Identification of DPPA4 and other genes as putative Sox2:Oct-3/4 target genes using a combination of in silico analysis and transcription-based assays.
Sox2 and Oct-3/4 function as master regulators during mammalian embryogenesis, where they are believed to regulate a critical gene regulatory network by cooperatively binding to DNA regulatory regions composed of adjacent HMG and POU motifs (HMG/POU cassettes). Previous studies have identified seven genes that contain highly active HMG/POU cassettes (referred to as Sox2:Oct-3/4 target genes). Importantly, nearly all known Sox2:Oct-3/4 target genes appear to be essential for embryogenesis. Recent genome-wide ChIP-chip studies identified over 300 genes that are co-occupied by Sox2 and Oct-3/4, which suggests that most Sox2:Oct-3/4 target genes remain to be identified. The work described here used a 3-step strategy for identifying additional Sox2:Oct-3/4 target genes. First, we employed in silico analysis to search for putative HMG/POU cassettes in 50 genes reported to be co-occupied by Sox2 and Oct-3/4 in embryonic stem cells. We identified 39 genes that contain putative HMG/POU cassettes. Next, we tested the activity of seven of the putative HMG/POU cassettes in a transcription-based assay and determined that nearly all are functional. Finally, as a proof-of-principle, we tested one of the seven cassettes (DPPA4) in the context of its endogenous promoter using a promoter/reporter gene construct. DPPA4 was tested in part because it was shown recently to play an important role in ES cell self-renewal. We determined that the 5' flanking region of the DPPA4 gene contains a functional HMG/POU cassette and behaves as a Sox2:Oct-3/4 target gene. Finally, we used a transcription-based assay to help develop a refined consensus sequence for HMG/POU cassettes.
Chakravarthy
Harini
H
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.
Boer
Brian
B
Desler
Michelle
M
Mallanna
Sunil Kumar
SK
McKeithan
Timothy W
TW
Rizzino
Angie
A
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051519:Fibroblast Growth Factor 4 / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D024002:HMGB Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D055748:SOXB1 Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C519287:Dppa4 protein, mouse; D051519:Fibroblast Growth Factor 4; D024002:HMGB Proteins; D009687:Nuclear Proteins; D050814:Octamer Transcription Factor-3; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D014157:Transcription Factors