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

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.

SESN3已被证实参与多种生物学过程，包括对抗氧化应激（oxidative stress）、调控糖脂代谢。然而，目前对其基因转录水平表达的调控因子与机制尚不清楚。我们对一组多样化的人类SESN3同源基因（orthologs）的5 kb上游区域开展了计算机模拟（in silico）系统发育足迹分析（phylogenetic footprinting analysis），以鉴定高可信度的保守结合基序（binding motifs, BMo）。我们进一步通过基序比对工具对预测得到的BMo进行分析，以识别可能结合这些发现基序的转录因子（transcription factors, TFs）。随后，我们将预测得到的TFs与已被实验证实的蛋白质相互作用数据进行整合，并通过实验验证，以厘清SESN3的关键转录调控因子。本研究在SESN3的上游调控区域中鉴定出了高可信度的BMo集合（整合了DNase I超敏位点（DNase I hypersensitivity sites）），这些基序可被多家族转录因子结合，包括叉头框蛋白家族（FOXOs）、SMAD蛋白家族（SMADs）、SOX蛋白家族（SOXs）、TCF蛋白家族（TCFs）以及HNF4A。转录因子-转录因子（TF-TF）网络分析确定了相互作用枢纽，包括SMAD3、TCF3、SMAD2、HDAC2、SOX2、TAL1以及TCF12，同时也揭示了它们之间可能形成的蛋白质复合物。我们通过染色质免疫沉淀-PCR（ChIP-PCR）、过表达与基因敲除实验证实，FOXO3与SOX2可转录调控SESN3基因的表达。本研究的发现为进一步理解SESN3的调控机制提供了重要的指导框架。