Hypermethylation of metallothionein-I promoter and suppression of its induction in cell lines overexpressing the large subunit of Ku protein.

We have shown previously that the heavy metal-induced metallothionein-I (MT-I) gene expression is specifically repressed in a rat fibroblast cell line (Ku-80) overexpressing the 80-kDa subunit of Ku autoantigen but not in cell lines overexpressing the 70-kDa subunit or Ku heterodimer. Here, we explored the molecular mechanism of silencing of MT-I gene in Ku-80 cells. Genomic footprinting analysis revealed both basal and heavy metal-inducible binding at specific cis elements in the parental cell line (Rat-1). By contrast, MT-I promoter in Ku-80 cells was refractory to any transactivating factors, implying alteration of chromatin structure. Treatment of two clonal lines of Ku-80 cells with 5-azacytidine, a potent DNA demethylating agent, rendered MT-I gene inducible by heavy metals, suggesting that the gene is methylated in these cells. Bisulfite genomic sequencing revealed that all 21 CpG dinucleotides in MT-I immediate promoter were methylated in Ku-80 cells, whereas only four CpG dinucleotides were methylated in Rat-1 cells. Almost all methylated CpG dinucleotides were demethylated in Ku-80 cells after 5-azacytidine treatment. To our knowledge, this is the first report that describes hypermethylation of a specific gene promoter and its resultant silencing in response to overexpression of a cellular protein.     

Chromatin modification and the endothelial-specific activation of the E-selectin gene

E-selectin plays a role in the binding and extravasation of leukocytes from the bloodstream. The E-selectin gene is rapidly and transiently expressed by endothelial cells activated by inflammatory stimuli. Despite the identification of factors critical for cytokine-induced activation of the E-selectin promoter, little is known about the mechanisms that restrict the gene expression to endothelial cells. We used in vivo approaches to characterize the E-selectin promoter in primary cultures of human umbilical vein endothelial cells and umbilical artery smooth muscle cells. In endothelial cells specifically, nucleosomes are remodeled after tumor necrosis factor (TNF) alpha induction. Chromatin immunoprecipitation analysis demonstrated the binding of the p65 (RelA) component of nuclear factor-kappa B (NF-kappa B) to the endogenous E-selectin promoter after TNFalpha stimulation along with IkappaB kinase alpha. Multiple coactivators, including p300, steroid receptor coactivator-1, and p300/cAMP-response element-binding protein (CREB)-binding protein (CBP)-associated factor localize differentially to the E-selectin promoter. Additionally, TNFalpha induced localized histone hyperacetylation, phosphorylation, and methylation in the E-selectin gene specifically in endothelial cells. Post-induction repression of E-selectin expression is associated with recruitment of multiple deacetylases. Collectively, these studies suggest a model for the selective induction of the E-selectin gene in which the core promoter chromatin architecture is specifically modified in endothelial cells.     

Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

We purified the KH-type splicing regulatory protein (KSRP) as a protein interacting with the 3'-untranslated region (3'-UTR) of the human inducible nitric oxide (iNOS) mRNA. Immunodepletion of KSRP enhanced iNOS 3'-UTR RNA stability in in vitro-degradation assays. In DLD-1 cells overexpressing KSRP cytokine-induced iNOS expression was markedly reduced. In accordance, downregulation of KSRP expression increases iNOS expression by stabilizing iNOS mRNA. Co-immunoprecipitations showed interaction of KSRP with the exosome and tristetraprolin (TTP). To analyze the role of KSRP binding to the 3'-UTR we studied iNOS expression in DLD-1 cells overexpressing a non-binding mutant of KSRP. In these cells, iNOS expression was increased. Mapping of the binding site revealed KSRP interacting with the most 3'-located AU-rich element (ARE) of the human iNOS mRNA. This sequence is also the target for HuR, an iNOS mRNA stabilizing protein. We were able to demonstrate that KSRP and HuR compete for this binding site, and that intracellular binding to the iNOS mRNA was reduced for KSRP and enhanced for HuR after cytokine treatment. Finally, a complex interplay of KSRP with TTP and HuR seems to be essential for iNOS mRNA stabilization after cytokine stimulation.     

Effect of 27nt small RNA on endothelial nitric-oxide synthase expression

We have reported previously that the 27nt repeat polymorphism in endothelial nitric-oxide synthase (eNOS) intron 4-a source of 27nt small RNA-inhibits eNOS expression. In the current study, we have investigated how 27nt small RNA suppresses eNOS expression. Using a chromatin immunoprecipitation assay, we examined histone acetylation in the 27nt repeat element of eNOS intron 4, the promoter region up to -1486 bp, and the 5' enhancer region (-4583/-4223bp) in human aortic endothelial cells (HAECs) treated with 27nt RNA duplex. 27nt RNA duplex induced hyperacetylation in H3 (lysine8, 12, and 23) and H4 (lysine 9 and 12) at the 27nt repeat element, which then interacted with nuclear actin, histone deacetylase 3 (HDAC3), and NonO proteins. In contrast, the histone H3 and H4 became hypoacetylated at the eNOS core promoter. HAECs treated with 27nt RNA duplex had reduced eNOS expression, but treatment with either HDAC3 small interfering RNA or NonO siRNA significantly attenuated the 27nt small RNA-induced suppression. We further found that 27nt small RNA induced DNA methylation in a region approximately 750nt upstream of the intron 4 repeats, and a methyltransferase inhibitor reversed the effect on methylation and eNOS expression. Our study demonstrates that 27nt small RNA may suppress eNOS expression by altering histone acetylation and DNA methylation in regions adjacent to the 27nt repeat element and core promoter.     

GAD1 mRNA expression and DNA methylation in prefrontal cortex of subjects with schizophrenia

Dysfunction of prefrontal cortex in schizophrenia includes changes in GABAergic mRNAs, including decreased expression of GAD1, encoding the 67 kDa glutamate decarboxylase (GAD67) GABA synthesis enzyme. The underlying molecular mechanisms remain unclear. Alterations in DNA methylation as an epigenetic regulator of gene expression are thought to play a role but this hypothesis is difficult to test because no techniques are available to extract DNA from GAD1 expressing neurons efficiently from human postmortem brain. Here, we present an alternative approach that is based on immunoprecipitation of mononucleosomes with anti-methyl-histone antibodies differentiating between sites of potential gene expression as opposed to repressive or silenced chromatin. Methylation patterns of CpG dinucleotides at the GAD1 proximal promoter and intron 2 were determined for each of the two chromatin fractions separately, using a case-control design for 14 schizophrenia subjects affected by a decrease in prefrontal GAD1 mRNA levels. In controls, the methylation frequencies at CpG dinucleotides, while overall higher in repressive as compared to open chromatin, did not exceed 5% at the proximal GAD1 promoter and 30% within intron 2. Subjects with schizophrenia showed a significant, on average 8-fold deficit in repressive chromatin-associated DNA methylation at the promoter. These results suggest that chromatin remodeling mechanisms are involved in dysregulated GABAergic gene expression in schizophrenia.