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

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.

Keywords: Differentiation; Herpesvirus; Histone modification; Thyroid hormone; Transcription factor.

Fig. 1.

Nucleotide sequences of nTRE regions from TSHα and wild-type and mutant HSV-1 TK with PiDNA computational analysis. The interactions of protein and DNA were investigated by PiDNA to determine which point mutations might reverse the T₃-mediated repression. (A) TSHα gene TATA box, TRE half-sites and transcription start site are depicted for comparison and reference to the TK TREs. (B) HSV-1 wt TK gene TATA box, TRE half-sites and transcription start site with random screening of PiDNA identified 5′-AGGTGA-3′ (TRE-1a) on the positive strand and 5′-AGGCCA-3′ (TRE-2) located at the reverse strand of the HSV-1 TK promoter as the best half-sites for protein binding. The orientation of these half-sites for TRE-1 and TRE-2 was suggested as palindromic TREs with a six nucleotide in between (Pal 6). (C) HSV-1 mutant TK gene TATA box, TRE half-sites and transcription start site with the point mutation on TRE-1a to 5′-AGGTGC-3′ and TRE-1b to 5′-GCGGCA-3′ on the reverse strand. This shift would allow TRE-1b′ and TRE-2 to form a direct repeat separated by three nucleotides (DR 3).

Fig. 2.

PMV and PDBSum analysis of HSV-1 wild-type and mutant TREs. The high-scored putative TRE half-sites were assessed for hydrogen bond interactions using Python molecule viewer (PMV) software and PDBSum. PMV was used to visualize and measure distances between the DNA-binding protein residues and the bases of the DNA TRE half-sites. PMV hydrogen bonding analysis of TRβ (top) and PDBSum RXRα hydrogen bonding analysis (bottom) to (A) 5′-AGGTGA-3′ (wt TRE-1a), (B) 5′-AGGTGC-3′ (mt TRE-1a′) and (C) 5′-GCGGCA-3′ (mt TRE-1b′). PDBSum calculates and illustrates putative hydrogen bonds between DNA and protein in the pdb files (bottom, green dashed lines). = and < are referencing the number of hydrogens bonds.

Fig. 3.

Transfection and DLuc assays of nTREs from TSHα and wild-type and mutant HSV-1 TK. Dual-luciferase assays of (A) undifferentiated LNCaP cells and (B) differentiated LNCaP cells transfected with TSHα and HSV-1 TK TRE luciferase reporter plasmids: wtPal6 and mtTRE treated with and without T₃. Mutant plasmids exhibit different T₃-mediated regulation compared with the wt and TSHα. (C) Dual-luciferase assays of differentiated LNCaP cells transfected with the reporter plasmids and dnTR overexpression vector in the presence or absence of T₃. Results are mean±s.d. of n=3 replicate experiments. Two-way ANOVA with Holm–Sidak post hoc analysis suggests difference in activity is statistically significant where *P<0.05; NS, not significant.

Fig. 4.

Electro-mobility shift assays. Labeled wtTRE oligo was incubated with undifferentiated (A) or differentiated (B) LNCaP cell protein extract and various anti-TRβ and T₃ treatments, followed by gel electrophoresis. (C) Differentiated LNCaP protein extract and mtTRE oligo. Lane 1, no protein; lanes 2–5 were incubated with cell protein extract; lanes 4 and 5 with anti-TRβ antibody; lanes 3 and 5 were treated with T₃. Protein–oligo complex can be seen at the top and free probe can be seen at the bottom of the membrane. Triplicate runs of lanes 2–5 were performed and analyzed using pixel densitometry using Bio-Rad image lab software and ANOVA with Holm–Sidak post hoc to determine statistical significance. Lane 5 (B) had a 15% statistically significant increase in pixel density over lane 4 (B) with P<0.0005.

Fig. 5.

Enrichment of repressive chromatin H3K9me3 at the HSV-1 TK TREs. ChIP assay measuring the enrichment of H3K9me3, a repressive chromatin protein marker, to HSV-1 TK TREs from differentiated LNCaP cells transfected with wtTRE and mtTRE1 treated with and without T₃ presented as percentage input with IgG background subtracted. T₃ treatment of wtTRE resulted in a 4.5-fold increase in comparison to no T₃ treatment. The mtTRE1, however, showed an opposite effect where T₃ treatment caused a reduction. As a control, immunoprecipitated chromatin was subjected to qPCR analysis for a region 1.5 kb downstream of the TK promoter. The differences between wtTRE TK promoter and mtTRE TK promoter treated or not treated with T3 are statistically significant (P<0.05 by ANOVA and Holm-Sidak post-hoc analysis). Results are mean±s.d. of n=3 replicate experiments.