E2 Promoter Binding Factor Research Articles

A transcriptionally active pRb–E2F1–P/CAF signaling pathway is central to TGFβ-mediated apoptosis

Transforming growth factor-β (TGFβ) modulates the expression of multiple apoptotic target genes; however, a common and central signaling pathway, acting downstream of TGFβ and leading to cell death, has yet to be uncovered. Here, we show that TGFβ-induced apoptosis in cancer cells requires the transcription factor E2F1 (E2 promoter-binding factor 1). Using the E2F1 knockout mouse model, we also found E2F1 to be required for TGFβ-mediated apoptosis in normal cells. Moreover, we found TGFβ to increase E2F1 protein stability, acting at the post-translational level. We further investigated the molecular mechanisms by which E2F1 contributes to TGFβ-mediated apoptosis and found that TGFβ treatment led to the formation of a transcriptionally active E2F1–pRb–P/CAF complex on multiple TGFβ pro-apoptotic target gene promoters, thereby activating their transcription. Together, our findings define a novel process of gene activation by the TGFβ-E2F1 signaling axis and highlight E2F1 as a central mediator of the TGFβ apoptotic program.

Positive regulation of minichromosome maintenance gene expression, DNA replication, and cell transformation by a plant retinoblastoma gene

Retinoblastoma-related (RBR) genes inhibit the cell cycle primarily by repressing adenovirus E2 promoter binding factor (E2F) transcription factors, which drive the expression of numerous genes required for DNA synthesis and cell cycle progression. The RBR-E2F pathway is conserved in plants, but cereals such as maize are characterized by having a complex RBR gene family with at least 2 functionally distinct members, RBR1 and RBR3. Although RBR1 has a clear cell cycle inhibitory function, it is not known whether RBR3 has a positive or negative role. By uncoupling RBR3 from the negative regulation of RBR1 in cultured maize embryos through a combination of approaches, we demonstrate that RBR3 has a positive and critical role in the expression of E2F targets required for the initiation of DNA synthesis, DNA replication, and the efficiency with which transformed plants can be obtained. Titration of endogenous RBR3 activity through expression of a dominant-negative allele with a compromised pocket domain suggests that these RBR3 functions require an activity distinct from its pocket domain. Our results indicate a cell cycle pathway in maize, in which 2 RBR genes have specific and opposing functions. Thus, the paradigm that RBR genes are negative cell cycle regulators cannot be considered universal.

Pro-apoptotic Bim Induction in Response to Nerve Growth Factor Deprivation Requires Simultaneous Activation of Three Different Death Signaling Pathways

Bim is a pro-apoptotic member of the Bcl-2 family that is induced and contributes to neuron death in response to nerve growth factor (NGF) deprivation. Past work has revealed that Bim is downstream of multiple independent transcriptional pathways in neurons, including those culminating in activation of the c-Jun, FoxO, and Myb transcription factors. This study addresses the issue of whether the three signaling pathways are redundant with respect to Bim induction or whether they act cooperatively. Examination of the proximal Bim promoter reveals binding sites for FoxO, Mybs, and, as shown here, c-Jun. We find that mutation of any one of these types of sites abolishes induction of a Bim promoter-driven reporter in response to NGF deprivation. Moreover, down-regulation of either c-Jun, FoxOs, or Mybs by short hairpin RNAs blocks induction of Bim promoter-reporter activity triggered by withdrawal of NGF. This was the case for reporters driven by either the proximal promoter or a promoter that also includes additional regulatory elements in the first intron of the Bim gene. Such short hairpin RNAs also suppressed the induction of endogenous Bim protein. These findings thus indicate that the Bim promoter acts as a coincidence detector that optimally responds to the simultaneous activation of three different pro-apoptotic transcriptional pathways. Such a mechanism provides a "fail-safe" that prevents neurons from dying by accidental activation of any single pathway. It also permits neurons to utilize individual pathways such as JNK signaling for other purposes without risk of demise.

