Figure 3 | Scientific Reports

Figure 3

From: Downregulation of PARP1 transcription by promoter-associated E2F4-RBL2-HDAC1-BRM complex contributes to repression of pluripotency stem cell factors in human monocytes

Figure 3

Quiescent state of differentiated cells entails the assembly of RBL2-E2F4-HDAC1-BRM repressor complex at PARP1 promoter and the HDAC1-BRM induced chromatin compaction. The effect of RBL2 silencing on PARP1 expression was determined with western blot (a) and real-time PCR (b) in human blood-derived monocytes 72 h after cell transient transfection with siRNA. Transcription regulating epigenetic marks (c,d,e) as well as HDAC1 (f), histone H3 density (g) and BRM (h) at PARP1 promoter were analyzed with ChIP. The interaction among repressive complex components was confirmed with immunoprecypitation (i) by pulling down HDAC1 and detection of co-immunoprecipitated proteins with western blot. In order to verify the contribution of HDACs (j,k) and BRM (l,m) in PARP1 repression blood-derived monocytes were incubated with the corresponding inhibitors (iHDAC – sodium butyrate – 0.5 mM and iBRM/iBRG1 – PFI-3 – 1 µM) for 24 h. PARP1 expression was monitored at the protein level with western blot (j,l)), while PARP1 mRNA was determined with real-time PCR (k,m). The effect of iHDAC on HDAC1, RBL2 and BRM association with chromatin (n,o,p) and the chromatin compaction (H3 density) in differentiated cells was assayed with ChIP (r).

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