FIG. 9.

dREAM/MMB represses group D/E genes by two independent mechanisms. (A) Western blot analysis of whole-cell extracts from cells treated with double-stranded RNA (dsRNA) targeting white (control) or Pc. The blot was probed with anti-PC or anti-β-tubulin antibodies. (B) Northern blot analysis using probes to group D/E genes or β-tubulin. Total RNA was isolated from white-, p55CAF1-, or Pc-depleted cells. (C) Changes in H3K27 acetylation levels at coding regions of group D/E genes in dE2F2- or E(Z)-depleted cells. ChIP assay was performed with anti-acetyl H3K27 antibodies in SL2 cells treated with dsRNA targeting white (control), dE2F2, or E(Z). (D) Histone H3K27me2 at coding regions of group D/E genes is not affected by HDAC inhibition. ChIP assay was performed with H3K27me2 antibodies on cells treated with PBS or NaB. (E) Histone acetylation at promoter regions of group D/E genes is not affected by the depletion of E(Z). ChIP assay was performed with panacetylated histone H3 (top panel) or panacetylated histone H4 (bottom panel) antibodies. Note that the panacetylated histone H3 antibody used in this experiment does not target the H3K27 site. (The antibody is raised against the peptide consisting of the first 20 amino acids of histone H3). (F) Northern blot analysis using probes to group D/E genes. Total RNA was isolated from cells incubated with dsRNA targeting white (control), dE2F2, E(Z), dRPD3, or both dRPD3 and E(Z) (top panel) or treated with E(Z) dsRNA or NaB or cotreated with E(Z) dsRNA and NaB (bottom panel).