Functional and Mechanistic Studies of XPC DNA-Repair Complex as Transcriptional Coactivator in Embryonic Stem Cells

We recently identified the DNA repair complex XPC-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4 and SOX2 transcriptional activation. Here we investigate genome-wide the role of SCC in murine ESCs by mapping regions bound by its RAD23B subunit in wild type and Xpc-/- ESCs, and analyzing transcriptional profiles of SCC-depleted ESCs. RAD23B ChIP-seq in wild type murine ESCs was performed in two replicates; normal IgG were used as controls. RAD23B ChIP-seq in Xpc-/- mESCs was compared to normal IgG. Antibodies to immunoprecipitate RAD23B were raised in guinea pigs against the 108-177 peptide of murine RAD23B protein (NP_033037). Anti-sera were affinity-purified. For RNA-seq, we compared wild type JM8.N4 cells with either Rad23b-/- cells, Rad23b-/-Xpc KD1 or Rad23b-/-Xpc KD2 cells. Xpc was knocked down with two independent shRNAs (KD1 and KD2).