We have examined the relationship between sequence-specific DNA-binding proteins that activate transcription of ElA-inducible adenovirus early promoters. Factors previously referred to as E4F1 and E2A-EF bind to the E4 and E2A promoters, respectively. We demonstrate here that E4F1 and E2A-EF have identical DNA-binding specificity. Moreover, E4F1 and E2A-EF both activate transcription of the E4 and E2A promoters in vitro. These findings demonstrate that E4F1 and E2A-EF are the same factor, which we have designated activating transcription factor, or ATF. In addition to the E4 and E2A promoters, ATF binds to an important functional element of the ElA-inducible E3 promoter. Interaction of a common activator protein, ATF, with multiple ElA-inducible early viral promoters, suggests a significant role for ATF in ElA-mediated transcriptional activation.The EIA gene of adenovirus produces closely related 13S and 12S mRNAs that encode nuclear-localized phosphoproteins with diverse transcriptional regulatory properties (1-3). The EMA 13S gene product coordinately activates a set of viral early genes (EIA, EIB, E2A, E3, and E4) during a productive infection of permissive human cells (4-7). The ElA 12S gene product encodes a transcriptional repression function that appears to act through transcriptional enhancer elements (8-10). In addition to regulating viral transcription, ElA activates or represses transcription of a limited number of cellular genes (11,12), and activates polymerase III-dependent promoters (for review, see ref. E4 and E3 transcription (18, 19). For the EIB promoter (22) and the cellular /-globin promoter (23), the "TATA" box has been implicated as an ElA-responsive promoter element. Thus activation of a variety of ElA-inducible promoters appears to involve different cellular factors and may occur through divergent pathways, ultimately linked by ElA. Two independent studies have identified additional factors that interact with early viral promoters. A factor referred to as E4F1 binds to the E4 promoter and also interacts with the EIA, E2A, and E3 promoters (18). Similarly, a factor referred to as E2A-EF binds to the E2A, EIA, E3, and E4 promoters (17). We show here that E4F1 and E2A-EF have the same DNA-binding specificity and that both factors activate transcription of the E4 and E2A promoters in vitro. These results demonstrate that E4F1 and E2A-EF are the same factor, which we refer to as ATF, for activating transcription factor. In addition to the E4 and E2A promoters, ATF interacts with an important functional element of the adenovirus E3 promoter. The interaction of ATF with multiple ElA-inducible promoters suggests a significant role for ATF in E1A-mediated transcriptional activation. MATERIALS AND METHODSPlasmids. pE4WT contains the adenovirus type 5 genome between map units 100 and 89, including the entire E4 gene cloned into pBR322 between the EcoRI and Pvu II sites.

One of the most consistent results in the epidemiology of human breast cancer is the inverse relationship of risk and early full-term parity. The goal of this study was to investigate the molecular mechanisms through which early full-term pregnancy protects the breast from cancer development. We used Wistar-Furth (WF) rats as our experimental system and mimicked pregnancy using estrogen and progesterone (E/P). Sexually mature female rats were treated with steroid hormones for 21 days and after 28 days of gland involution, the rats were administered MNU. Rats that received a high dose of 20 microg E and 20 mg P exhibited an 82% reduction in the incidence of mammary adenocarcinomas as compared to the rats receiving only blank pellets. Decreasing doses of E/P were partially protective suggesting that complete differentiation of the gland was not required for refractoriness. We measured the RNA expression levels of several target genes involved in the regulation of mammary cell proliferation and/or differentiation including estrogen receptor (ER) and progesterone receptor (PR), cyclins D1 and D2, the cell cycle inhibitors p16, p21 and p27, and the tumor suppressor p53. At the time of MNU treatment we found no significant differences in the expression of these genes, with the possible exception of p21, indicating that hormone treatment did not result in constitutive changes in expression levels. The numbers of apoptotic cells were low and comparable in the hormone exposed and age-matched virgin gland (AMV) at the time of carcinogen challenge and remained low for 8 days after MNU treatment. The number of BrdU-labeled cells at the time of carcinogen challenge were also low in both the AMV (1.8%) and hormone exposed (0.8%) animals. In contrast, cell proliferation in the AMV (5.7%) was significantly different from both the parous involuted (1.2%) and the E/P-treated involuted (1.5%) animals 8 days after MNU treatment. We interpret these data to indicate that hormone treatment results in mammary epithelial cells that have persistent alterations in intracellular pathways governing proliferation responses to carcinogens.

