PLURIPOTENCY REGULATOR ZIC3 IS AN EARLY LINEAGE SPECIFIER. Mus musculus

Sustained expression of the key pluripotency transcriptional regulators and proliferative factors is needed for the maintenance of embryonic stem cells and early progenitors of various lineages. Zic3 is one of those factors which has been shown to be important for embryonic stem cell pluripotency. Through combinatorial analysis of transcription factor binding sites and the corresponding gene-regulation, we show that Zic3 not only activates key pluripotency genes but also acts cooperatively with them in connecting important circuits of regulation in ES cell in proliferation and maintenance of early progenitors. Overall design: Chromatin immunoprecipitation was performed as previously described (Loh, Y.H. et al, Nat Genet 38, 431-440 (2006)). Briefly, cells were treated with 1% formaldehyde for 10 mins at RT. The reaction was quenched with 125 mM glycine. Chromatin was harvested from nuclear extracts. Chromatin extracts were sonicated to an average size of 500 bp. Immunoprecipitation was performed using 10 µg of Zic3 antibody previously generated (Lim, L.S. et al., Mol Biol Cell 18, 1348-1358 (2007)). Immunoprecipitated chromatin extracts were subjected to sequencing.