FIGURE 4.

HIRA mediates H3acK56 incorporation at the Vegfr1 locus. A, YSECs were starved overnight in the presence or absence of hydroxyurea (HU) and treated with FGF2/EGF for 3 h, and H3acK56 incorporation at the Vegfr1 locus was determined by ChIP in both hydroxyurea-treated and untreated cells. sup, FGF2/EGF. B, Western blots showing expression of histone chaperones in YSECs. C, Western blots showing induction of HIRA in starved HUVECs and BRMVECs upon FGF2/EGF treatment. D, Western blots (WB) showing specific knockdown of HIRA expression in YSECs. YSECs were infected with lentiviral vectors expressing shRNAs to knock down Hira, starved, and treated with FGF2/EGF for 3 h. E, RT-PCR and Western blot (WB) analysis showing strong inhibition of Vegfr1 expression in HIRAkd YSECs. F, ChIP analysis showing complete loss of FGF2/EGF-induced H3acK56 incorporation at the Vegfr1 locus in HIRAkd YSECs. G, Western blot analysis showing loss of total cellular levels of Lys-56 acetylated histone H3 but maintenance of FGF2/EGF-induced CBP expression in HIRAkd YSECs. H, Western blots showing rescue of H3acK56 levels in HIRAkd YSECs upon Na-Bu treatment. I, ChIP and RT-PCR analysis showing that Na-Bu treatment does not rescue loss of FGF2/EGF-induced H3acK56 incorporation and transcription at the Vegfr1 locus. PI, preimmune rabbit serum.