Journal: Nucleic Acids Research
Article Title: Gene-specific factors determine mitotic expression and bookmarking via alternate regulatory elements
Figure Lengend Snippet: MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. ( A ) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA + ) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. ( B–E ) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against CREB, RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and IgG (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.
Article Snippet: Reagents α-RFX5 was from Rockland, α-NFY-A (H-209) (sc-10779), α-NFYA (G-2) (sc-17753), α-NFY-B (FL-207) (sc-13045), α-CREB (C-21) (sc-186), α-RNA Polymerase II (N-20) (sc-899), normal rabbit IgG (sc-2027), normal mouse IgG (sc-2025) were from Santa Cruz Biotechnology, α-TBP (ab28175) and α-H3 (ab1791) were from Abcam, α-acetyl H3 (06-599), α-acetyl H4 (06-866), α-H3K4me2 (07-030), α-H3K4me3 (07-473), α-H3S10-Phos (06-570), α-PP2Ac (Cat.# 05-421) and α-CREB (17-600) were from Millipore, α-PP2Ac (#2028) was from Cell Signaling, α-β-tubulin (T4026) was from Sigma-Aldrich, α-GFP was from Minotech, α-JellyRed (α-KillerRed, Cat.# AB962) was from Evrogen, mouse monoclonal α-myc antibody was secreted from the hybridoma cell line Myc-9E10.2 ( ) and anti-CIITA antibody was described earlier ( ).
Techniques: Expressing, Activation Assay, Flow Cytometry, Staining, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction