Figure 1

Histone deacetylase 1 and 2 (Hdacs1,2) localize to replication origins and target histone deposition marks. A. Western blot analysis of immunoprecipitated (IP) samples to determine interaction of Hdac1 and Hdac2 with proliferating cell nuclear antigen (PCNA). Nuclear extracts from HeLa cells were used in IP with anti-PCNA antibody or IgG (negative control). Percentage ratio of IP over input for Hdac1 and Hdac2 are shown. B. Hdac1 and Hdac2 occupancies at α-globin locus (early replicating origin) in NIH3T3 cells synchronized in S-phase were determined using chromatin immunoprecipitation (ChIP) assays. Average fold enrichment of immunoprecipitated DNA over input DNA from three independent experiments is shown. C. Western analysis of nuclear extracts prepared from NIH3T3 cells transfected with either non-targeting (NT) or Hdac1,2 siRNA (H12si) at 72 h post-siRNA transfection to determine changes in histone modifications. D-E. Hdac1 or Hdac2 or Hdac3 immunoprecipitated from nuclear extracts following treatment of NIH3T3 cells with either DMSO (D), 898 (panel D) or 233 (panel E) were used in enzyme assays. Enzyme inhibition was measured using Fluor-de-Lys HDAC fluorimetric activity assay. AFU, arbitrary fluorescence units; dotted line, denotes background/baseline signal obtained from using rabbit IgG in IP. F-G. Western analysis to determine changes in histone acetylation in NIH3T3 cells following 24 h treatment with DMSO or increasing concentrations of 898 (F) or 233 (G). H-I. Western analysis of whole cell lysates prepared from Hdac1^Fl/FlHdac2^Fl/Fl or Hdac3^Fl/Fl fibrosarcoma cells following Ad-Cre infection and treatment with 898 or 233. Cells were treated with 3 μM 898 or 233 for 24 h following a 48 hr Ad-Cre infection.