Identification of a chromatin-bound ERRα interactome network in mouse liver [ChIP-Seq]. Identification of a chromatin-bound ERRα interactome network in mouse liver [ChIP-Seq]

Objective Estrogen-related-receptor α (ERRα) plays a critical role in the transcriptional regulation of cellular bioenergetics and metabolism, and perturbations in its activity have been associated with metabolic diseases. While several coactivators and corepressors of ERRα have been identified to date, a knowledge gap remains in understanding the extent to which ERRα cooperates with coregulators in the control of gene expression. Herein, we mapped the primary chromatin-bound ERRα interactome in mouse liver. Methods RIME (Rapid Immuno-precipitation Mass spectrometry of Endogenous proteins) analysis using mouse liver samples from two circadian time points was used to catalog ERRα-interacting proteins on chromatin. The genomic crosstalk between ERRα and its identified cofactors in the transcriptional control of precise gene programs was explored through cross-examination of genome-wide binding profiles from chromatin immunoprecipitation-sequencing (ChIP-seq) studies. The dynamic interplay between ERRα and its newly uncovered cofactor Host cell factor C1 (HCFC1) was further investigated by loss-of-function studies in hepatocytes Results Characterization of the hepatic ERRα chromatin interactome led to the identification of 48 transcriptional interactors of which 42 were previously unknown including HCFC1. Interrogation of available ChIP-seq binding profiles highlighted oxidative phosphorylation (OXPHOS) under the control of a complex regulatory network between ERRα and multiple cofactors. While ERRα and HCFC1 were found to bind to a large set of common genes, only a small fraction showed their co-localization, found predominately near the transcriptional start sites of genes particularly enriched for components of the mitochondrial respiratory chain. Knockdown studies demonstrated inverse regulatory actions of ERRα and HCFC1 on OXPHOS gene expression ultimately dictating the impact of their loss-of-function on mitochondrial respiration. Conclusions Our work unveils a repertoire of previously unknown transcriptional partners of ERRα comprised of chromatin modifiers and transcription factors thus advancing our knowledge of how ERRα regulates metabolic transcriptional programs. Overall design: Chromatin immunoprecipitation followed by sequencing (ChIP-seq) for HCFC1 or IgG control was conducted in Hepa-6 cells treated with 200nM of siRNAs targeting ERRα (siERRa, siE), HCFC1 (siHCFC1, siH) or control siRNAs (siC) for 96h.

雌激素相关受体α（ERRα）在细胞能量代谢与转录调控中发挥关键作用，其活性异常与代谢疾病密切相关。尽管目前已鉴定出ERRα的多种共激活因子与共抑制因子，但学界对于ERRα与共调控因子协同调控基因表达的具体程度仍存在认知空白。本研究针对小鼠肝脏中结合于染色质的ERRα核心互作组进行了定位分析。
方法：本研究采用内源蛋白快速免疫沉淀质谱分析法（Rapid Immuno-precipitation Mass spectrometry of Endogenous proteins，RIME），采集两个昼夜时间点的小鼠肝脏样本，对染色质上与ERRα互作的蛋白质进行编目鉴定。此外，本研究通过整合染色质免疫共沉淀测序（Chromatin Immunoprecipitation-Sequencing，ChIP-seq）获得的全基因组结合谱数据，探究了ERRα与其已鉴定共因子在特定基因程序转录调控中的基因组串扰机制。同时，本研究还通过肝细胞功能丧失实验，进一步解析了ERRα与新发现的共因子宿主细胞因子C1（Host cell factor C1，HCFC1）之间的动态互作关系。
结果：对肝脏ERRα染色质互作组的表征分析共鉴定出48个转录互作蛋白，其中42个为此前未报道的互作因子，包括HCFC1。对现有ChIP-seq结合谱的分析显示，氧化磷酸化（oxidative phosphorylation，OXPHOS）相关基因处于ERRα与多种共因子构成的复杂调控网络的调控之下。尽管ERRα与HCFC1可结合大量共有基因，但仅少数基因区域存在二者的共定位，且这些共定位区域主要集中于基因转录起始位点附近，其中显著富集线粒体呼吸链复合体相关组分。基因敲低实验结果表明，ERRα与HCFC1对OXPHOS相关基因的表达具有相反的调控作用，这最终决定了二者功能丧失对线粒体呼吸功能的影响。
结论：本研究揭示了一批此前未被报道的ERRα转录互作伙伴，涵盖染色质修饰因子与转录因子，从而加深了学界对ERRα调控代谢相关转录程序机制的认知。
实验整体设计：在Hepa-6细胞中，分别使用浓度为200nM的靶向ERRα的小干扰RNA（small interfering RNA，siRNA，siERRα，简称siE）、靶向HCFC1的小干扰RNA（siHCFC1，简称siH）以及阴性对照小干扰RNA（siC）处理细胞96小时，随后针对HCFC1或IgG对照开展染色质免疫共沉淀测序（ChIP-seq）。