Functional Identification of Api5 as a Suppressor of E2F-Dependent Apoptosis In Vivo

Retinoblastoma protein and E2-promoter binding factor (E2F) family members are important regulators of G1-S phase progression. Deregulated E2F also sensitizes cells to apoptosis, but this aspect of E2F function is poorly understood. Studies of E2F-induced apoptosis have mostly been carried out in tissue culture cells, and the analysis of the factors that are important for this process has been restricted to the testing of a few candidate genes. Using Drosophila as a model system, we have generated tools that allow genetic modifiers of E2F-dependent apoptosis to be identified in vivo and developed assays that allow effects on E2F-induced apoptosis to be studied in cultured cells. Genetic interactions show that dE2F1-dependent apoptosis in vivo involves dArk/Apaf1 apoptosome-dependent activation of both initiator and effector caspases and is sensitive to levels of Drosophila inhibitor of apoptosis-1 (dIAP1). Using these approaches, we report the surprising finding that apoptosis inhibitor-5/antiapoptosis clone-11 (Api5/Aac11) is a critical determinant of dE2F1-induced apoptosis in vivo and in vitro. This functional interaction occurs in multiple tissues, is specific to E2F-induced apoptosis, and is conserved from flies to humans. Interestingly, Api5/Aac11 acts downstream of E2F and suppresses E2F-dependent apoptosis without generally blocking E2F-dependent transcription. Api5/Aac11 expression is often upregulated in tumor cells, particularly in metastatic cells. We find that depletion of Api5 is tumor cell lethal. The strong genetic interaction between E2F and Api5/Aac11 suggests that elevated levels of Api5 may be selected during tumorigenesis to allow cells with deregulated E2F activity to survive under suboptimal conditions. Therefore, inhibition of Api5 function might offer a possible mechanism for antitumor exploitation.

Bim Is a Direct Target of a Neuronal E2F-Dependent Apoptotic Pathway

The inappropriate expression/activation of cell-cycle-related molecules is associated with neuron death in many experimental paradigms and human neuropathologic conditions. However, the means whereby this links to the core apoptotic machinery in neurons have been unclear. Here, we show that the pro-apoptotic Bcl-2 homology 3 domain-only molecule Bcl-2 interacting mediator of cell death (Bim) is a target of a cell-cycle-related apoptotic pathway in neuronal cells. Induction of Bim in NGF-deprived cells requires expression and activity of cyclin-dependent kinase 4 (cdk4) and consequent de-repression of E2 promoter binding factor (E2F)-regulated genes including members of the myb transcription factor family. The Bim promoter contains two myb binding sites, mutation of which abolishes induction of a Bim promoter-driven reporter by NGF deprivation or E2F-dependent gene de-repression. NGF deprivation significantly increases endogenous levels of C-myb and its occupancy of the endogenous Bim promoter. These findings support a model in which apoptotic stimuli lead to cdk4 activation, consequent de-repression of E2F-regulated mybs, and induction of pro-apoptotic Bim.

RBR3, a member of the retinoblastoma-related family from maize, is regulated by the RBR1/E2F pathway.

Retinoblastoma-related (RBR) proteins regulate cell division in higher eukaryotes by controlling the adenovirus E2 promoter binding factor (E2F)/dimerization partner (DP) family of transcription factors that regulate expression of many genes involved in cell-cycle progression. We identified a previously undescribed member of the maize RBR family, RBR3, which has the characteristic structure and binding activities of pocket proteins, where interaction depends on a LxCxE motif in the partner proteins and a critical cysteine within the B pocket domain. Like other RBR proteins, RBR3 appears to be regulated by phosphorylation mediated by cyclin-dependent kinases. During endosperm development, RBR3 expression is restricted to the mitotic stage preceding the onset of endoreduplication. This finding suggests a role distinct from RBR1, which is constitutively expressed. Two sites in the RBR3 promoter bind to complexes containing maize E2F1 and DP proteins. Expression of wheat dwarf virus RepA protein, which blocks RBR1 activity and stimulates cell proliferation, dramatically up-regulates RBR3, but not RBR1, RNA in embryogenic maize calli. The results indicate that RBR3 expression is controlled by RBR1 through the activity of E2F/DP and that RBR3 is the maize equivalent of mammalian p107. Furthermore, maize and related grasses might have evolved a compensatory mechanism among distinct types of RBR proteins to ensure robust control of pocket protein activity.

