NF-Y Dependent Epigenetic Modifications Discriminate between Proliferating and Postmitotic Tissue (original) (raw)
Figure 4
Lack of NF-Y DNA binding activity induces hypermethylation of histones and target gene inactivation.
(A) RT-PCR amplification of the indicated genes from uninfected MEFs (lane 1), MEFs 24hours after infection with Ad-GFP (lane 2), MEFs 24hours and 48hours after infection with Ad-DN-NFYA (lanes 3 and 4). (B) Western blot analysis performed on total lysates from: Mef after 24hours post infection with Ad-GFP (lane1), Mef after 24 hours post infection with Ad-DN-NFYA (lane 2), Mef after 48 hours post infection with Ad-DN-NFYA (lane 3). The extracts were probed with rabbit polyclonal with anti-NFY-A. To normalised protein's loading, the filter was stained with a monoclonal antibody anti-actin protein. (C) Q-ChIP analysis, of the indicated NF-Y target promoters on MEF after 16, 24, 48 hours (h) from Ad-GFP (white bars), or Ad-DN-NFYA after 16 h from infection (dark grey bars), 24 h (black bars), 48 h (grey bars), performed with anti-NFY-A, -p300, -H4K20me3, -H3K9me3 and -H3K9ac antibodies. The fold difference value in each case compares the sample performed after Ad-DN-NFYA infection to the corresponding control sample performed after Ad-GFP infection at the same time point (defined as 1). One of two independent experiments performed in triplicate is represented. (D) Q-ChIP analysis, performed with anti-HP1α antibody, of the indicated NF-Y target promoters on MEF after 24 h after infection with Ad-GFP (white bars) and Ad-DN-NFYA (black bars). It is shown one of two independent experiments performed in triplicate. (E) ChIPs were performed on proliferating MEFs 24 hours after Ad-GFP and wtNF-YA infections. No antibody was used as control (No Ab). PCR analysis were performed on the immunoprecipitated DNA samples with anti-NFY-A, -H3K9ac and -PAN-H4ac-Pan antibodies, using specific primers for the indicate promoters.