Fig. 5.

E47 directly binds to the Cdk6 gene and represses its transcription. (A) Sorted PD31 cells transduced with the ER-E47 retroviral vector were cultured with ethanol (vehicle control) or tamoxifen (4-OH) for 6 h. RNA was harvested, and Cdk6 and Cdk4 mRNA levels were measured by quantitative RT-PCR normalized to hprt. Error bars indicate the standard deviations of triplicate samples. (B) Nuclear extracts from 220-8 cells that were cultured for the indicated times or for 24 h (+) in 2 μM STI571 or in the absence of STI571 (-) were incubated with a ³²P-labeled oligonucleotide containing the E2A binding site μE5 from the IgH intronic enhancer and analyzed in an EMSA. A dried gel phosphorimage is shown. Lanes labeled α-Ig and α-E47 contain control and E47-specific antisera. The arrow indicates the specific E47-DNA complex. (C) A 3-kb region encompassing the first exon of Cdk6 (upper). The black box represents the first exon, and the arrow indicates the transcriptional start site. The letters indicate putative E2A binding sites. The alignment of putative E2A binding sites in the murine Cdk6 promoter and first exon to the E-box consensus binding site are indicated by the box (lower). (D) Results of an EMSA performed as described above, using nuclear extracts from 220-8 cells cultured in the presence (+) or absence (-) of 2 μM STI571 and a 24- to 30-bp radiolabeled probe representing each of the putative E2A binding sites compared to the μE5 probe. The arrow indicates the specific E2A-DNA complex. (E) HF4 cells (expressing Flag-tagged endogenous E2A) cultured in the presence or absence of 1 μM STI571 were subjected to chromatin immunoprecipitation with anti-Flag or control IgG antibody. Precipitates were analyzed by quantitative PCR using primer pairs encompassing region A, region D, region EX1, Cd19 (negative control), and Cd79a (positive control). The results are presented as enrichment over input. (F) 7G-S cells were cultured in the presence (+) or absence (-) of 2 μM STI571 for 16 h and then analyzed by quantitative RT-PCR for E47, E12, Id2, and Id3 mRNA. All values were normalized to hprt levels, and error bars represent standard deviations of triplicates. (G) 7G-S cells were cultured in the presence of 2 μM STI571 for 16 h followed by whole-cell lysis. Lysates were analyzed by immunoblotting with antibodies to Id2 and tubulin.