Chromatin Remodeling Is a Key Mechanism Underlying Cocaine-Induced Plasticity in Striatum

Genome-wide Analysis of Chromatin Regulation by Cocaine Reveals a Role for Sirtuins

Trophoblast cell lineage is established through the first cellular differentiation in mammalian embryogenesis, and its developmental potential is restricted to the extraembryonic tissues contributing solely to the placenta. Several lines of evidence suggest a relative lack of importance of DNA methylation in gene regulation in the extraembryonic tissues when compared with embryonic ones. Here we analyzed the dynamics of epigenetic status in the upstream region of mouse Ddah2 gene, which was found to be specifically repressed in a stem cell population of trophoblast cell lineage. We found a tissue-dependent differentially methylated region in the regulatory region of the Ddah2 gene. This region was hypermethylated in trophoblast stem cells and was hypomethylated in differentiated cells both in vivo and in vitro. This change was well correlated with Ddah2 expression. In addition, in vitro methylation confined to the differentially methylated region was sufficient to repress promoter activity in the reporter assay. Furthermore, a repressive pattern of histone modifications was formed around the differentially methylated region in undifferentiated trophoblast stem cells with repressed Ddah2. Our data suggest that DNA methylation-mediated chromatin remodeling is involved in the regulation of the Ddah2 gene expression and thus is important even in trophoblast cell lineage.

15-Lipoxygenase-1 (15-LOX-1) contributes significantly to inflammation regulation and terminal cell differentiation. 15-LOX-1 is transcriptionally silenced in cancer cells, and its transcriptional reactivation (e.g. via histone deacetylase inhibitors (HDACIs)) is essential for restoring terminal cell differentiation to cancer cells. STAT-6 acetylation via the histone acetyltransferase KAT3B has been proposed to be necessary for 15-LOX-1 transcriptional activation. However, the exact mechanism underlying 15-LOX-1 transcriptional reactivation in cancer cells is still undefined, especially in regard to the contribution of 15-LOX-1 promoter histone modifications. We therefore examined the relative mechanistic contributions of 15-LOX-1 promoter histone modifications and STAT-6 to 15-LOX-1 transcriptional reactivation by HDACIs in colon cancer cells. We found that: 1) histone H3 and H4 acetylation in the 15-LOX-1 promoter through KAT3B was critical to 15-LOX-1 transcriptional activation; 2) 15-LOX-1 transcription was activated independently from STAT-6; and 3) dimethyl-histone H3 lysine 9 (H3K9me2) demethylation in the 15-LOX-1 promoter via the histone lysine demethylase KDM3A was an early and specific histone modification and was necessary for activation of transcription. These findings demonstrate that histone modification in the 15-LOX-1 promoter is important to 15-LOX-1 transcriptional silencing in colon cancer cells and that HDACIs can activate gene transcription via KDM3A demethylation of H3K9me2.

Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of beta-casein gene expression involves two principal regulatory regions, a proximal promoter and a distal enhancer located in the mouse approximately -6 kb upstream of the transcription start site. Using a chromosome conformation capture assay and quantitative real time PCR, we demonstrate that a chromatin loop is created in conjunction with the recruitment of specific transcription factors and p300 in HC11 mammary epithelial cells. Stimulation with both prolactin and hydrocortisone is required for the induction of these long range interactions between the promoter and enhancer, and no DNA looping was observed in nontreated cells or cells treated with each of the hormones separately. The lactogenic hormone-induced interaction between the proximal promoter and distal enhancer was confirmed in hormone-treated primary three-dimensional mammary acini cultures. In addition, the developmental regulation of DNA looping between the beta-casein regulatory regions was observed in lactating but not in virgin mouse mammary glands. Furthermore, beta-casein mRNA induction and long range interactions between these regulatory regions were inhibited in a progestin-dependent manner following stimulation with prolactin and hydrocortisone in HC11 cells expressing human PR-B. Collectively, these data suggest that the communication between these regulatory regions with intervening DNA looping is a crucial step required to both create and maintain active chromatin domains and regulate transcription.

