Interaction of retinoblastoma gene product with transcription factors ATF-a and ATF-2

The retinoblastoma gene product (pRb) plays an important role in constraining cellular proliferation and in regulating the cell cycle. pRb's regulation of the expression of transforming growth factor β2 (TGF-P2), an inhibitory growth factor gene, was suggested to be mediated by Activating Transcription Factor 2 (ATF-2)(Kim et al., 1992), Factor a (ATF-a) (Gong et al., 1995), and their DNA target element (ATF site: CGTCA) in the TGF-P2 promoter. The differential regulation of TGF-P2 promoter activity (first observed by Gong et al., 1995) by pRb has been explored in more depth and the functional interaction sites of ATF-2 and ATFa on pRb have been mapped and compared in this study. Optimal transient transfection conditions using lipofectamine as transfection inducer for the introduction of a foreign TGF-β2 CAT reporter construct into CHO cells have been selected. Under these conditions, the activation by co-transfected ATF-a, ATF-2 and the differential regulation mediated by pRb on the TGF-β2 promoter have been explored further in terms of varying input DNA concentrations and the time course for the expression of the regulated TGF-β2 CAT. Although the regulatory pattern was similar on days 1, 2 and 3, the greatest response was observed 48 hr after transient transfection. The functional interaction sites for both ATF-2 and ATF-a on pRb were probed by cotransfecting cells with a series of truncated pRb mutants and ATF-2 or ATF-a. Our results demonstrate that ATF-2 and ATF-a share the same interaction sites on pRb in vivo: primarily in the so called A/B pocket domains of pRb, since deletions of amino acid sequences in these domains caused dramatic decreases in the ability of pRb to affect the TGF-P2 promoter with or without the overexpression of ATF's. The interaction sites also appear to involve the C-terminal domain of pRb (referred to as the C pocket), although the degree of effect was less substantial than that seen with the A/B pocket mutants. The levels of expression of foreign wild type (wt) and mutant (mt) pRb's in the presence or absence of overexpression of ATF-2 and ATF-a have been monitored using an antibody against the HA tag located on the C-terminus of pRb. All mutant forms of pRb have been shown to be expressed at levels similar to wt pRb and overexpression of ATF-2 and ATF-a did not effect the levels of expression of wt and mt pRb's. We also studied the differential regulation of ATF-2's and ATF-a's transcriptional activations by pRb in an alternative cell line, the human uterine mesodermal tumor cell line SKUT-1 which lacks endogenous pRb. The introduction of foreign pRb does not cause cellular apoptosis in SKUT-1 cells. The same regulatory pattern as in CHO cells has been observed, although there was minor variation in the degree of stimulation or inhibition.