The transcriptional program of terminal granulocytic differentiation

AbstractTo characterize the transcriptional program that governs terminal granulocytic differentiation in vivo, we performed comprehensive microarray analyses of human bone marrow populations highly enriched in promyelocytes (PMs), myelocytes/metamyelocytes (MYs), and neutrophils (bm-PMNs). These analyses identified 11 310 genes involved in differentiation, of which 6700 were differentially regulated, including previously unidentified effector proteins and surface receptors of neutrophils. Differentiation of PMs toward MYs was accompanied by a marked decline of proliferative and general cellular activity as defined by down-regulation of E2 promoter binding factor (E2F) target genes; cyclin dependent kinases 2, 4, and 6; and various metabolic, proteasomal, and mitochondrial genes. Expression patterns of apoptosis genes indicated death control by the p53 pathway in PMs and by death receptor pathways in bm-PMNs. Effector proteins critical for host defense were expressed successively throughout granulocytic differentiation, whereas receptors and receptor ligands essential for the activation of the host defense program were terminally up-regulated in bm-PMNs. The up-regulation of ligand-receptor pairs, which are defined inducers as well as target genes of nuclear factor-κB (NF-κB), suggests a constitutive activation of NF-κB in bm-PMNs by autocrine loops. Overall, these results define a granulocytic differentiation model governed by a highly coordinated fail-safe program, which promotes completion of differentiation before cells gain responsiveness toward activating stimuli that accompany infections. (Blood. 2005; 105:1785-1796)

B-myb and C-myb play required roles in neuronal apoptosis evoked by nerve growth factor deprivation and DNA damage.

Activation of cell cycle elements plays a required role in neuronal apoptosis associated with both development and neurodegenerative disorders. We demonstrated previously that neuron survival requires gene repression mediated by the cell cycle transcription factor E2F (E2 promoter binding factor) and that apoptotic stimuli lead to de-repression of E2F-regulated genes and consequent death. However, the downstream mediators of such death have been unclear. The transcription factors B- and C-myb are E2F-regulated genes that are induced in neurons by apoptotic stimuli. Here, we examine the role of B- and C-myb induction in neuron death. Antisense and siRNA constructs that effectively block the upregulation of B- and C-myb provide substantial protection against death of cultured neuronal PC12 cells, sympathetic neurons, and cortical neurons elicited by either NGF withdrawal or DNA damage. There is also significant protection from death induced by direct E2F-dependent gene de-repression. Our findings thus establish required roles for B- and C-myb in neuronal apoptosis.

Phytocalpain controls the proliferation and differentiation fates of cells in plant organ development.

Calpain, a calcium-dependent cysteine protease, plays an essential role in basic cellular processes in animal cells, including cell proliferation, apoptosis, and differentiation. NbDEK encodes the calpain homolog of N. benthamiana. In this study, virus-induced gene silencing (VIGS) of NbDEK resulted in arrested organ development and hyperplasia in all the major plant organs examined. The epidermal layers of the leaves and stems were covered with hyperproliferating cell masses, and stomata and trichome development was severely inhibited. During flower development, a single dome-like structure was grown from the flower meristem to generate a large cylinder-shaped flower lacking any floral organs. At the cellular level, cell division was sustained in tissues that were otherwise already differentiated, and cell differentiation was severely hampered. NbDEK is ubiquitously expressed in all the plant tissues examined. In the abnormal organs of the NbDEK VIGS lines, protein levels of D-type cyclins (CycD)2, CycD3, and proliferating cell nuclear antigen (PCNA) were greatly elevated, and transcription of E2F (E2 promoter binding factor), E2F-regulated genes, retinoblastoma (Rb), and KNOTTED1 (KN1)-type homeobox genes was also stimulated. These results suggest that phytocalpain is a key regulator of cell proliferation and differentiation during plant organogenesis, and that it acts partly by controlling the CycD/Rb pathway.

Differential stage-specific regulation of cyclin-dependent kinases during cambial dormancy in hybrid aspen.

