Identification of E2F4 targets using a CpG island microarray. Presented is a flow chart describing the methodology used to examine E2F targets. A portion of the CpG island microarray probed with E2F4 (red:Cy5) and the control no antibody (green:Cy3) chromatin is also shown.

Confirmation of E2F targets. A representative chromatin immunoprecipitation experiment using HeLa cells is shown (A–C). Antibodies against E2F1 (lane 1), E2F4 (lane 2), RNA polymerase II (lane 3), or no antibody (lane 5) were used. Following DNA purification, samples were subjected to PCR with primers designed to the individual loci. A fixed portion of the total input (0.2%) was also examined by PCR (lane 4). (A) Examination of 16 randomly chosen positive clones identified as E2F targets in the CpG island microarray by ChIP. The first five clones contain a consensus E2F site, the next five clones contain a 7/8-bp match to the consensus, and the last six clones contain no recognizable E2F site. (*) Loci that were multiple hits in the microarray analysis. (B) Examination of five randomly chosen negative clones by ChIP. (C) Loci identified by multiple hits in the CpG island microarray are examined in a ChIP experiment.

In vitro confirmation of E2F binding to newly identified target promoters. (A) An electromobility shift competition experiment was performed using the E2F site from the B-myb promoter as a probe. Lane 1 contains the probe alone. In lanes 2–10, the probe was incubated with HeLa nuclear extract and either no competitor DNA (lanes 2,10), the unlabeled B-myb E2F site oligonucleotide (lane 3), fragments containing E2F sites from the identified promoters indicated above the gel image (lanes 4,6,8,9), or fragments which do not contain E2F sites (lanes 5,7). NC-A (negative control A) and NC-B (negative control B) are PCR fragments generated using the same method as the specific competitors. These fragments were derived from promoters that contain a consensus E2F site (NC-A) or a 7 out of 8-bp match to the consensus E2F-binding site promoter (NC-B), but the fragments lack the region containing the E2F site. Arrows to the right of the gel image in A and B indicate the specific E2F/DP complex. (B) An EMSA competition experiment was performed using the E2F site from the B-myb promoter as a probe. Lane 1 contains the probe alone. In lanes 2–9, the probe was incubated with HeLa nuclear extract and either no competitor DNA (lane 2), the unlabeled B-myb E2F site oligonucleotide (lane 3), or two different concentrations of fragments from the identified promoters UXT (lanes 4,5), DBPA (lanes 6,7), and NASP (lanes 8,9). Increasing competitor concentrations are indicated above the gel image.

Characterization of UXT promoter. (A) Fragments of the UXT, ChET8, c-Rel, or CMV promoters were cloned upstream of the luciferase reporter and used in transient transfection experiments. 3T3 cells were transfected with either a promoter luciferase reporter construct alone or with an E2F1 expression plasmid as indicated on the x-axis. The specific promoter used in each transfection is also indicated on the x-axis. The y-axis represents the relative activity of each construct transfected alone or with E2F1 overexpression. Ratios are relative to the individual construct activity without E2F overexpression. (B) A ChIP experiment utilizing antibodies to E2F1 (lane 1), E2F2 (lane 2), E2F3 (lane 3), E2F4 (lane 4), E2F5 (lane 5), Rb (lane 6), p107 (lane 7), p130 (lane 8), or a no-antibody control (lane 9) are shown. PCR was performed with a primer set specific to the UXT promoter. A standardized aliquot of the total input chromatin is also shown (lane 10).

Characterization of E2F targets. A summary of the results obtained after sequence analysis of the positive clones. The results of the ChIP assay for the first 16 randomly chosen clones is presented, as is a list of loci represented by multiple hits, a list of known E2F targets, and a list of targets involved in chromatin structure and DNA replication/repair/recombination.