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

Robert W Figliozzi,Shaochung V Hsia,Feng Chen

doi:10.1242/jcs.204222

Abstract

Thyroid hormones (THs) 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 or orientation within TR binding sites - the TH response 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 retinoid X receptor (RXR) revealed a single TRE point mutation sufficient to reverse the TRE orientation. In vitro experiments showed that the TRE point mutation had distinct impacts on promoter activity, sufficient to reverse the TH-dependent negative regulation in neuroendocrine differentiated cells. This point mutation altered the promoter's regulatory mechanism by discrete changes in transcription factor TR occupancy and altered enrichment of the repressive chromatin modification of histone-3-lysine-9-trimethyl (H3K9Me3). Insights relating to this negative TRE (nTRE) mechanism aids our understanding of other nTREs and TRE mutations associated with TH and herpes diseases.

Highlights

Thyroid hormones [THs; thyroxine (T4) and triiodothyronine (T3)] and their nuclear receptor family members, thyroid hormone receptors (TRs), have the ability to increase or decrease the rate of transcription of target genes (Lazar, 1993)
Computer analyses of putative binding of TR to alternative TH response elements (TREs) Swiss PDB viewer (SPV) was used to generate two pdb files from 2NLL, each with either the DNA binding domain of TRβ or RXRα bound to its half-site
Bioinformatics analyses have demonstrated a pair of non-traditional palindromic thymidine kinase (TK) TREs located between the TATA box and the transcription initiation site, in thyroid-stimulating hormone alpha (TSHα) TREs, one of the most well-characterized negative TREs (Carr et al, 1992; Hollenberg et al, 1995; Jacobs and Kühn, 1992; Kohn et al, 1992; Rentoumis et al, 1990)

Summary

Introduction

Thyroid hormones [THs; thyroxine (T4) and triiodothyronine (T3)] and their nuclear receptor family members, thyroid hormone receptors (TRs), have the ability to increase or decrease the rate of transcription of target genes (Lazar, 1993). A host of criteria such as T3 binding to TRs, TR isoform binding to target gene promoter regions as monomers, homo- or hetero-dimers, and the number, arrangement and sequence of TREs impact the type of regulation (Yen, 2001) These criteria and their outcomes are extensively studied but still poorly understood for many TH-sensitive genes, especially those downregulated by T3. The thyroid-stimulating hormone α-subunit (TSHα) gene contains a palindrome depicted in Fig. 1A (Chatterjee et al, 1989) For these negative (n)TREs, the non-ligand-bound TR somehow activates gene expression and confers repression upon binding T3; this type of regulation is not well described, possibly because of the conflicting circumstances for different genes that contain similar nTREs. There is argument over whether TR–DNA binding is maintained and what cofactors are involved because there is evidence to support many different hypotheses depending on the system or cell line and gene being studied

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