A dual role for the dREAM/MMB complex in the regulation of differentiation-specific E2F/RB target genes

Abstract

E2F and RB proteins regulate the expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. Recent studies indicate that they function as part of an evolutionarily conserved multiprotein complex termed dREAM/DREAM/LINC. Here we characterize the role of the Drosophila complex, dREAM, in the regulation of differentiation-specific E2F target genes in actively proliferating cells. These genes are regulated differently from cell cycle-related E2F targets, they do not depend on E2F activation, and E2F/RB repression is maintained throughout the cell cycle. In proliferating cells, their repression is dependent on dREAM. We find that dREAM plays a dual role in their regulation. First, it is required for the stability of the repressive dE2F2/RBF complexes at their promoters during S phase. Second, we find that dREAM is indispensable for both transcriptional repression mechanisms employed at these genes.

Fig 1

E2F binding at target gene promoters in dREAM-disrupted cells. (A and B) Chromatin immunoprecipitation (ChIP) performed with antibodies recognizing dE2F1 (A) or dE2F2 (B) in cells incubated with double-stranded RNA (dsRNA) targeting white (control), dE2F2, or Mip40. The amount of coprecipitated DNA was quantified by quantitative real-time PCR. Results are normalized to a nonspecific sequence (promoter of RP49) and represent the average of three independent experiments. Promoter sequences analyzed are DNA Pol α and PCNA (cell cycle-regulated genes) and Arp53D, CG2887, and CG3505 (group E genes). (C) RT-PCR analysis of mRNA levels from cells incubated with dsRNA targeting white (control), dE2F2, or Mip40. (D) ChIP assay performed with antibodies recognizing dE2F2 in cells incubated with dsRNA targeting white (control), Mip130/TWT, or p55/CAF1. (E) Western blot analysis of whole-cell extracts from SL2 cells incubated with dsRNA targeting white (control), dE2F2, Mip40, p55/CAF1, or Mip130/TWT. Numbers at the bottom represent quantified dE2F2 protein levels. ChIP results in panels B and D are corrected for total dE2F2 protein levels.

Fig 2

dREAM is required for dE2F2 binding at group E gene promoters during S phase. (A) Cell cycle profiles of asynchronously growing SL2 cells or cells synchronized in S phase and treated with dsRNA targeting either white (control) or Mip40. (B) Percentage of cells in G₁, S, and G₂/M phases of the cell cycle as determined by two-dimensional (2D) FACS analysis. Numbers under graph represent the percentage of S-phase cells. (C) Western blot analysis of whole-cell extracts from SL2 cells incubated with dsRNA targeting white (control), dE2F2, or Mip40. (D) RT-PCR analysis of mRNA levels from cells incubated with dsRNA targeting white (control), dE2F2, or Mip40. (E) ChIP assay performed with antibodies recognizing dE2F2 in asynchronously growing cells (black bars) and in cells synchronized in S phase and incubated with dsRNA targeting white (control), dE2F2, or Mip40. (F) ChIP assay performed with antibodies recognizing RBF1 in asynchronously growing cells (black bars) and in cells synchronized in S phase and incubated with dsRNA targeting white (control), dE2F2, or Mip40.

Fig 3

dREAM is not required for dE2F2 binding outside S phase. (A) Cell cycle profiles of cells incubated with dsRNA targeting white (control), Mip40, dE2F1, and dE2F1+Mip40 (cotreated). (B) Percentage of cells in G₁, S, and G₂/M phases of the cell cycle as determined by 2D FACS analysis. (C) ChIP assay performed with antibodies recognizing dE2F2 in cells incubated with dsRNA targeting white (control), Mip40, dE2F1, and dE2F1+Mip40 (cotreated). (D) ChIP assay performed with antibodies recognizing RBF1 in cells incubated with dsRNA targeting white (control), Mip40, dE2F1, and dE2F1+Mip40 (cotreated).

