QUANTITATIVE DETERMINATION OF THE ASSEMBLY OF THE OCT4-SOX2-DNA TERNARY COMPLEX AND ITS RELEVANCE TO PRE-IMPLANTATION DEVELOPMENT

Abstract

Sox2 and Oct4 partner to regulate the transcription of many genes in the pluripotent lineage through binding composite Sox-Oct cis-regulatory elements (SO-CREs). While sequence variation within this SO-CRE is evident across multiple target genes, strong conservation within a specific SO-CRE across species argues for functional constraints within the specific SO-CRE sequences. Given the dynamically changing expression levels of Sox2 in pre-implantation development and its role in cell fate decisions I sought to identify the quantitative differences in Oct4-Sox2-DNA interaction on target genes important for blastocyst biology. I used a combination of electrophoretic mobility shift assay (EMSA) and fluorescence correlation spectroscopy (FCS) on full-length fluorescently tagged Sox2 and Oct4 to generate quantitative binding data for Sox2-Oct4 complex formation on different SO-CREs. On key target genes, I found differences in dissociation constants that correspond with pre-implantation Sox2 protein levels and target gene transcriptional output. Specifically, the expression of Fgf4, a growth factor that is itself essential for pre-implantation development, parallels expression of Sox2. Fgf4 expression, though mediated through a cooperative interaction between Oct4 and Sox2, is more sensitive to Sox2 levels than other target genes. I also used my developed quantitative assay to analyze the complexity in dimer formation of Oct4, highly expressed in embryonic stem (ES) cells and Oct6, abundantly expressed in neuronal stem (NS) cells in the presence of Sox2. I showed that preferential transcriptional complex formation of POU factors in the presence of Sox2 accounts for the difference. Oct6 tends to form a homodimer on palindromic or more palindromic Oct regulatory elements (PORE/MORE) rather than forming a heterodimer with Sox2 on SO-CRE. This pattern of complex formation provides a molecular and biochemical explanation for the mechanism of induced pluripotent stem cells generation from NS cells. Indeed, such quantitative analyses will be the key to achieve a system level of understanding into developmental and cellular processes.