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 ChlP-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.