Stemness-related Factor Sall4 Interacts with Transcription Factors Oct-3/4 and Sox2 and Occupies Oct-Sox Elements in Mouse Embryonic Stem Cells

A small number of transcription factors, including Oct-3/4 and Sox2, constitute the transcriptional network that maintains pluripotency in embryonic stem (ES) cells. Previous reports suggested that some of these factors form a complex that binds the Oct-Sox element, a composite sequence consisting of closely juxtaposed Oct-3/4 binding and Sox2 binding sites. However, little is known regarding the components of the complex. In this study we show that Sall4, a member of the Spalt-like family of proteins, directly interacts with Sox2 and Oct-3/4. Sall4 in combination with Sox2 or Oct-3/4 simultaneously occupies the Oct-Sox elements in mouse ES cells. Overexpression of Sall4 in ES cells increased reporter activities in a luciferase assay when the Pou5f1- or Nanog-derived Oct-Sox element was included in the reporter. Microarray analyses revealed that Sall4 and Sox2 bound to the same genes in ES cells significantly more frequently than expected from random coincidence. These factors appeared to bind the promoter regions of a subset of the Sall4 and Sox2 double-positive genes in precisely similar distribution patterns along the promoter regions, suggesting that Sall4 and Sox2 associate with such Sall4/Sox2-overlapping genes as a complex. Importantly, gene ontology analyses indicated that the Sall4/Sox2-overlapping gene set is enriched for genes involved in maintaining pluripotency. Sall4/Sox2/Oct-3/4 triple-positive genes identified by referring to a previous study identifying Oct-3/4-bound genes in ES cells were further enriched for pluripotency genes than Sall4/Sox2 double-positive genes. These results demonstrate that Sall4 contributes to the transcriptional network operating in pluripotent cells together with Oct-3/4 and Sox2. Background: Oct-3/4 and Sox2 form a complex to regulate gene expression and maintain pluripotency. Results: Sall4 directly interacts with Oct-3/4 or Sox2. Sall4 and Sox2 occupy the same promoter regions of genes active in ES cells. Conclusion: Sall4 is involved in transcriptional networks retaining pluripotency, in combination with Oct-3/4 or Sox2. Significance: This study provides a molecular understanding of how ES cells maintain pluripotency.