Effect of 5-Aza-2´-Deoxycytidine and Trichostatin A on Endogenous Versus Ectopic Expression of Placental Members of the Human Growth Hormone Gene Family

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Ganguly, Esha
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Background: The genes coding for human (h) chorionic somatomammotropin (CS), hCS-A and hCS-B, and placental growth hormone (GH-V), hGH-V are located at a single locus on chromosome 17q22-24. Local regulatory (5´ P and 3´ enhancer) sequences and a remote locus control region (LCR) containing a placenta-specific hypersensitive site (HS) IV, have been implicated in the efficient expression of the placental hCS/GH-V genes, in part through gene transfer studies in placental and non-placental tumor cell lines. However, low levels of endogenous expression are reported in placental tumor cells compared to normal term placenta. Thus it was hypothesized that the hCS/GH-V chromatin structure in human choriocarcinoma cells is less accessible to regulatory regions essential for efficient expression due to DNA and/or histone modifications, specifically methylation and acetylation, respectively. Approach: To assess individual hCS-A, hCS-B and hGH-V gene expression in placental and non-placental tumor cells, and assess the effect of increasing “chromatin accessibility” on hCS/GH-V RNA levels by inhibiting DNA methylation and histone deacetylation using 5-aza-2´-deoxycytidine (azadC) and trichostatin A (TSA). Principal Findings: Low levels of hCS-A, hCS-B and hGH-V RNA were detected in placental and non-placental tumor cells compared to term placenta. A significant >5-fold increase in promoter activity was seen in placental but not non-placental cells transfected with hybrid hCS promoter luciferase genes containing 3´-enhancer sequences. Placental JEG-3 cells pretreated with azadC and TSA resulted in a significant >10-fold increase in hCS-A, hCS-B and hGH-V RNA levels compared to TSA treatment alone, however, a modest ~3-fold effect was seen in non-placental MCF-7 cells. By contrast to the effect of pretreatment with azadC, post-treatment with azadC mutes the stimulatory effects of TSA on hCS/GH-V transcripts. The specificity of the response suggests that azadC treatment, and presumably hypomethylation of DNA, results in an increase in response to TSA and histone hyperacetylation at the hGH/CS locus. An assessment of histone H3/H4 hyperacetylation in JEG-3 cells treated with azadC and TSA versus TSA alone revealed significant increases consistent with a more open chromatin structure including the hCS 3´-enhancer sequences and LCR. Conclusions: These observations suggest that accessibility of remote and local regulatory regions required for efficient placental hGH/CS expression can be restricted by DNA methylation and histone acetylation status. This includes restricting access of the hCS 3´-enhancer sequences to available placental enhancer transcription factors.
5-Aza-2´-Deoxycytidine , Trichostatin A, Acetylation, DNA Methylation, Placental, GH-CS