Homeostatic balance of histone acetylation and deconstruction of repressive chromatin marker H3K9me3 during adipocyte differentiation of 3T3-L1 cells

Background Adipocyte differentiation is completed by changing gene expression. Chromatin is closely related to gene expression. Therefore, its structure might be changed for adipocyte differentiation. Mouse 3T3-L1 preadipocytes have been used as a cell model to study molecular mechanisms of adipogenesis. Objective To examine changes of chromatin modification and expression of histone modifying enzymes during adipocyte differentiation. Methods Microscopic analysis and Oil Red O staining were performed to determine distinct phenotype of adipocyte differentiation. RT-PCR and Western blot analysis were used to examine expression levels of histone modifying enzymes during adipocyte differentiation. Histone modifications were examined by immunostaining analysis. Results Expression levels of P300 and cbp were increased during adipocyte differentiation. However, acetylation of histones was not quantitatively changed postdifferentiation of 3T3-L1 cells compared to that at pre-differentiation. RT-PCR and Western blot analyses showed that expression levels of hdac2 and hdac3 were increased during adipocyte differentiation, suggesting histone acetylation at chromatin level was homeostatically controlled by increased expression of both HATs and HDACs. Tri-methylation level of H3K9 (H3K9me3), but not that of H3K27me3, was significantly decreased during adipocyte differentiation. Decreased expression of setdb1 was consistent with reduced pattern of H3K9me3. Knock-down of setdb1 induced adipocyte differentiation. This suggests that setdb1 is a key chromatin modifier that modulates repressive chromatin. Conclusion These results suggest that there exist extensive mechanisms of chromatin modifications for homeostatic balance of chromatin acetylation and deconstruction of repressive chromatin during adipocyte differentiation.     

Cyclin D1 inhibits peroxisome proliferator-activated receptor gamma-mediated adipogenesis through histone deacetylase recruitment

The cyclin D1 gene encodes the labile serum-inducible regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. Overexpression of cyclin D1 promotes cellular proliferation and normal physiological levels of cyclin D1 function to inhibit adipocyte differentiation in vivo. We have previously shown that cyclin D1 inhibits peroxisome proliferator-activated receptor (PPAR)gamma-dependent activity through a cyclin-dependent kinase- and retinoblastoma protein-binding-independent mechanism. In this study, we determined the molecular mechanism by which cyclin D1 regulated PPARgamma function. Herein, murine embryonic fibroblast (MEF) differentiation by PPARgamma ligand was associated with a reduction in histone deacetylase (HDAC1) activity. Cyclin D1-/- MEFs showed an increased propensity to undergo differentiation into adipocytes. Genetic deletion of cyclin D1 reduced HDAC1 activity. Reconstitution of cyclin D1 into the cyclin D1-/- MEFs increased HDAC1 activity and blocked PPARgamma-mediated adipogenesis. PPARgamma activity was enhanced in cyclin D1-/- cells. Reintroduction of cyclin D1 inhibited basal and ligand-induced PPARgamma activity and enhanced HDAC repression of PPARgamma activity. Cyclin D1 bound HDAC in vivo and preferentially physically associated with HDAC1, HDAC2, HDAC3, and HDAC5. Chromatin immunoprecipitation assay demonstrated that cyclin D1 enhanced recruitment of HDAC1 and HDAC3 and histone methyltransferase SUV39H1 to the PPAR response element of the lipoprotein lipase promoter and decreased acetylation of total histone H3 and histone H3 lysine 9. Collectively, these studies suggest an important role of cyclin D1 in regulation of PPARgamma-mediated adipocyte differentiation through recruitment of HDACs to regulate PPAR response element local chromatin structure and PPARgamma function.     

Ligand-dependent activation of the farnesoid X-receptor directs arginine methylation of histone H3 by CARM1

In this study we demonstrate that the class II nuclear hormone receptor, farnesoid X-receptor (FXR), incorporates histone methyltransferase activity within the gene locus for bile salt export pump (BSEP), a well established FXR target gene that functions as an ATP-dependent canalicular bile acid transporter. This methyltransferase activity is directed specifically to arginine 17 of histone H3. We demonstrate that FXR is directly associated with co-activator-associated arginine methyltransferase 1 (CARM1) activity. Furthermore, we show by chromatin immunoprecipitation that the ligand-dependent activation of the human BSEP locus is associated with a simultaneous increase of FXR and CARM1 occupation. The increased occupation of the BSEP locus by CARM1 also corresponds with the increased deposition of Arg-17 methylation and Lys-9 acetylation of histone H3 within the FXR DNA-binding element of BSEP. Consistent with these findings, CARM1 led to increased BSEP promoter activity with an intact FXR regulatory element, whereas CARM1 failed to transactivate the BSEP promoter with a mutated FXRE. Induction of endogenous BSEP mRNA and Arg-17 methylation by FXR regulatory element ligand, CDCA, requires CARM1 activity. Therefore, histone methylation at Arg-17 by CARM1 is a downstream target of signaling through ligand-mediated activation of FXR. Our studies provide evidence that FXR directly recruits specific chromatin modifying activity of CARM1 necessary for full potentiation of the BSEP locus in vivo.     

Methylation at arginine 17 of histone H3 is linked to gene activation

The nuclear hormone receptor co-activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro. The methyltransferase activity of CARM1 is necessary for its co-activator functions in transient transfection assays. However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on histones. We have raised an antibody that specifically recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1. Using this antibody we show that methylated R17 exists in vivo. Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor-regulated pS2 gene is activated. Coincident with the appearance of methylated R17, CARM1 is found associated with the histones on the pS2 gene. Together these results demonstrate that CARM1 is recruited to an active promoter and that CARM1-mediated R17 methylation on histone H3 takes place in vivo during this active state.     

Histone Acetylation Regulation in Sleep Deprivation-Induced Spatial Memory Impairment

Sleep disorders negatively affect cognition and health. Recent evidence has indicated that chromatin remodeling via histone acetylation regulates cognitive function. This study aimed to investigate the possible roles of histone acetylation in sleep deprivation (SD)-induced cognitive impairment. Results of the Morris water maze test showed that 3 days of SD can cause spatial memory impairment in Wistar rats. SD can also decrease histone acetylation levels, increase histone deacetylase 2 (HDAC2) expression, and decrease histone acetyltransferase (CBP) expression. Furthermore, SD can reduce H3 and H4 acetylation levels in the promoters of the brain-derived neurotrophic factor (Bdnf) gene and thus significantly downregulate BDNF expression and impair the activity of key BDNF signaling pathways (pCaMKII, pErk2, and pCREB). However, treatment with the HDAC inhibitor trichostatin A attenuated all the negative effects induced by SD. Therefore, BDNF and its histone acetylation regulation may play important roles in SD-induced spatial memory impairment, whereas HDAC inhibition possibly confers protection against SD-induced impairment in spatial memory and hippocampal functions.