Definition of the fibroblast SRF-mediated immediate-early transcriptional response. Mus musculus

To control transcription, SRF recruits signal-regulated co-activators, the Ternary Complex Factors (TCFs) and the Myocardin-related Transcription Factors (MRTFs), which compete for a common site on its DNA-binding domain. The TCFs - SAP-1, Elk-1 and Net - are Ets proteins that link SRF activity to Ras-ERK signalling. In contrast, the two MRTFs, MRTF-A and MRTF-B, link SRF activity to Rho-actin signalling. In this novel signalling pathway, the actin-binding MRTF RPEL domain acts as a G-actin sensor, controlling MRTF nuclear accumulation in response to signal-induced depletion of the G-actin pool. Previous studies have suggested that the Ras-ERK signalling and Rho-actin pathways control specific subsets of SRF target genes. We used ChIP-seq and RNA-seq to analyse the immediate-early transcriptional response in NIH3T3 fibroblasts, using pathway-specific inhibitors to identify the contributions of Ras-ERK and Rho-actin signalling Overall design: Chromatin immunoprecipitation and sequencing (ChIP-seq) in NIH3T3 fibroblast after serum stimulation in presence or absence of LatrunculinB or U0126 drugs and using antibodies against SRF, MRTF-A, MRTF-B, SAP1, ELK1, NET, Pol II, PolII S5P, PolII S2P and total H3. Validation by ChIP-PCR. Strand specific total-RNA-seq following DSN normalisation and validation by qRT-PCR from NIH3T3 stimulated by serum or Cytochalasin D in presence or absence of LatrunculinB and/or U0126 drugs.

为调控转录，血清反应因子（Serum Response Factor, SRF）会招募信号调控型共激活因子：三元复合物因子（Ternary Complex Factors, TCFs）与心肌素相关转录因子（Myocardin-related Transcription Factors, MRTFs），这两类因子会在SRF的DNA结合结构域上争夺同一结合位点。其中，TCFs家族成员包括SAP-1、Elk-1与Net，均属于Ets家族蛋白，可将SRF的活性与Ras-ERK信号通路相偶联。与之相对，MRTFs家族的两个亚型MRTF-A与MRTF-B则可将SRF活性与Rho-肌动蛋白信号通路相偶联。在这一新型信号通路中，具备肌动蛋白结合能力的MRTF RPEL结构域可作为G-肌动蛋白感受器，响应信号诱导的G-肌动蛋白库耗竭，进而调控MRTF的核聚集过程。既往研究表明，Ras-ERK与Rho-肌动蛋白这两条信号通路可分别调控SRF靶基因的特定子集。

本研究采用染色质免疫沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）与RNA测序（RNA sequencing, RNA-seq）技术，分析NIH3T3成纤维细胞中的即刻早期转录应答，并通过通路特异性抑制剂明确Ras-ERK与Rho-肌动蛋白信号通路的各自调控贡献。

整体实验设计：
对经血清刺激（分别添加或不添加拉春库林B（Latrunculin B）与U0126药物）的NIH3T3成纤维细胞进行染色质免疫沉淀测序，所用抗体包括抗SRF、MRTF-A、MRTF-B、SAP1、ELK1、NET、RNA聚合酶II（Pol II）、磷酸化S5位点的RNA聚合酶II（PolII S5P）、磷酸化S2位点的RNA聚合酶II（PolII S2P）以及总组蛋白H3抗体；并通过染色质免疫沉淀PCR（ChIP-PCR）对实验结果进行验证。采用经过双链特异性核酸酶（Duplex-Specific Nuclease, DSN）标准化的链特异性总RNA-seq技术，对经血清或细胞松弛素D（Cytochalasin D）刺激、且分别添加或不添加拉春库林B及/或U0126药物的NIH3T3细胞进行测序，并通过实时荧光定量聚合酶链反应（qRT-PCR）对测序结果进行验证。