Epigenetic regulation of oligodendrocyte identity

Decoding the Epigenetic Language of Neuronal Plasticity

Heat shock transcription factors (HSFs) regulate gene expression in response to heat shock and in physiological conditions. In mammals, HSF1 is required for heat-mediated induction of classic heat shock genes; however, we do not know the molecular mechanisms by which HSF4 regulates gene expression or the biological consequences of its binding to chromatin. Here, we identified that HSF4 binds to various genomic regions, including the introns and distal parts of protein-coding genes in vivo in mouse lenses, and a substantial numbers of the regions were also occupied by HSF1 and HSF2. HSF4 regulated expression of some genes at a developmental stage when HSF1 and HSF2 expression decreased. Although HSF4 binding did not affect expression of many genes, it induces demethylated status of histone H3K9 on the binding regions. Unexpectedly, a lot of HSF4 targets were induced by heat shock treatment, and HSF4 is required for induction of a set of non-classic heat shock genes in response to heat shock, in part by facilitating HSF1 binding through chromatin modification. These results suggest novel mechanisms of gene regulation controlled by HSF4 in non-classic heat shock response and in lens development.

Lipin 1 plays a crucial role in lipid metabolism in adipose tissue, skeletal muscle, and liver. Its physiological role involves two cellular functions: regulation of phosphatidate phosphatase activity and regulation of fatty acid oxidation. In this study, we have demonstrated that lipin 1 gene (LPIN1) expression is regulated by cellular sterols, which are key regulators of lipid metabolism. We have also characterized the sterol-response element and nuclear factor Y-binding sites in the human LPIN1 promoter. Using a luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of LPIN1 gene, mediated by SREBP-1 (sterol regulatory element-binding protein 1) and nuclear factor Y. Furthermore, we investigated whether lipin 1 is involved in lipogenesis by transfection of LPIN1 small interfering RNA. We infer that sterol-mediated regulation of lipin 1 gene transcription modulates triglyceride accumulation. This modulation involves changes in the activity of phosphatidate phosphatase.

Estrogen-related receptor alpha (ERRalpha), a member of the nuclear receptor superfamily, is closely related to the estrogen receptors (ERalpha and ERbeta). The ERRalpha gene is estrogen-responsive in several mouse tissues and cell lines, and a multiple hormone-response element (MHRE) in the promoter is an important regulatory region for estrogen-induced ERRalpha gene expression. ERRalpha was recently shown to be a negative prognostic factor for breast cancer survival, with its expression being highest in cancer cells lacking functional ERalpha. The contribution of ERRalpha in breast cancer progression remains unknown but may have important clinical implications. In this study, we investigated ERRalpha gene expression and chromatin structural changes under the influence of 17beta-estradiol in both ER-positive MCF-7 and ER-negative SKBR3 breast cancer cells. We mapped the nucleosome positions of the ERRalpha promoter around the MHRE region and found that the MHRE resides within a single nucleosome. Local chromatin structure of the MHRE exhibited increased restriction enzyme hypersensitivity and enhanced histone H3 and H4 acetylation upon estrogen treatment. Interestingly, estrogen-induced chromatin structural changes could be repressed by estrogen antagonist ICI 182 780 in MCF-7 cells yet were enhanced in SKBR3 cells. We demonstrated, using chromatin immunoprecipitation assays, that 17beta-estradiol induces ERRalpha gene expression in MCF-7 cells through active recruitment of co-activators and release of co-repressors when ERRalpha and AP1 bind and ERalpha is tethered to the MHRE. We also found that this estrogen effect requires the MAPK signaling pathway in both cell lines.

