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
✍ Scribed by Harini Chakravarthy; Brian Boer; Michelle Desler; Sunil Kumar Mallanna; Timothy W. McKeithan; Angie Rizzino
- Publisher
- John Wiley and Sons
- Year
- 2008
- Tongue
- English
- Weight
- 198 KB
- Volume
- 216
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
Abstract
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. J. Cell. Physiol. 216: 651–662, 2008, © 2008 Wiley‐Liss, Inc.