Fig. 3.

hnRNPK has little role in SOX2 transcriptional regulation by facilitating transcription elongation. A EMSA of Cy5-labeled Fgf4 enhancer DNA (50 nM) with SOX2 (100 nM) and increasing concentrations (100–400 nM) of hnRNPK. Concentrations of SOX2 and hnRNPK are labelled above each lane. Free DNA and protein-DNA complex are marked. ‘-’ denotes the absence of the corresponding protein in the reaction mix. B ChIP analysis for SOX2 in E14 ESCs with or without hnRNPK KD. Relative enrichments to IgG-ChIP control are shown. C A dual-luciferase reporter assay validated hnRNPK’s impact on the transcriptional activation of SOX2 via an Fgf4 enhancer-containing reporter plasmid. Schematic showing luciferase reporter with Fgf4 enhancer region containing SOX2 binding motif (upper panel) and relative expression of luciferase in control (shscramble), Sox2-KD (shSox2-1 and shSox2-2) and hnRNPK-KD (shhnRNPK-1 and shhnRNPK-2) E14 ESCs (lower panel). Data are represented as mean ± SD (n = 3). ANOVA was used to assess significance (**** indicates P-value of < 0.0001; ns indicates not significant). Pr: SV40 promoter. D Co-IP analysis verifying interactions between SOX2, hnRNPK, HEXIM1, and CYCLINT1. Endogenous SOX2 was immunoprecipitated from E14 ESCs whole cell lysate using anti-SOX2 antibody or IgG. IP products were blotted with anti-Sox2, anti-hnRNPK, anti-HEXIM1 and anti-CYCLINT1 antibodies. E In vitro pull-down assays of GST-HEXIM1 with His-hnRNPK. E. coli lysates expressing His-hnRNPK were incubated with either GST-HEXIM1 or GST coupled with glutathione sepharose beads, and the resulting pull-down products were detected using anti-His or anti-GST antibodies. The symbols ‘ + ’ and ‘-’ denote the presence and absence, respectively, of the corresponding protein in the reaction mix. F Co-IP of SOX2 and CYCLINT1. Whole cell lysates of E. coli expressing HA-SOX2 and/or Flag-CYCLINT1 were immunoprecipitated with anti-Flag antibody. IP products were blotted with anti-HA and anti-Flag antibodies. G Scheme of 7SK snRNA and its 5’ stem loop (SL1). The sequence of SL1 used is highlighted. H-I. In vitro RIP with 1.2 nmol HEXIM1 and 1.2 nmol 7SK snRNA constructs with increasing amount of hnRNPK. The names of proteins and RNA substrates are as indicated above each panel. J Analysis of differentially expressed genes obtained through RNA-seq. The Venn diagram displays the number of genes that exhibit differential expression in Sox2-KD, hnRNPK-KD, and Cdk9-KD E14 ESCs compared to control (shscramble) E14 ESCs