Identification of direct serum-response factor gene targets during Me2SO-induced P19 cardiac cell differentiation.
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.
Zhang
Shu Xing
SX
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Garcia-Gras
Eduardo
E
Wycuff
Diane R
DR
Marriot
Suzanne J
SJ
Kadeer
Nijiati
N
Yu
Wei
W
Olson
Eric N
EN
Garry
Daniel J
DJ
Parmacek
Michael S
MS
Schwartz
Robert J
RJ
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019992:Databases as Topic; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D016678:Genome; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006579:Heterozygote; D017403:In Situ Hybridization; D007763:Lac Operon; D008156:Luciferases / Q000378:metabolism; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009132:Muscles / Q000378:metabolism; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D000090243:Nucleophosmin; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D026362:Serum Response Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000378:metabolism
D002843:Chromatin; D018840:HSP70 Heat-Shock Proteins; C097210:Hspa1a protein, rat; C000715570:Npm1 protein, mouse; D026362:Serum Response Factor; D000090243:Nucleophosmin; D012313:RNA; D004247:DNA; D008156:Luciferases; D001616:beta-Galactosidase; D004121:Dimethyl Sulfoxide