reelin promoter in neural progenitor cells (NT2). NT2 cells were treated with the histone deacetylase inhibitors, trichostatin A (TSA) and valproic acid (VPA), and the methylation inhibitor aza-2'-deoxycytidine (AZA) for various times. All three drugs reduced the methylation profile of the reelin promoter relative to untreated cells. The acetylation status of histones H3 and H4 increased following treatment with VPA and TSA at times as short as 15 min following treatment; a result consistent with the reported mode of action of these drugs. Chromatin immunoprecipitation experiments showed that these changes were accompanied by changes occurring at the level of the reelin promoter as well. Interestingly, AZA decreased reelin promoter methylation without concomittantly increasing histone acetylation. In fact, after prolonged treatments with AZA, the acetylation status of histones H3 and H4 decreased relative to untreated cells. We also observed a trend towards reduced methylated H3 after 18 h treatment with TSA and VPA. Our data indicate that while TSA and VPA act to increase histone acetylation and reduce promoter methylation, AZA acts only to decrease the amount of reelin promoter methylation.     

DNA methylation as an epigenetic regulator of neural 5-lipoxygenase expression: evidence in human NT2 and NT2-N cells

Increased expression of 5-lipoxygenase is associated with various neuropathologies and may be related to epigenetic gene regulation. DNA methylation in promoter regions is typically associated with gene silencing. We found that human NT2 cells, which differentiate into neuron-like NT2-N cells, express 5-lipoxygenase and we investigated the relationship between 5-lipoxygenase expression and the methylation state of the 5-lipoxygenase core promoter. We used the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor valproate to alter DNA methylation and to induce histone modifications. 5-Lipoxygenase expression and DNA methylation were assayed with RT-PCR and bisulfite genomic sequencing, respectively. Neuronal differentiation of proliferating NT2 precursors decreased 5-lipoxygenase expression. 5-Aza-2'-deoxycytidine increased 5-lipoxygenase mRNA levels only in proliferating cells, whereas valproate increased 5-lipoxygenase mRNA levels in a cell cycle-independent manner. In both precursors and differentiated cells, CpG dinucleotides of the promoter were poorly methylated. In precursors, both 5-aza-2'-deoxycytidine and valproate further reduced the number of methylated CpGs. Moreover, we found evidence for cytosine methylation in CpWpG (W=adenine or thymine) and other asymmetrical sequences; CpWpG methylation was reduced by valproate in NT2-N but not in NT2 cells. This is the first report demonstrating that the dynamics of DNA methylation relates to neural 5-lipoxygenase gene expression.     

Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line.

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP-1-like motif and other upstream control elements play an important role in the high-level constitutive expression of NT/N in the human endocrine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/N expression and to better understand the mechanisms responsible for the terminal differentiation of the N-cell lineage in the gut.     

Down-regulation of IL-7Ralpha expression in human T cells via DNA methylation

IL-7 is critical for the development and survival of T cells. Recently, we found two subsets of human CD8+ T cells expressing IL-7Ralpha(high) and IL-7Ralpha(low) with different cell survival responses to IL-7. Although these CD8+ T cell subsets have differential IL-7Ralpha gene expression, the mechanism for this is unknown. DNA methylation is an important gene regulatory mechanism and is associated with the inactivation of gene expression. Thus, we investigated a role for DNA methylation in differentially regulating IL-7Ralpha gene expression in human CD8+ T cells and Jurkat T cells. IL-7Ralpha(high)CD8+ T cells had decreased methylation in the IL-7Ralpha gene promoter compared with IL-7Ralpha(low)CD8+ T cells and Jurkat T cells with low levels of IL-7Ralpha. Treating Jurkat T cells with 5-aza-2'-deoxycytidine, which reduced DNA methylation, increased IL-7Ralpha expression. Plus, the unmethylated IL-7Ralpha gene promoter construct had higher levels of promoter activity than the methylated one as measured by a luciferase reporter assay. These findings suggest that DNA methylation is involved in regulating IL-7Ralpha expression in T cells via affecting IL-7Ralpha gene promoter activity, and that the methylation of this gene promoter could be a potential target for modifying IL-7-mediated T cell development and survival.     

Regulation of RANKL promoter activity is associated with histone remodeling in murine bone stromal cells

Receptor activator of NFkappa-B ligand (RANKL) is essential for osteoclast formation, function, and survival. Although RANKL mRNA and protein levels are modulated by 1,25(OH)2D3 and other osteoactive factors, regulatory mechanisms remain unclear. In this study, we show that 2 kb or 2 kb plus exon 1 of a RANKL promoter sequence conferred neither 1,25(OH)2D3 response nor tissue specificity. The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (SB), however, strongly increased RANKL promoter activity. A series of 5'-deleted RANKL promoter constructs from 2,020 to 110 bp showed fourfold increased activity after TSA treatment. TSA also dose dependently enhanced endogenous RANKL mRNA expression with 50 microM of TSA treatment causing equivalent RANKL expression to that seen with 1 nM 1,25(OH)2D3. Using a chromatin immunoprecipitation (ChIP) assay we showed that TSA significantly enhanced association of both acetylated histone H3 and H4 on the RANKL promoter, with H4 > H3. A similar increase in acetylated histone H4 on the RANKL gene locus was seen after 1,25(OH)2D3 treatment, but ChIP assay did not reveal localization of VDR/RXR heterodimers on the putative VDRE of the RANKL promoter. To explore the role of H4 acetylation of 1,25(OH)2D3 stimulated RANKL, we added both TSA and 1,25(OH)2D3 together. While the combination further increased acetylation of H4 on the RANKL locus, surprisingly, TSA inhibited 1,25(OH)2D3-induced RANKL mRNA expression by 70% at all doses of 1 ,25(OH)2D3 studied. These results suggest that TSA increases of endogenous expression of RANKL involve enhanced acetylation of histones on the proximal RANKL promoter. Preventing deacetylation, however, blocks 1,25(OH)2D3 action on this gene. Chromatin remodeling is therefore involved in RANKL expression.     

TLR4 expression in mouse embryonic stem cells and in stem cell-derived vascular cells is regulated by epigenetic modifications

Embryonic stem (ES) cells and ES cell-derived differentiated cells can be used in tissue regeneration approaches. However, inflammation may pose a major hurdle. To define the inflammatory response of ES and ES cell-derived vascular cells, we exposed these cells to LPS. With the exception of MIF no significant cytokine mRNA levels were observed either at baseline or after stimulation. Further experiments revealed that these cells do not express TLR4. Analysis of the DNA methylation status of the TLR4 upstream region showed increased methylation. Moreover, in vitro methylation suppressed TLR4 promoter activity in reporter gene assays. ChIP assays showed that in this region histones H3 and H4 are hypoacetylated in ES cells. Interestingly, 5-aza-dC or TSA partially relieves this gene repression. Finally, the increased levels of TLR4 observed in ES cells after treatment with 5-aza-dC or TSA confer responsiveness to LPS, as induction of IL-6 and TNFalpha mRNA was detected in endotoxin stimulated ES cells.