Molecular Mechanism Of Orphan Nuclear Receptor HB1F Transcriptional Activation

Orphan nuclear receptor human B1 binding factor (hB1F, also known as LRH-1, FTF, CPF, and nominated as NR5A2) is a member of the FTZ-F1 subfamily of nuclear receptor superfamily. It was firstly cloned as a novel transcription factor in our previous study of the enhancer II (ENII) of hepatitis B virus (HBV). Accumulating data on the biological function of hB1F has shown in recent years that hB1F plays important roles in regulating the expression of a number of cellular and viral genes which are actively involved in a wide range of biological processes such as the bile acid biosynthesis, liver specific gene regulatory network and hepatitis B virus replication However, experimental evidences on the functional domains of hB1F and the molecular mechanism underlying hB1F transcriptional activation are limited. In this dissertation, the functional domains and potential regulation mechanisms of hB1F transcriptional activation have been studied.Initially, multiple domains that are essential or important for transactivation of hB1F were revealed by reporter gene assays of progressive N-terminal deletions as well as point mutation analysis. A proximal helix downstream of the hinge region, (the helix 1 of LBD according to structure prediction) (aa257-264), was identified to be essential for the transcriptional activity of receptor. A conserved domain in the central of hinge region (aa219-227) was also characterized, which imposed a strong repression effect on the activity of receptor. Moreover, two other domains (aa 186-218 and 236-243 respectively) that apparently regulated the transcriptional activity of hB1F were also identified. Their functions might be mediated by the phosphorylation modification of protein, prompted by point mutation analysis. Subsequently, a directed physical and functional interaction between hB1F and p160 coactivator SRC-1 was determined. This interaction employs a novel pattern that has not been previously described between SRC-1 and nuclear receptors, which requires a region containing the glutamine-rich domain of SRC-1, and the helix 1 and AF-2 of the hB1F LBD. Furthermore, the activation domain 1 (AD1) of SRC-1 was required for the coactivation of hB1F, and that was synergied by the general coactivator CREB-binding protein (CBP). Additionally, we also demonstrated that corepressor SMRT specifically repressed the transcriptional activity of hB1F via an indirect interaction.Finally, a specific auto-activation domain (AF-3) of hB1F was investigated. The minimal domain of AF-3 was delimited in the residues 236-270 by deletion mapping analysis, which located at the boundary of hinge region and LBD. It was comprised of a typical amphipathic α-helix (helix 1 of LBD) and proximal sequences, and the hydrophobic residues of helix 1 and a potential phosphorylation site (S242) were pivotal for its activity. The function of AF-3 was apparently enhanced by the general coactivators CBP/p300, and repressed by SRMT. Moreover, the interaction between AF-3 and C/H3 domain of CBP/p300 was demonstrated by two-hybrid assays in Huh7 cells. Despite its physiological significance needed further exploration, the identification of AF-3 of hB1F will facilitate us to understand the elaborated mechanism of hB1F transcriptional activation.Taken together, the work presented here facilitates the understanding of the molecular basis on hB1F-mediated transcription and provides new information for the molecular mechanism of transcriptional activation of FTZ-F1 related receptors.