The cambium of woody plants cycles between active and dormant states. Dormancy can be subdivided into eco- and endodormant stages. Ecodormant trees resume growth upon exposure to growth-promotive signals, while the establishment of endodormant state results in loss of the ability to respond to these signals. In this paper, we analysed the regulation of cyclin-dependent kinases (CDKs) to understand the differential response of cell division machinery to growth-promotive signals during the distinct stages of dormancy in hybrid aspen. We show that 4 weeks of short-day (SD) treatment causes termination of the cambial cell division and establishment of the ecodormant state. This coincides with a steady decline in the histone H1 kinase activity of the PSTAIRE-type poplar CDKA (PttCDKA) and the PPTTLRE-type PttCDKB kinase complexes. However, neither the transcript nor the polypeptide levels of PttCDKA and PttCDKB are reduced during ecodormancy. In contrast, 6 weeks of SD treatment establishes endodormancy, which is marked by the reduction and disappearance of the PttCDKA and PttCDKB protein levels and the PttCDKB transcript levels. The transition to endodormancy is preceded by an elevated E2F (adenosine E2 promoter binding factor) phosphorylation activity of the PttCDKA kinase that reduces the DNA-binding activity of E2F in vitro. The transition to endodormancy is followed by a reduction of retinoblastoma (Rb) phosphorylation activity of PttCDKA protein complexes. Both phosphorylation events could contribute to block the G1 to S phase transition upon the establishment of endodormancy. Our results indicate that eco- and endodormant stages of cambial dormancy involve a stage-specific regulation of the cell cycle effectors at multiple levels.

α-Pal/NRF-1 Regulates the Promoter of the Human Integrin-associated Protein/CD47 Gene

Integrin-associated protein (IAP or CD47) is expressed in a variety of tissues, including the nervous system and immune system. To understand how cells control the expression of the IAP gene, we cloned the 5'-proximal region of the human IAP gene and investigated IAP promoter activity by transient transfection. RT-PCR confirmed the expression of IAP transcripts in human neuroblastoma IMR-32 and hepatoma HepG2 cells. Deletion analysis identified a core promoter of the human IAP gene located between nucleotide positions -232 and -12 relative to the translation initiation codon in these two cell lines. Site-directed mutagenesis and gel electrophoretic mobility shift assay identified a alpha-Pal/NRF-1 binding element within the IAP core promoter. Supershift assays using the alpha-Pal/NRF-1 antiserum confirmed the binding of this transcription factor on the alpha-Pal/NRF-1 site. Overexpression of the DNA binding domain of alpha-Pal/NRF-1 in cells enhanced DNA-alpha-Pal/NRF-1 binding in vitro. Furthermore, overexpression of full-length alpha-Pal/NRF-1 significantly enhanced IAP promoter activity while overexpression of dominant-negative mutant reduced promoter activity both in the cultured human cell lines and primary mouse cortical cells. These results revealed that alpha-Pal/NRF-1 is an essential transcription factor in the regulation of human IAP gene expression.

Histological Study of Genesis of the Mice Oral Vestibule

Background: Both the vestibular lamina (VL) and dental lamina (DL) develop from the primary epithelial band of the oral cavity. Previous studies have reported that VL and DL were initiated by certain epithelial-mesenchymal interactions between the primary epithelial band of the oral cavity and underlying mesenchyme; however, the exact mechanisms remain to be elucidated. Aims: Here we studied how the retinoblastoma (Rb) family and E2 promoter binding factor (E2F) regulate signals that control and regulate the proliferation and differentiation of the VL leading to the formation of the oral vestibule. Methods: Immunohistochemical light microscopy by the enhanced polymer one-step staining(EPOS) method and confocal laser scanning microscopy by the LsAB labelled streptavidin biotin (LSAB) method were performed on the developing vestibular lamina(VL) of fetal mice at stages from 11 days after fertilization E11 to E14.The immunohistochemical findings were compared with the findings in the developing dental lamina (DL) and tooth germ. Conventional transmission electron microscopy was performed on the E13 fetuses, in an effort to elucidate the differentiation of keratocytes in the developing VL. Results: We found that: 1) EGF, TGF , EGFR, PCNA, FGF2, pRb, and FGFR1-4 were immunolocalized in the developing tissues. 2) Cytokine expression patterns indicated that the EGF family and FGF2 essentially induced VL generation and cell proliferation. 3) FGFR1 as diffusely localized in the primary epithelial band, but was strongly expressed in the E12-14 VL and DL/enamel organ. By contrast, EGFR internalization was observed in the differentiating E13 VL. 4) Expression of pRb was intensely localized in the suprabasal layer of the E13 VL corresponding to CK-10 expression and keratohyaline-containing structures in the VL stratum germinativum cells. Conclusions: We suggest a mechanism in which FGFR1 regulates pRb to induce proliferation of cells in the VL and DL/enamel organ, and, in particular, to incite keratocyte differentiation and subsequent exfoliation of keratinizing VL cells.

