Transcriptional control of human glucocorticoid receptor. (Doctoral thesis)

The hypothalamus-pituitary-adrenocortical (HPA) axis is one of the major stress response systems in human body, it maintains homeostasis and adaptation during challenges. Cortisol, the key stress mediators in human, exerts profound effects on a wide range of physiological and developmental processes that are crucial for the adaptation to stress. Cortisol action is mediated by two corticosteroid receptors including the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). In the human brain, the co-expression of GR and MR in the hippocampus plays an important role in regulating the HPA negative feedback loop and the balance between both receptors may be involved in vulnerability to disease. GR is ubiquitously distributed, but expression levels vary widely between tissues. The complicated promoter region of the GR plays a pivotal role in the regulation of GR levels.
Among the multiple promoters, in particular, promoter 1F, is susceptible to methylation by adverse early life events. CpG methylation in promoter 1F is thought to interfere with transcription factor binding, subsequently inhibiting transcription and consequently lowering mRNA and protein levels. The research presented in this thesis explored several layers of complexity in GR transcriptional regulation. The promoter activity of multiple alternative GR first exons which are located in an upstream CpG island was investigated. Subsequently, the susceptibility of GR promoters to epigenetic modification was examined. Furthermore, the new SNPs occurring in upstream promoter regions and their functions were tested by genotyping 221 donors, revealing a new promoter-specific haplotype and 5 new SNPs lowering promoter activity. The role of NGFI-A and several other transcription factors on exon 1F regulation and the epigenetic sensitivity of promoter 1F by performing the single CpG dinucleotide methylation was studied. In addition, the distribution patterns of GR first exon transcripts, 5’ splice variants, GR/MR ratio and methylation status in GR promoters in different human health brain regions was determined.
The findings in this thesis showed that GR first exons are independently controlled by a unique promoter located directly upstream and promoter activities were cell type-specific, and varied considerably between cell types. These promoters were susceptible to silencing by methylation and the activity of the individual promoters was also modulated by sequence variants (SNPs). We provide evidence that E2F1 is a major element in the transcriptional complex capable of driving the expression of GR 1F transcripts and that single CpG dinucleotide methylation can not mediate the inhibition of transcription in vitro. We showed that the GR first exons distribution is expressed throughout the human brain with no region specific alternative first exon usage. GR 3’ splice variants (GR_ and GRP) were equally distributed in all the brain regions. These data mirrored the consistently low levels of methylation in the brain, and the observed homogeneity throughout the studied regions.