Reversing the Thyroid Hormone Mediated Repression of a HSV-1 Promoter through Computationally Guided Site Directed Mutagenesis

Abstract

Thyroid hormones (TH or T3) and their DNA binding nuclear receptors (TRs), direct transcriptional regulation in different ways depending on the host cell environment and specific promoter characteristics of TH sensitive genes. This study sought to elucidate the impact on repression of nucleotide sequence/orientation of TR binding sites, TR elements, (TREs) within TH sensitive 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 promoters regulatory mechanism through discrete changes in transcription factor Sp1 and TR occupancy and altered enrichment of repressive chromatin, histone-3-lysine-9-trimethyl (H3K9Me3). Incites relating to this negative TRE (nTRE) mechanism impacts the understanding of other nTREs and TRE mutations associated with TH and herpes diseases.