Youths, Careers Combined With RAD001
To address this possibility we performed RNAseq on mESCs that were transduced with a PHF13 specific shRNA or a scrambled Biperiden HCl shRNA control (Figure 6B and C). The efficiency of the knockdown was controlled 12 days later by qPCR and western blot and showed an approximate 80% reduction (Figure 6��figure supplement 1). Differential gene expression analysis showed a total of 1386 genes (Figure 6��source data 3) that were up or down regulated at an adjusted p-value less than 0.05. Of these 807 genes went down and 579 genes went up after PHF13 depletion, supporting the idea that PHF13 can promote both gene activation and repression. Several up and down regulated genes identified by RNA sequencing were confirmed by qPCR in control and PHF13 shRNA depleted E14 mESCs and in a Doxycyclin inducible PHF13 shRNA mESC cell RAD001 cost line that expresses a completely independent shRNA (Figure 6��figure supplement 1). Of the 1386 genes that were affected by PHF13 depletion, 845 genes were also identified as PHF13 targets by the ChIPseq (Figure 6B) supporting a direct correlation between the occupancy of PHF13 at these targets and their expression levels. Of these 845 genes, 487 were upregulated and 358 were downregulated. Gene set enrichment analysis of the genes affected by PHF13 depletion further revealed a similar overrepresentation of associated functional terms to ChIPseq and mass spectrometry targets and identified transcription, cell cycle, chromosome organization, differentiation, selleck chemicals DNA binding, RNA binding and chromatin binding (Figure 6C and Figure 6��source data 4 and 5). Together these findings support that PHF13 interacts with transcriptional regulatory proteins, that it binds to genes influencing transcription, cell cycle, chromosome organization and differentiation and that its depletion alters genes with similarly associated functional terms, all of which is consistent with a transcriptional co-regulatory role of PHF13. PHF13 depletion leads to upregulation of Polycomb repressed genes and the down regulation of active genes To further explore the relationship between the differentially expressed genes and the ChIPseq levels of PHF13, H3K4me1, H3K4me2, H3K4me3, H3K27me3, EZH2, SUZ12, and the differently phosphorylated forms of RNA PolII we compared their normalized levels to the corresponding levels in the unchanged genes (Figure 7A). We found that the genes that were both up- and down- regulated had significantly higher PHF13 enrichment than the genes that were unchanged (pvalue
