Functional promoter polymorphisms direct the expression of cystathionine gamma-lyase gene in mouse models of essential hypertension

Vinayak Gupta; Piyushkumar R Kapopara; Abrar A Khan; Vikas Arige; Lakshmi Subramanian; Parshuram J Sonawane; Binu K Sasi; Nitish R Mahapatra

doi:10.1016/j.yjmcc.2016.11.005

Functional promoter polymorphisms direct the expression of cystathionine gamma-lyase gene in mouse models of essential hypertension

J Mol Cell Cardiol. 2017 Jan:102:61-73. doi: 10.1016/j.yjmcc.2016.11.005. Epub 2016 Nov 16.

Authors

Vinayak Gupta¹, Piyushkumar R Kapopara¹, Abrar A Khan¹, Vikas Arige¹, Lakshmi Subramanian¹, Parshuram J Sonawane¹, Binu K Sasi¹, Nitish R Mahapatra²

Affiliations

¹ Cardiovascular Genetics Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India.
² Cardiovascular Genetics Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India. Electronic address: [email protected].

PMID: 27865915
DOI: 10.1016/j.yjmcc.2016.11.005

Abstract

Despite the well-known role of cystathionine γ-lyase (Cth) in cardiovascular pathophysiology, transcriptional regulation of Cth remains incompletely understood. Sequencing of the Cth promoter region in mouse models of genetic/essential hypertension (viz. Blood Pressure High [BPH], Blood Pressure Low [BPL] and Blood Pressure Normal [BPN] mice) identified several genetic variations. Transient transfections of BPH/BPL-Cth promoter-reporter plasmids into various cell types revealed higher promoter activity of BPL-Cth than that of BPH-Cth. Corroboratively, endogenous Cth mRNA levels in kidney and liver tissues were also elevated in BPL mice. Computational analysis of the polymorphic Cth promoter region predicted differential binding affinity of c-Rel, HOXA3 and IRF1 with BPL/BPH-Cth promoter domains. Over-expression of c-Rel/HOXA3/IRF1 modulated BPL/BPH-Cth promoter activities in a consistent manner. Gel shift assays using BPH/BPL-Cth-promoter oligonucleotides with/without binding sites for c-Rel/HOXA3/IRF1 displayed formation of specific complexes with c-Rel/HOXA3/IRF1; addition of antibodies to reaction mixtures resulted in supershifts/inhibition of Cth promoter-transcription factor complexes. Furthermore, chromatin immunoprecipitation (ChIP) assays proved differential binding of c-Rel, HOXA3 and IRF1 with the polymorphic promoter region of BPL/BPH-Cth. Tumor necrosis factor-α (TNF-α) reduced the activities of BPL/BPH-Cth promoters to different extents that were further declined by ectopic expression of IRF1; on the other hand, siRNA-mediated down-regulation of IRF1 rescued the TNF-α-mediated suppression of the BPL/BPH-Cth promoter activities. In corroboration, ChIP analysis revealed enhanced binding of IRF1 with BPH/BPL-Cth promoter following TNF-α treatment. BPL/BPH-Cth promoter activity was diminished upon exposure of hepatocytes and cardiomyoblasts to ischemia-like pathological condition due to reduced binding of c-Rel with BPL/BPH-Cth-promoter. Taken together, this study reveals the molecular basis for the differential expression of Cth in mouse models of essential hypertension under basal and pathophysiological conditions.

Keywords: Cystathionine γ-lyase promoter; Gene regulation; Ischemia; Transcription factor; Tumor necrosis factor-α.

MeSH terms

Animals
Base Sequence
Binding Sites
Blood Pressure
Chromosome Mapping
Computational Biology / methods
Cystathionine gamma-Lyase / genetics*
Disease Models, Animal
Essential Hypertension
Gene Expression Regulation*
Genomics / methods
Hypertension / genetics*
Hypertension / physiopathology*
Mice
Nucleotide Motifs
Organ Specificity / genetics
Polymorphism, Genetic*
Promoter Regions, Genetic*
Protein Binding
Quantitative Trait Loci
Rats
Transcription Factors / metabolism
Transcription, Genetic
Tumor Necrosis Factor-alpha / metabolism

Substances

Transcription Factors
Tumor Necrosis Factor-alpha
Cystathionine gamma-Lyase