BMP-4 suppresses progesterone production by inhibiting histone H3 acetylation of StAR in bovine granulosa cells in vitro

This study examined the molecular mechanism by which BMP-4 inhibits progesterone production and the expression of genes involved in steroidogenesis. Granulosa cells were cultured in medium with or without BMP-4 for 0-96 h. BMP-4 inhibited progesterone secretion in granulosa cells and suppressed the expression of steroidogenic acute regulatory protein (StAR) at the mRNA and protein levels, whereas BMP-4 did not affect the proliferation of granulosa cells. In addition, we found that BMP-4 affected the expression of SR-B1 mRNA but not LDL-R in granulosa cells. To examine the protein-DNA interaction at specific sites within the StAR gene promoter, we used the quantitative real-time PCR and the ChIP technique. We demonstrated that BMP-4 suppresses the acetylation of histone H3 associated with the StAR promoter region at 48 and 72 h of culture in bovine granulosa cells. Our results showed for the first time that BMP-4 inhibited the acetylation of histone H3 associated with the StAR promoter region in bovine granulosa cells. Taken together, we propose that the inhibition of the acetylation of histone H3 associated with the StAR promoter region by BMP-4 may be one of the inhibitory molecular mechanisms of progesterone synthesis in granulosa cells. Our data suggested that theca cell-derived BMP-4 is important as a regulator of steroid hormone synthesis in granulosa cells during follicular development in the mammalian ovary.     

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.     

State of histone modification in the rat Ig-beta/growth hormone locus.

The state of acetylation in H3 and H4 histones and dimethylation in the H3 histone Lys4 residue were examined by chromatin immunoprecipitation (ChIP) at 11 targets in the rat Ig-beta/growth hormone locus. Marked enhancement of the acetylation of histones H3 and H4 and the dimethylation of H3 Lys4 was observed in the chromatin situated close to the promoter of an actively transcribed gene. Chromatin positioned near a cell-type-specific DNase I-hypersensitive site with enhancer activity had the same histone modifications as the active promoter. In one transcribed intron, chromatin with fewer histone modifications was found, and in another transcribed intron, chromatin with markedly enhanced modifications was found. In most cases, no appreciable difference in the acetylation of histones H3 and H4 was found at prominently enhanced targets. However, different acetylation levels of H3 and H4 were found at one target. The targets with enhanced dimethylation of the H3 Lys4 residue coincided with those with prominently enhanced acetylation of histones H3 and H4.     

Expression Levels of the BDNF Gene and Histone Modifications Around Its Promoters in the Ventral Tegmental Area and Locus Ceruleus of Rats During Forced Abstinence from Morphine

Brain-derived neurotrophic factor (BDNF) plays a role in mediating molecular, cellular, and behavioral adaptations underlying drug addiction. Here, we examined the influence of withdrawal from repeated morphine treatment on the expression of BDNF mRNA in the ventral tegmental area (VTA) and locus coeruleus (LC) of the rat brain. We also studied whether alternations in mRNA levels of BDNF in these tissues are associated with histone modifications around promoters II and III of the BDNF gene. Thus, chromatin immunoprecipitation (CHIP) and quantitative (q)-PCR were employed to assess acetylation of histone H3 at K9/K14 and trimethylation of histone H3 at K9. Results of qRT-PCR showed that levels of BDNF mRNA in both VTA and LC were significantly increased 7 days rather than 2 h or 24 h following the last injection of morphine. Consistently, CHIP and qPCR analysis revealed that on day 7 of morphine abstinence, both VTA and LC levels of histone methylation at BDNF promoters II and III of morphine treated rats were significantly lower than control animals. Morphine withdrawal caused only a significant increase in H3 acetylation at the promoter II in the LC. These data demonstrate the involvement of histone H3 methylation in the regulation of gene expression in the VTA and LC of rats during forced abstinence of morphine.