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. Author manuscript; available in PMC: 2015 Jul 3.
Published in final edited form as: Nat Commun. 2014 Dec 18;5:5872. doi: 10.1038/ncomms6872

Figure 6. Brd1-Hbo1 complex acetylates H3K14 at the Cd8 gene loci.

Figure 6

(a) Levels of acetylation at histone H3 and H4 in total thymocytes from Tie2-Cre (Brd1 +/+) and Tie2-Cre;Brd1fl/fl (Brd1Δ/Δ) thymus. Histones purified from thymocytes were analysed by western blotting using the indicated antibodies. (b) Levels of H3K14 acetylation at the enhancers and promoters of the Cd8a and Cd8b1 genes. Schematic representation of the Cd8a and Cd8b1 gene loci and the location of the primer sets are depicted (upper panel). The binding of Brd1 and Hbo1 to the Cd8a and Cd8b1 gene loci was determined by ChIP using an anti-Flag antibody in AKR1/3 × Flag-Brd1 cells and anti-HA antibody in AKR1/HA-Hbo1 cells, respectively. The relative amount of immunoprecipitated DNA is depicted as a percentage of input DNA. An isotype control IgG was used as a negative control. The Cd4 silencer element and p16 promoter served as controls. The data are shown as the mean±s.d. for triplicate PCRs (middle panel). ChIP analyses were performed using Brd1 +/+ and Brd1Δ/Δ DP cells and Brd1Δ/Δ CD4 +CD8 −TCRβ−/low cells purified by cell sorting and an anti-acetylated H3K14 antibody. The relative amount of immunoprecipitated DNA is depicted as a percentage of input DNA. The Cd4 silencer element served as a control. The data are shown as the mean±s.d. for triplicate PCRs (lower panel). (c) Brd1 and Hbo1 directly interact with Runx proteins. 293T cells were transfected with Flag-tagged Runx1 or Runx3 together with either HA-tagged Brd1 or Hbo1. Runx proteins were immunoprecipitated using an anti-Flag antibody, and then immunoprecipitates were detected by immunoblotting using anti-Flag and anti-HA antibodies (upper and middle panels). The direct interactions between Runx proteins with Brd1 and Hbo1 were confirmed by GST-pull down assays using GST-Runx1 and GST-Runx3 fusion proteins and in vitro translated HA-tagged Brd1 and Hbo1 following immunoblotting using an anti-HA antibody (lower panels). (d) Brd1 and Hbo1 directly interact with Ikaros proteins. 293T cells were transfected with Flag-tagged Ikaros together with either HA-tagged Brd1 or Hbo1. Ikaros protein was immunoprecipitated using an anti-Flag antibody, and then immunoprecipitates were detected by immunoblotting using anti-Flag and anti-HA antibodies.