Reversing thyroid hormone mediated repression of a HSV-1 promoter via computationally guided mutagenesis

Abstract

Thyroid hormones (TH or T3) and their DNA binding nuclear receptors (TRs), direct transcriptional regulation in diverse ways depending on the host cell environment and specific promoter characteristics of TH sensitive genes. This study sought to elucidate the impact on transcriptional repression of nucleotide sequence/orientation within TR binding sites, TR elements, (TREs) of TH sensitive promoters, to better understand ligand dependent transcriptional repression of wild-type promoters. Computational analysis of the HSV-1 thymidine kinase (TK) gene TRE bound by TR and RXR revealed a single TRE point mutation sufficient to reverse the TRE orientation. In vitro experiments corroborated that the TRE point mutation exhibited distinct impacts on promoter activity, sufficient to reverse the TH dependent negative regulation in neuro-endocrine differentiated cells. EMSA and ChIP experiments suggest that this point mutation altered the promoter's regulatory mechanism through discrete changes in transcription factor TR occupancy and altered enrichment of repressive chromatin modification, histone-3-lysine-9-trimethyl (H3K9Me3). Insights relating to this negative TRE (nTRE) mechanism impacts the understanding of other nTREs and TRE mutations associated with TH and herpes diseases.