Prediction and Validation of Transcription Factors Modulating the Expression of Sestrin3 Gene Using an Integrated Computational and Experimental Approach.

Rajneesh Srivastava,Xiwen Xiong,Yang Zhang,X Charlie Dong,Suthat Liangpunsakul,Xiaoyan Pan,Sarath Chandra Janga,Xiaoning Zhang

doi:10.1371/journal.pone.0160228

Abstract

SESN3 has been implicated in multiple biological processes including protection against oxidative stress, regulation of glucose and lipid metabolism. However, little is known about the factors and mechanisms controlling its gene expression at the transcriptional level. We performed in silico phylogenetic footprinting analysis of 5 kb upstream regions of a diverse set of human SESN3 orthologs for the identification of high confidence conserved binding motifs (BMo). We further analyzed the predicted BMo by a motif comparison tool to identify the TFs likely to bind these discovered motifs. Predicted TFs were then integrated with experimentally known protein-protein interactions and experimentally validated to delineate the important transcriptional regulators of SESN3. Our study revealed high confidence set of BMos (integrated with DNase I hypersensitivity sites) in the upstream regulatory regions of SESN3 that could be bound by transcription factors from multiple families including FOXOs, SMADs, SOXs, TCFs and HNF4A. TF-TF network analysis established hubs of interaction that include SMAD3, TCF3, SMAD2, HDAC2, SOX2, TAL1 and TCF12 as well as the likely protein complexes formed between them. We show using ChIP-PCR as well as over-expression and knock out studies that FOXO3 and SOX2 transcriptionally regulate the expression of SESN3 gene. Our findings provide an important roadmap to further our understanding on the regulation of SESN3.

Highlights

Sestrins belong to a small family of evolutionally conserved proteins
We identified the set of binding sites and corresponding Transcription factors (TFs) controlling the SESN3 gene by performing motif discovery based on phylogenetic alignments of orthologous sequences from a diverse set of primates and rodents using the human SESN3 gene as a reference
We observed that most of the established binding motif Position Weight Matrices (PWM) in publically available databases ranges in length between 4 bp to 30 bp (See Materials and Methods) we believe that the discovered motifs in current study would be able to capture most of these recognition sequences, including large co-complex TF binding sites or palindrome motifs, if they are present in the SESN3 upstream

Summary

Introduction

Sestrins belong to a small family of evolutionally conserved proteins They are distinct from any other characterized eukaryotic protein families because they do not have any previously identified domain structures[1]. Sestrins do not contain any known structural domains/catalytic motifs; only a partial homologous sequence to bacterial oxidoreductases is identified, suggesting an antioxidant function of this protein[1]. Sestrins reduce oxidative stress through either their intrinsic oxidoreductase activity or NRF2 (nuclear factor erythroid derived 2 like 2)-regulated pathway [4,5]. Sestrins modulate glucose and lipid metabolism through AMPK (AMP-activated protein kinase) and mTORC1 (mechanistic target of rapamycin complex 1)[1]. We have observed that ethanol suppresses SESN3 gene expression and function in hepatocytes and mouse livers. Understanding the complex regulatory mechanisms that regulate the SESN3 is of importance, as new therapeutic targets for metabolic diseases might be discovered

Methods

Results

Conclusion