Fig 4

Phosphomimic RBF1 mutant cannot bind at both cell cycle-regulated and group E gene promoters. (A) Schematic representation of the 7 putative CDK phosphorylation sites of RBF1. (B) ChIP assay using antibodies that recognize dE2F2 or nonspecific antibodies in cell lines expressing either RBF1 WT (R1WT) or RBF1 S/T→E phosphomimic (R1E). Left panels, cell cycle-regulated gene promoters; right panels, group E gene promoters. (C) ChIP assay using antibodies that recognize RBF1 or nonspecific antibodies in cell lines expressing either RBF1 WT (R1WT) or RBF1 S/T→E phosphomimic (R1E). Left panels, cell cycle-regulated gene promoters; right panels, group E gene promoters.

Fig 5

Phosphomutant RBF1 suppresses dE2F2 binding defects in dREAM disrupted cells. (A) RT-PCR an analysis of mRNA levels from cells expressing RBF1 WT (R1WT) or RBF1 phosphomutant (R1A) and incubated with dsRNA targeting white (control) or Mip40. (B) Percentage of cells in S phase of the cell cycle expressing RBF1 WT (R1WT) or RBF1 phosphomutant (R1A) and incubated with dsRNA targeting white (control) or Mip40; cells incubated with dsRNA targeting white (control) or dE2F1 are shown as comparison. (C) ChIP assay using antibodies that recognize dE2F2 in cells expressing RBF1 WT (R1WT) or RBF1 phosphomutant (R1A) and incubated with dsRNA targeting white (control) or Mip40.

Fig 6

Phosphomutant RBF1 restores dE2F2 binding in S-phase enriched cells lacking dREAM. (A) Cells were treated with hydroxyurea-aphidicolin and collected 3 h after release. Expression of RBF1 WT or R1A mutant was induced 24 h prior to collection. Percentage of cells in S phase of cells expressing RBF1 WT (R1WT) or RBF1 phosphomutant (R1A) and incubated with dsRNA targeting white (control) or Mip40. (B) ChIP assay using antibodies that recognize dE2F2 in S-phase enriched cells, expressing RBF1 WT (R1WT) or RBF1 phosphomutant (R1A) and incubated with dsRNA targeting white (control) or Mip40. (C) ChIP assay using antibodies that recognize RBF1 in S-phase enriched cells, expressing RBF1 WT (R1WT) or RBF1 phosphomutant (R1A) and incubated with dsRNA targeting white (control) or Mip40. (D) Western blot analysis of whole-cell extracts from S-phase enriched cells incubated with dsRNA targeting white (control), RBF1, or Mip40; hyper- and hypophosphorylated forms of RBF1 (black and white arrowheads, respectively) were separated using a bis-Tris SDS-PAGE system; blots were probed with anti-RBF1 and anti-β-tubulin (loading control) antibodies. Numbers under blots represent the ratio of quantified levels of slower-migrating/faster-migrating forms of RBF1. (E) Coimmunoprecipitation of RBF1 with p55/CAF1 and dE2F2. Anti-dE2F2, anti-p55, or antibodies were used in immunoprecipitations from whole-cell extracts. Blots were probed with anti-RBF1 antibodies.

Fig 7

dREAM is required for the repression of group E genes in both R1WT- and R1A-expressing cells. (A and B) ChIP assay performed with antibodies recognizing pan-acetylated histone H3 (A) or pan-acetylated histone H4 (B) in cells expressing RBF1 WT (R1WT) or phosphomutant (R1A) and incubated with dsRNA targeting white (control) or Mip40. (C) ChIP assay performed with antibodies recognizing histone H2 K27 dimethylation (H3K27me2) in cells expressing RBF1 WT (R1WT) or phosphomutant (R1A) and incubated with dsRNA targeting white (control) or Mip40. (D) Northern blot analysis of total RNA isolated from cells expressing RBF1 WT (R1WT) or phosphomutant (R1A) and incubated with dsRNA targeting white (control) or Mip40.