Transcriptional and epigenetic regulation of labour associated inflammatory genes in the amnion

Research Doctorate - Doctor of Philosophy (PhD)Inflammatory genes are activated in the pregnant uterus at term labour producing an “acute inflammation gene expression signature” even in the absence of infection or chorio-amnionitis. The mechanisms that activate the inflammatory pathways and determine the timing of labour are unknown. The primary objective of my research presented in the thesis was to determine the molecular mechanisms that regulate the expression of labour-promoting inflammatory genes in the human fetal membranes at term parturition in vivo. Previous work in cell culture models suggested the involvement of the NFĸB system and glucocorticoids in the upregulation of these genes including the prototypical proinflammatory gene, PTGS2, in the amnion. Epigenetic mechanisms, such as DNA methylation and histone modifications, were also hypothesised to participate in the control of proinflammatory gene activity in the fetal membranes during pregnancy. Chromatin immunoprecipitation with freshly delivered, term amnion tissue identified TBP, NFkB p65 and p50 subunit binding at the proximal promoter of the PTGS2 gene, but binding was not associated with transcriptional stimulation. However these transcription factors stimulated IĸBα gene expression in the same samples indicating the activation of NFκB signalling. Increased histone-3 and -4 acetylation was found in the proximal 1000 bp region of the PGHS-2 promoter suggesting that an open chromatin structure, permissive of gene expression, was present at term and after labour. In short term amnion explants (≤24 h) the glucocorticoid dexamethasone decreased PTGS2 gene activity and mRNA levels. Glucocorticoid receptor-α (GRα) binding to the PTGS2 promoter decreased initiating (Ser-5) and elongating (Ser-2) pol-II phosphorylation. Accumulation of pol II in the 5’-region of the PTGS2 gene indicated post-initiation pausing. Acetylation of Histones -3 and -4 decreased, but histone-3 methylation was unchanged. The data indicated rapid PTGS2 mRNA turnover <i>in vivo</i>, which slowed quickly in the explants due to declining transcription rate and increased mRNA stability. The transcriptional mechanism thus alters profoundly <i>in vitro</i>, even in short term explant cultures. DNA methylation of the inflammatory genes PTGS2, BMP2, NAMPT and CXCL2, and the glucocorticoid, progesterone and oestrogen receptor genes, was examined using the Methyl Profiler PCR system and bisulfite sequencing with fresh tissue samples. Variable proportions of inflammatory and steroid receptor gene copies, to a maximum of 50.9%, were densely methylated in both amnion and decidua tissues consistent with repression. Densely methylated copy proportions were significantly different between genes showing no relationship with varying expression during pregnancy, between tissues and in individuals. Methylated copy proportions of all genes in amnion and most genes in decidua were highly correlated in individuals. DNMT1 and -3A were expressed in both tissues with significantly higher levels in the amnion at 11–17 weeks than at term. Collectively, the data generated by my PhD research suggest that PTGS2 expression is suppressed in term amnion <i>in vivo</i> and endogenous glucocorticoid may be involved in this process. Epigenetic changes occur rapidly <i>in vitro</i> potentially influencing gene ``expression. Pol-II pausing limits PTGS2 transcriptional activity in the amnion and this regulatory mechanism is associated with the changing phosphorylation of Pol-II. The DNA methylation study found the unmethylated portion of gene copies is responsible for the regulated expression in the amnion. Dense methylation of individually variable gene copy proportions likely occurs in the first trimester amnion, influenced by DNA sequence context and affected strongly by individual circumstances. The experiments described in the thesis provide important insights into the control of inflammatory gene activation in pregnancy, emphasising the need for techniques and approaches that detect or preserve the <i>in vivo</i> condition of the gestational tissues. Information obtained by these approaches supplements and places into context the results generated by experimental models describing molecular mechanisms potentially underpinning pregnancy maintenance and parturition