Figure 9

Loss of histone deacetylase 1 and 2 (Hdacs1,2) affects nascent chromatin structure. A-B. Nuclei were isolated from cells treated with either DMSO or 898 (A), or from cells transfected with non-targeting (NT) siRNA or Hdacs1,2-siRNA (B), bromodeoxyuridine (BrdU)-labeled nuclei were digested performed with various micrococcal nuclease (MNase) concentrations (0, 0.5, 2 and 8 units) for 5 min at 37°C. Equal amount of purified DNA was subjected to agarose gel electrophoresis. Pixel intensity of mono, di- and trinucleosomes obtained from 0.5 units MNase for control and knockdown samples and 2 units MNase for DMSO or inhibitor-treated samples were measured using the ImageJ software. Quantitative data for nucleosome levels and input levels were obtained using densitometry from four independent experiments for inhibitor treated samples and two independent experiments for knockdown samples. Nucleosome intensities were normalized to input intensities. The average intensity for control was set as 1 and fold-increase in nucleosome intensities in treated samples were determined in comparison to the control. Error bars denote standard error of the mean from multiple experiments. NTsi and H12si refer to non-targeting and Hdacs1,2-siRNA, respectively. C. Mononucleosome sensitivity was determined in S-phase synchronized cells as described in the Methods section. Fold-change in MNase sensitivity for a given region in S-phase cells was determined by qRT-PCR and normalized to the sensitivity of the locus in serum-starved samples. The plotted data represents an average +/− standard error of 3 independent treatments and the experiment was repeated two times. D, 8 and 2 refer to DMSO, 898 and 233 treatments, respectively. EtBr, ethidium bromide.