Proteomic Analysis of Sox2-Associated Proteins During Early Stages of Mouse Embryonic Stem Cell Differentiation Identifies Sox21 as a Novel Regulator of Stem Cell Fate  

Abstract

Small increases in the levels of master regulators, such as Sox2, in embryonic stem cells (ESC) have been shown to promote their differentiation. However, the mechanism by which Sox2 controls the fate of ESC is poorly understood. In this study, we employed Multidimensional Protein Identification Technology and identified >60 nuclear proteins that associate with Sox2 early during ESC differentiation. Gene ontology analysis of Sox2-associated proteins indicates that they participate in a wide range of processes. Equally important, a significant number of the Sox2-associated proteins identified in this study have been shown previously to interact with Oct4, Nanog, Sall4 and Essrb. Moreover, we examined the impact of manipulating the expression of a Sox2-associated protein on the fate of ESC. Using ESC engineered for inducible expression of Sox21, we show that ectopic expression of Sox21 in ESC induces their differentiation into specific cell types, including those that express markers representative of neurectoderm and heart development. Collectively, these studies provide new insights into the range of molecular processes through which Sox2 is likely to influence the fate of ESC, and provide further support for the conclusion that the expression of Sox proteins in ESC must be precisely regulated. Importantly, our studies also argue that Sox2, along with other pluripotency-associated transcription factors, is woven into highly interconnected regulatory networks that function at several levels to control the fate of ESC.

Heterogeneity in Oct4 and Sox2 Targets Biases Cell Fate in 4-Cell Mouse Embryos

“…Focusing on the transcriptomes of each and every cell in 16 4-cell stage embryos uncovered consistent patterns of highly heterogeneous gene expression, with the most heterogeneously expressed genes being targets of Sox2 and Oct4, among which is the gene for the transcription factor Sox21. The role of Sox21 in the early embryo has not been previously investigated, but studies in ES cells indicated its involvement in stem cell differentiation, the inhibition of Cdx2 expression, and cellular reprogramming (Mallanna et al, 2010, Kuzmichev et al, 2012. The regulatory regions of the Sox21 gene are targets of Oct4 binding, and its expression is regulated by Sox2 (Chakravarthy et al, 2011; Gö ke et al, 2011; Kuzmichev et al, 2012;Mallanna et al, 2010).…”

“…The role of Sox21 in the early embryo has not been previously investigated, but studies in ES cells indicated its involvement in stem cell differentiation, the inhibition of Cdx2 expression, and cellular reprogramming (Mallanna et al, 2010, Kuzmichev et al, 2012. The regulatory regions of the Sox21 gene are targets of Oct4 binding, and its expression is regulated by Sox2 (Chakravarthy et al, 2011; Gö ke et al, 2011; Kuzmichev et al, 2012;Mallanna et al, 2010). Among other target genes of Oct4 and/ or Sox2 showing similar heterogeneity of expression, we identified those for pluripotency transcription factors such as Nanog and Esrrb; however, their expression peaked later than that of Sox21, suggesting that Sox21 could play a role very early in development.…”

“…One of the most highly heterogeneous genes in all embryos analyzed at the 4-cell stage is the gene encoding the transcription factor Sox21 (Figure 2A), which is involved in regulating ES cell-fate downstream of Sox2 (Kuzmichev et al, 2012;Mallanna et al, 2010). Sox21 has not been previously studied in the early mouse embryo but is known to inhibit expression of the trophectoderm (TE) master gene Cdx2 in ES cells and is important for reprogramming (Kuzmichev et al, 2012).…”

“…Sox21 has not been previously studied in the early mouse embryo but is known to inhibit expression of the trophectoderm (TE) master gene Cdx2 in ES cells and is important for reprogramming (Kuzmichev et al, 2012). Furthermore, Sox21 expression is directly regulated by Sox2 (Chakravarthy et al, 2011;Kuzmichev et al, 2012;Mallanna et al, 2010), and its regulatory region is bound by Oct4 (Chakravarthy et al, 2011;Gö ke et al, 2011), which has heterogeneous nuclear-cytoplasmic kinetics at the 4-cell stage (Plachta et al, 2011). Together, this suggests that the heterogeneous Sox21 expression may itself be regulated by heterogeneous Oct4 and/or Sox2 activity in the embryo.…”

Mitotic phosphorylation of SOX2 mediated by Aurora kinase A is critical for the stem-cell like cell maintenance in PA-1 cells

“…6 Comparing interactomes of SOX2 in ESC and ESC beginning to differentiate, it indicates that the SOX2-interactomes changes dramatically within 24 hours. 7 It is unclear why the SOX2-interactome changes so rapidly. The attractive changes are the post-translational modifications of interacting proteins.…”