Effects of HDAC inhibitor 106 on frataxin mRNA and protein levels and on histone acetylation in KIKI mice.
(A) The effect 106 on histone acetylation persists for at least 24 hours in KIKI mice. Mice were sacrificed, then extraction of brain acid-soluble nuclear proteins, SDS-PAGE, and western blotting with antibodies to total histone H3 and to acetylated histone H3 were performed. Sample in lane a is from the brain of a 106-treated mouse sacrificed after four hours, in lane b is from the brain of a vehicle-treated mouse, in lane c is from the brain of a 106-treated mouse sacrificed after 24 hours. (B) Frataxin mRNA levels in WT mice and in KIKI mice treated with either vehicle or HDAC inhibitor 106. Frataxin levels were determined by quantitative real-time RT-PCR relative to RER1 and β2m mRNAs, both unaffected by the HDACI. The WT frataxin mRNA level in each tissue was set to 100. Brain: n = 12. Cerebellum and Heart: n = 8. Data from 3 separate experiments were pooled and bars indicate SD. (** P<0.001; * P<0.05). (C) Western blot of frataxin in brain extracts of KIKI mice that had been treated with daily injections of 150 mg/Kg of 106 (lanes B5–B8) or vehicle (lanes B1–B4) for three days. Mice were sacrified 24 hours after the last injection. Actin was detected for normalization. (D) Densitometric analysis of the frataxin western blot shown in (C). (E) Levels of H4K5, H4K8, H4K16 and H3K14 acetylation of H3K9 trimethylation in KIKI mice treated with one subcutaneous injection per day of 150 mg/kg of 106 for 3 consecutive days compared to vehicle-treated KIKI littermates and WT (n = 6). We performed ChIP experiments with antibodies for murine histones H3 and H4 carrying each modification. Primer pairs corresponded to the first intron of the mouse frataxin gene just upstream of the point of insertion of the GAA repeat in KIKI mice. Relative recovery, determined by quantitative real-time PCR, is expressed in relation to GAPDH and the recovery in samples from WT animals is set to 100 for each antibody. Error bars are SEM of four independent immunoprecipitations, each quantified in triplicate.