Full-term pregnancy early in reproductive life is protective against breast cancer in women. Pregnancy also provides protection in animals against carcinogen-induced breast cancer, and this protection can be mimicked by using the hormones estrogen and progesterone. The molecular mechanisms that form the basis for this protective effect have not been elucidated. On the basis of our results, we propose a cell-fate hypothesis. At a critical period in adolescence the hormonal milieu of pregnancy affects the developmental fate of a subset of mammary epithelial cells and its progeny, which results in persistent differences in molecular pathways between the epithelial cells of hormone-treated and mature virgin mammary glands. These changes in turn dictate the proliferative response to carcinogen challenge and include a block in carcinogen-induced increase in mammary epithelial cell proliferation and an increased and sustained expression of nuclear p53 in the hormone-treated mammary gland. This hormone-induced nuclear p53 is transcriptionally active as evidenced by increased expression of mdm2 and p21 (CIP1͞WAF1). Importantly, exposure to perphenazine, a compound that induces mammary gland differentiation but does not confer protection, does not induce p53 expression, indicating that p53 is not a differentiation marker. The proliferative block and induction of p53 are operative in both rats and mice, results that support the generality of the proposed hypothesis.T he lifetime risk of developing breast cancer among Western women is Ϸ10%, and despite advances in therapeutic strategies, breast cancer remains the leading cause of cancer deaths in women in most developed countries (1). Prevention of the disease can be achieved with better understanding of the etiological factors contributing to the development of the disease. There is significant evidence that the timing of normal developmental events like menarche, menopause, and age of first parity have a significant impact on an individual's susceptibility to breast cancer (2, 3). In particular, there is strong epidemiological evidence that women who experience a full-term pregnancy early in their lives have a significantly reduced risk for developing breast cancer (3-5). This is recapitulated in rat models that demonstrate that early full-term pregnancy confers resistance to chemical carcinogen-induced mammary tumorigenesis (6-11). This protection can be mimicked with the hormones estrogen (E) and progesterone (P; refs. 9 and 12) or human CG (13) given either before or immediately after carcinogen challenge to induce a refractory state.Despite a wealth of literature supporting the role of endocrinological processes in mediating parity-related refractoriness, the cellular and molecular mechanisms that underlie hormone-induced refractoriness are largely unresolved. The utility of the rodent models in which a defined hormonal regimen can be used to mimic the protective effect of pregnancy is well documented (9-17).Differing hypotheses to explain the protective effects have ...

Utilizing the gel electrophoresis/DNA binding assay, a factor specific for the upstream transcriptional control sequence of the EIA-inducible adenovirus EIIA-early promoter has been detected in HeLa cell nuclear extract. We have analyzed the EIIA-E promoter by a linkerscanning (LS) mutagenesis procedure (20). These studies not only helped us to rule out the direct interaction of the.EIA gene product with the EIIA-E promoter sequences during the EIA-stimulated transcription but also allowed us to identify two transcriptional control sequences upstream from the cap site, namely, regions I and II (see Fig. 1). Region I is located closest to the cap site and appears to be analogous to the "TATA" box, and its sequence is 5' CTTAAGAGT 3'. Region II is 17 nucleotides long and maps upstream from the cap site, and its sequence is 5' TGGAGATGACGTAGlTTl 3'. Mutations in either region I or II result in a drastic reduction of transcription (20)

We previously reported the identification of a host factor (EIIA-EF) specific for an upstream transcriptional control sequence (-82 to -66) of the EIA-inducible adenovirus EIIA early promoter. The levels of this factor remained unchanged after virus infection of human cells. Another study also identified a factor (EIIF) specific for this same promoter, but the activity of this second factor was shown to increase severalfold after virus infection. We now show that these dramatically different results, both based on gel shift assays on the same promoter, may be explained by variations in protocol details and actually identify two distinct factors. When synthetic DNA copolymers [poly(dI)-poly(dC) or poly(dI-dC)-poly(dI-dC)J are used as competitors in gel shift assays, a factor specific for DNA sequences between -82 and -66 can be identified, whereas when natural eukaryotic DNAs (salmon sperm or calf thymus) are used as competitors a different factor specific for DNA sequences between -69 and -33 can be identified. We have mapped the DNA-protein contact residues for the EIIF by analyzing a series of linker scan mutants in gel shift assays and methylation interference experiments. The EIIA-EF and EIIF bind to two distinct but adjacent sequences. Competition experiments indicate that these two activities are due to two different factors. Consistent with the earlier reports, the levels of one (EIIA-EF) do not change after virus infection of human cells, whereas the levels of the other (EIIF) are increased severalfold.Eukaryotic RNA polymerase TI promoters contain a complex array of cis-acting genetic elements that regulate basal, induced, and repressed transcription rates. These cis-acting regulatory sequences interact with a variety ofgeneral as well as gene-specific transcription factors. The interaction of the cis regulatory elements of promoters with the trans-acting promoter-specific DNA-binding proteins is important in control of tissue-specific, hormone-induced, viral-induced, and growth-related gene expression.Adenovirus (Ad) provides a useful model system to study the control of eukaryotic gene expression. In human cells infected with Ad type 2 or 5, a set offive early viral promoters are coordinately expressed (1). Efficient transcription of these early viral promoters and of several cellular promoters is dependent on the 32-kDa phosphoprotein encoded by the viral pre-early EIA gene. Recent negative results, including failure to detect either DNA-binding properties for the EIA protein (2) or sequence elements in the EIA responsive promoters recognized by the EIA gene product, have raised the possibility that the transcriptional activation by EIA may be mediated by promoter-specific host factors (for review, see ref.3). Viral infection of human cells may modify or increase the synthesis of host transcription factors. Alternatively, the transcription factors that are sequestered with the host promoters may upon infection be diverted to viral promoters by an as yet unidentified mechanism.Utilizing gel s...