In budding yeast and human cells, ING (inhibitor of growth) tumor suppressor proteins play important roles in response to DNA damage by modulating chromatin structure through collaborating with histone acetyltransferase or histone deacetylase complexes. However, the biological functions of ING family proteins in fission yeast are poorly defined. Here, we report that Png1p, a fission yeast ING homolog protein, is required for cell growth under normal and DNA-damaged conditions. Png1p was further confirmed to regulate histone H4 acetylation through collaboration with the MYST family histone acetyltransferase 1 (Mst1). Additionally, both fission yeast PNG1 and MST1 can functionally complement their budding yeast correspondence homologs YNG2 and ESA1, respectively. These results suggest that ING proteins in fission yeast might also conserve function, similar to ING proteins in budding yeast and human cells. We also showed that decreased acetylation in Deltapng1 cells resulted in genome-wide down-regulation of 756 open reading frames, including the central DNA repair gene RAD22. Overexpression of RAD22 partially rescued the png1 mutant phenotype under both normal and DNA-damaged conditions. Furthermore, decreased expression of RAD22 in Deltapng1 cells was confirmed to be caused by decreased H4 acetylation at its promoter. Altogether, these results indicate that Png1p is required for histone H4 acetylation and functions upstream of RAD22 in the DNA damage response pathway.

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

Cocaine Regulates MEF2 to Control Synaptic and Behavioral Plasticity

The human T-cell leukemia virus type 1 (HTLV-1) is integrated into the host cell DNA and assembled into nucleosomes. Within the repressive chromatin environment, the virally encoded Tax protein mediates the recruitment of the coactivators CREB-binding protein/p300 to the HTLV-1 promoter, located within the long terminal repeats (LTRs) of the provirus. These proteins carry acetyltransferase activity that is essential for strong transcriptional activation of the virus in the context of chromatin. Consistent with this, the amino-terminal tails of nucleosomal histones at the viral promoter are acetylated in Tax-expressing cells. We have developed a system in which we transfect Tax into cells carrying integrated copies of the HTLV-1 LTR driving the luciferase gene to analyze changes in "activating" histone modifications at the LTR. Unexpectedly, Tax transactivation led to an apparent reduction of these modifications at the HTLV-1 promoter and downstream region that correlates with a similar reduction in histone H3 and linker histone H1. Micrococcal nuclease protection analysis showed that less LTR-luciferase DNA is nucleosomal in Tax-expressing cells. Furthermore, nucleosome depletion correlated with RNA polymerase II recruitment and loss of SWI/SNF. The M47 Tax mutant, deficient in HTLV-1 transcriptional activation, was also defective for nucleosome depletion. Although this mutant formed complexes with CREB and p300 at the HTLV-1 promoter in vivo, it was unable to mediate RNA polymerase II recruitment or SWI/SNF displacement. These results support a model in which nucleosomes are depleted from the LTR and transcribed region during Tax-mediated transcriptional activation and correlate RNA polymerase II recruitment with nucleosome depletion.

Previous biochemical studies have demonstrated that Lys-123 ubiquitination of histone H2B is globally required for up-regulation of mono-, di, and trimethylation of Lys-4 of histone H3. However, recent studies have implicated H2B-Lys-123 ubiquitination in the regulation of di- and trimethylation, but not monomethylation, of H3-Lys-4 in vivo. Using a formaldehyde-based cross-linking and chromatin immunoprecipitation assay, we show that H3-Lys-4 trimethylation, but not dimethylation, is up-regulated by H2B-Lys-123 ubiquitination in vivo at the coding sequences of a set of transcriptionally active genes such as ADH1, PHO84, and PYK1. Both the ubiquitination of H2B-Lys-123 and the methylation of H3-Lys-4 are dispensable for recruitment of RNA polymerase II to the coding sequences of these genes, and hence, their transcription is not altered in the absence of these covalent modifications. However, recruitment of RNA polymerase II to the coding sequence of a galactose-inducible gene, GAL1, is significantly reduced in the absence of H2B-Lys-123 ubiquitination but not H3-Lys-4 methylation. Consistently, transcription of GAL1 is altered in the H2B-K123R point mutant strain. Finally, we show that H3-Lys-4 methylation does not regulate H3-Lys-9/14 acetylation. Collectively, our data reveal a "trans-tail" regulation of H3-Lys-4 tri- but not dimethylation by H2B-Lys-123 ubiquitination, and these modifications are dispensable for transcription of a certain set of genes in vivo.

IBD Genetics: Focus on (Dys) Regulation in Immune Cells and the Epithelium