Fig. 4.

Transcription activation requires the histone binding and ATPase activity of CHD4. (A) The PHD1 and CD2 domains bind histones. (Top) CHD4 truncations comprising the PHD fingers (PHD1 and PHD2) and chromodomains (CD1 and CD2) were incubated with histones and associated histones were visualized on stained PAA gels (Top) and on immunoblots with α-histone H3 antibody (Middle). (Bottom) Amount of histones. Scheme illustrates the position of PHD fingers (PHD), chromodomains (CD), and ATPase/helicase domain. Point mutations are indicated by asterisks. (B) CD2 is required for CHD4 binding to rDNA. ChIP monitoring rDNA promoter occupancy of wild-type CHD4 and the indicated mutants is shown. Binding of mutant proteins was normalized to wild-type CHD4 (n = 3). Western blot shows the expression level of wild-type and mutant CHD4. (C) H3K4me3 is required for CHD4 binding to chromatin. ChIP comparing rDNA occupancy of CHD4, UBF, and H3K4me3 in WDR5-depleted (siWDR5) and control (siCtrl) cells (n = 3). (D) Chromatin remodeling and histone binding activity of CHD4 are required for transcriptional activation. Pre-rRNA levels in NIH 3T3 cells expressing wild-type or mutant CHD4 compared with mock transfected cells are shown (n = 3). Western blot shows the level of Flag-tagged wild-type and mutant CHD4. (E) Wild-type but not mutant CHD4 rescues pre-rRNA synthesis in CHD4-depleted cells. CHD4-deficient NIH 3T3 cells were infected with retroviruses expressing wild-type CHD4 or the indicated mutants. Bars represent the relative levels of pre-rRNA normalized to GAPDH mRNA (n = 3). (F) Depletion of CHD4 shifts NucD to the position of NucU. Nucleosome positions were analyzed in NIH 3T3 cells expressing shRNA against CSB, TIP5, or CHD4 (lanes 1–4). To rescue nucleosome positions, CHD4-depleted cells were infected with retroviruses expressing wild-type or mutant CHD4 (lanes 5–9). Bar diagram shows the ratio of NucU (gray) to NucD (dark).