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    • It is widely acknowledged that CpG methylation of

    2021-09-16

    It is widely acknowledged that CpG methylation of the promoter regions of some genes plays an important regulatory role in transcriptional regulation and in the establishment and maintenance of cell type-specific gene expression (Li, 2002, Jaenisch and Bird, 2003). This study obtained multiple lines of evidence that GHSR1A transcription is in fact influenced by methylation of the promoter region. First, a defined CpG island spanning the proximal promoter and exonic regions was identified in the rat Ghsr gene. This CpG island is highly structured and conserved in, at a minimum, several mammalian species, including mice and humans. Second, in multiple cell types including RC-4B/C-derived sublines, MSP and bisulfite sequencing revealed a strong inverse correlation between mRNA expression level and methylation status in the promoter region. Third, in a transient transfection reporter assay, in vitro methylation of the proximal Ghsr promoter sequence resulted in a significant reporter activity decrease. Fourth, in Pifithrin-α with low or absent Ghsr1a expression, epigenetic modifications by DNMT and/or HDAC inhibitors effectively induced re-expression of Ghsr1a mRNA. Finally, the patterns of histone H3 post-translational modifications occurring at the Ghsr proximal promoter were cell type-specific, and an active pattern of histone H3 modifications was obvious in Ghsr1a-expressing RC-4B/C subclonal cells and vice versa. It is noteworthy that the effects of 5-aza-dC and TSA differed significantly among cell types. In pituitary GH-secreting GH3 cells, treatment with 5-aza-dC, but not with TSA, resulted in a concentration-dependent re-expression of Ghsr1a transcription, whereas TSA alone induced a slight but significant upregulation in pancreatic acinar AR42J cells. In L6 myoblasts, the individual agents had a minimal effect on Ghsr1a expression, but a remarkable synergistic effect was demonstrated using a combination of these two agents. These results suggest that DNA methylation and histone acetylation contribute differently to Ghsr silencing in different cell types, and that DNA methylation is probably dominant over HDAC activity because the histone deacetylation by TSA alone appeared to be insufficient to “unlock” the Ghsr gene from the silenced chromatin state in cells with a densely methylated promoter (GH3 and L6), and 5-aza-dC had no effect on cells which displayed a relatively low level of promoter methylation (AR42J). In line with previous studies showing that the presence of DNA methylation can modify local histone modification patterns (Jaenisch and Bird, 2003, Lande-Diner et al., 2007), our ChIP results indicated that Ghsr gene silencing in cell lines results from increased levels of heterochromatin-associated H3K9 and H3K27 trimethylation in the proximal promoter region of Ghsr. In contrast, Ghsr1a-expressing RC-4B/C-H1 and H2 subclones showed depletions of H3K9 and H3K27 trimethylation, but enrichment of H3K4 trimethylation in the same region, thus displaying a modification pattern associated with transcriptionally permissive/active chromatin. Next, we examined whether the relationship between GHSR gene expression patterns and promoter DNA methylation, both of which occur in an essentially all-or-none fashion in cultured cell lines, is reproducibly detectable in normal tissues. However, our methylation analysis results on rat tissues in relation to a tissue-specific pattern or age-associated changes in Ghsr1a mRNA expression were apparently inconclusive. We did observe a non-significant trend for more methylation in non-expressing tissues (heart and liver), and found that, in the hypothalamus, an age-related decline in Ghsr1a mRNA is accompanied by increased methylation. However, age-related methylation changes were not reproducible in the pituitary, instead being seen in cardiac tissue, in which Ghsr1a mRNA was undetectable at all ages examined. These observations raise the possibility of altered methylation status being independent of gene expression changes, although in the hypothalamus they might contribute to or accelerate changes in gene expression. It should be noted that, in this study, only a small number of rat tissues were tested. Thus, there is a clear need to analyze more tissues, preferably at different developmental stages, to determine whether there are clear associations between Ghsr1a mRNA expression patterns and promoter DNA methylation. However, there is a major challenge in that the “normal” tissues used in our present experiments were likely comprised of DNA from a heterogeneous cell population with variable Ghsr1a mRNA expression levels, as well as from other sources such as connective tissue. Therefore, more elaborate methods analyzing methylation in a pure population of cells expressing Ghsr11 are also needed.