Zhang Shu Xing, Garcia-Gras Eduardo, Wycuff Diane R, Marriot Suzanne J, Kadeer Nijiati, Yu Wei, Olson Eric N, Garry Daniel J, Parmacek Michael S, Schwartz Robert J
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 2005 May 13;280(19):19115-26. doi: 10.1074/jbc.M413793200. Epub 2005 Jan 28.
Serum-response factor (SRF) is an obligatory transcription factor, required for the formation of vertebrate mesoderm leading to the origin of the cardiovascular system. Protein A-TEV-tagged chromatin immunoprecipitation technology was used to collect direct SRF-bound gene targets from pluripotent P19 cells, induced by Me2SO treatment into an enriched cardiac cell population. From 242 sequenced DNA fragments, we identified 188 genomic DNA fragments as potential direct SRF targets that contain CArG boxes and CArG-like boxes. Of the 92 contiguous genes that were identified, a subgroup of 43 SRF targets was then further validated by co-transfection assays with SRF. Expression patterns of representative candidate genes were compared with the LacZ reporter expression activity of the endogenous SRF gene. According to the Unigene data base, 84% of the SRF target candidates were expressed, at least, in the heart. In SRF null embryonic stem cells, 81% of these SRF target candidates were greatly affected by the absence of SRF. Among these SRF-regulated genes, Raf1, Map4k4, and Bicc1 have essential roles in mesoderm formation. The 12 regulated SRF target genes, Mapk10 (JNK3), Txnl2, Azi2, Tera, Sema3a, Lrp4, Actc1, Myl3, Hspg2, Pgm2, Hif3a, and Asb5, have been implicated in cardiovascular formation, and the Ski and Hes6 genes have roles in muscle differentiation. SRF target genes related to cell mitosis and cycle, E2f5, Npm1, Cenpb, Rbbp6, and Scyl1, expressed in the heart tissue were differentially regulated in SRF null ES cells.
血清反应因子（SRF）是一种必需的转录因子，对于脊椎动物中胚层的形成至关重要，而中胚层的形成会导致心血管系统的起源。采用蛋白A-TEV标签染色质免疫沉淀技术，从多能性P19细胞中收集直接与SRF结合的基因靶点，通过二甲基亚砜（Me2SO）处理将其诱导为富集的心肌细胞群体。从242个测序的DNA片段中，我们鉴定出188个基因组DNA片段为潜在的直接SRF靶点，这些片段包含CArG盒和类CArG盒。在鉴定出的92个连续基因中，通过与SRF共转染实验进一步验证了43个SRF靶点组成的亚组。将代表性候选基因的表达模式与内源性SRF基因的LacZ报告基因表达活性进行了比较。根据Unigene数据库，84%的SRF靶点候选基因至少在心脏中表达。在SRF缺失的胚胎干细胞中，这些SRF靶点候选基因中有81%受到SRF缺失的极大影响。在这些受SRF调控的基因中，Raf1、Map4k4和Bicc1在中胚层形成中具有重要作用。12个受调控的SRF靶点基因，即Mapk10（JNK3）、Txnl2、Azi2、Tera、Sema3a、Lrp4、Actc1、Myl3、Hspg2、Pgm2、Hif3a和Asb5，与心血管形成有关，而Ski和Hes6基因在肌肉分化中发挥作用。与细胞有丝分裂和周期相关的SRF靶点基因E2f5、Npm1、Cenpb、Rbbp6和Scyl1在心脏组织中表达，在SRF缺失的胚胎干细胞中受到差异调控。
