In vivo detection of promoter occupancy by the E2F and pRB family using chromatin immunoprecipitations. (A) Outline of the in vivo cross-linking and chromatin immunoprecipitation (IP) protocol. (B) Promoters examined in this study. Solid ovals represent E2F-binding sites on the basis of previous studies; small arrows indicate primers used in PCR amplification reactions. Large arrows represent published major transcription start site. (C) Chromatin immunoprecipitations from asynchronously growing T98G cells. Input corresponds to PCR reactions containing 0.5% of total amount of chromatin used in immunoprecipitation reactions. Mock immunoprecipitations performed with irrelevant, control antibodies (anti-T Antigen pAb101) and control reactions lacking antibodies (No Ab) are shown. Amplified products were detected by autoradiography.

Cell cycle synchronization and gene expression analysis. (A) FACS analysis of T98G cells that were rendered quiescent by serum starvation (0 hr) and were subsequently restimulated with serum for the indicated lengths of time and allowed to enter the cell cycle. DNA content (propidium iodide intensity) is plotted vs. cell number. (B) Northern blot analysis of expression of the indicated genes during the synchronization experiment in A. To ensure equal loading of RNA, each blot was subsequently stripped and rehybridized with an ARPP P₀ gene fragment, and a representative filter is shown. (C) Gel mobility shift analysis of E2F and pRB complexes. Whole cell extracts at each stage of the cell cycle (indicated at top) were incubated with a labeled probe containing an E2F site and each of the designated antibodies. The positions of E2F-pRB/p107/p130 complexes and free E2F (E2F) are shown at right. Each of these complexes was abrogated by a specific competitor oligonucleotide (data not shown). Asynchronous (AS) samples were prepared from cells entering a second cell cycle (with roughly equal percentages of G₁, S, and G₂ cells). Lanes marked − and control contained no antibody and 12CA5 (anti-flu hemagglutinin antibody), respectively. α-E2F-4 lanes contain two different monoclonal antibodies specific for E2F-4 (WUF3 and LLF4, respectively).

The binding of E2F family of proteins changes during the cell cycle. Chromatin immunoprecipitations were performed with antibodies specific for individual E2F family members as indicated, and the resulting immunoprecipitates were amplified with primer pairs corresponding to the genes shown. G₀, serum-starved cells; early G₁, late G₁, G₁/S, and S-phase cells were stimulated with serum 4–8, 16, 20, and 24 hr, respectively. Antibodies tested in each case are listed in Materials and Methods.

Analysis of in vivo binding by the pRB family. Antibodies (listed in Materials and Methods) specific for each member of the pRB family were used to immunoprecipitate chromatin, and PCR was performed with primer pairs for each of the indicated genes. Cell cycle populations were identical to those in Fig. 3.

Antibodies against each pRB family member immunoprecipitate their chromatin-bound targets. (A) Schematic representing the position of chromatin in gradients relative to free DNA and protein under conditions in which cells were either fixed with formaldehyde (+ cross-linker) or left untreated (− cross-linker). Brackets indicate chromatin fractions selected for further analysis in B. (B) Fractions in A were immunoprecipitated with anti-pRB, anti-p107, and p130 antibodies as indicated and the resulting precipitates were probed with the same antibodies as indicated. (C) p107 and p130 are detected at similar levels in T98G cells entering a second cell cycle (32-hr time point in Fig. 2). Chromatin immunoprecipitations were performed with the indicated antibodies, and E2F-1, cyclin A, and actin promoter fragments were amplified as described in the legend to Fig. 1. Distinct monoclonal and polyclonal anti-pRB antibodies were tested as shown.

Histone acetylation levels of E2F-responsive genes change during the cell cycle. (A) Chromatin was prepared from synchronized T98G cells (as described in Fig. 2A) and immunoprecipitated with antibodies specific for acetylated histone H3 and acetylated H4 as described in the legend to Fig. 1. Cell cycle stages were identical to those described in Fig. 3. Parallel immunoprecipitations without antibody or with an irrelevant antibody control failed to yield detectable signals after an equivalent autoradiographic exposure (data not shown). (B) Data in A were quantitated by PhosphorImager analysis and normalized vs. input levels. The y-axis indicates fold acetylation (in arbitrary units). (Light-colored bars) acetylated H3; (dark bars) acetylated H4.

Model for in vivo occupancy by E2F and pRB family members during cell cycle progression. (A) In vivo occupation of E2F-1, Cdc25A, cyclin A, Cdc6, and p107 promoters by the E2F and pRB family during cell cycle progression and transcriptional consequences. (B) In vivo binding of B-myb promoter. In A and B, promoter binding by E2F-4 correlates with transcriptional repression, whereas binding by E2F-1 and E2F-3 occurs at a time when each promoter is activated. However, the B-myb promoter is transcriptionally active after E2F is no longer bound, consistent with Zwicker et al. (1996). (D) HDAC activity; (H) nucleosomes; (Ac) acetylated histones; (HAT) histone acetyltransferase. These promoters are likely to be regulated by additional trans-activator proteins proximal to E2F, including Sp1. Recruitment of deacetylase and HAT activities could occur by direct interactions with E2F complexes or could require additional factors (X) yet to be defined.