Figure 3

YY1 binding to region of P2 promoter is Xi-specific and upregulates P1 transcription. A) Chromatin immunoprecipitation (ChIP) of YY1 binding to P2 shown relative to input, at P2, a negative control region within XIST, and a positive control region (HCFC1 gene promoter) for female (IMR-90), HT1080XISTi transgenic XIST, and Xi and Xa-containing hybrids. A representative ChIP is shown ± standard deviation of q-PCR triplicates; with replicate experiments showing the same pattern but variable levels of IP relative to input. B) Q-PCR following 72 h of siRNA mediated knockdown of YY1 in IMR-90 female cells demonstrates a decrease in expression of XIST, both upstream (19, 18) and downstream of P2 (200d.2, qXIST5) relative to cells with a control (EGFP) knockdown. Levels of YY1 dropped to less than 20% confirming successful knockdown. C) FISH for XIST RNA following YY1 knockdown. Left panel shows representative IMR-90 female cells treated with lipofectamine alone (Lipo) and right panel shows cells after YY1 knockdown. Arrows indicate location of XIST RNA signal (green) which was substantially reduced after YY1 knockdown.