The transcriptional repressor gene Mad3 is a novel target for regulation by E2F1.

Mad family proteins are transcriptional repressors that antagonize the activity of the c- Myc proto-oncogene product. Mad3 is expressed specifically during the S-phase of the cell cycle in both proliferating and differentiating cells, suggesting that its biological function is probably linked to processes that occur during this period. To determine the mechanisms that regulate the cell-cycle-specific transcription of Mad3, we used reporter gene assays in stably transfected fibroblasts. We show that the activation of Mad3 at the G1-S boundary is mediated by a single E2F (E2 promoter binding factor)-binding site within the 5'-flanking region of the gene. Mutation of this element eliminated transcriptional activation at S-phase, suggesting that the positively acting E2F proteins play a role in Mad3 regulation. Using electrophoretic mobility-shift assays and chromatin immunoprecipitation, we show that E2F1 binds to the Mad3 5'-flanking region both in vitro and in vivo. We thus identify Mad3 as a novel transcriptional target of E2F1.

Reproductive cycle regulation of nuclear import, euchromatic localization, and association with components of Pol II mediator of a mammalian double-bromodomain protein.

Fsrg1 (female sterile homeotic-related gene 1) is the mouse homolog of the human RING3 protein, which has been shown to associate with the E2 promoter binding factor (E2F) transcription factor and to have a possible role in cell cycle-linked transcriptional regulation. The Fsrg1 protein is 60% identical in sequence to the RNA polymerase II mediator subunit Fsrg4, another member of this subfamily of double bromodomain-containing proteins that are homologs of Drosophila female sterile homeotic. Antibodies against murine Fsrg1 were generated and used in immunoblot and immunoprecipitation experiments to identify proteins interacting with Fsrg1 and RING3. In the presence of acetylated but not nonacetylated histone H3 and H4 peptides, RING3 was shown to interact with E2F, mediator components cyclin-dependent kinase 8 and thyroid receptor-associated protein 220, and the RNA polymerase II large subunit. Fsrg1 mRNA had been previously shown to be expressed at high levels in the epithelium of the adult mouse mammary gland. To determine the physiological relevance of these potential associations, we examined the patterns of expression of Fsrg1 mRNA and protein in the adult mammary epithelia during the reproductive cycle as the tissue is responding to estrogen, progesterone, and prolactin. Changes in the nuclear vs. cytoplasmic localization of Fsrg1 were observed and correlated with physiological changes in mammary gland function. The observations suggested that Fsrg1 may be involved in the transcriptional activities of genes involved in proliferation of the mammary epithelia during pregnancy and in orchestrating postlactation involution and apoptosis. Localization of Fsrg1 on euchromatin, the transcribed portion of the chromosomes, is consistent with its hypothesized function as a transcription regulator.

Characterization of protein kinase C beta isoform's action on retinoblastoma protein phosphorylation, vascular endothelial growth factor-induced endothelial cell proliferation, and retinal neovascularization.

Retinal neovascularization is a major cause of blindness and requires the activities of several signaling pathways and multiple cytokines. Activation of protein kinase C (PKC) enhances the angiogenic process and is involved in the signaling of vascular endothelial growth factor (VEGF). We have demonstrated a dramatic increase in the angiogenic response to oxygen-induced retinal ischemia in transgenic mice overexpressing PKC beta 2 isoform and a significant decrease in retinal neovascularization in PKC beta isoform null mice. The mitogenic action of VEGF, a potent hypoxia-induced angiogenic factor, was increased by 2-fold in retinal endothelial cells by the overexpression of PKC beta 1 or beta 2 isoforms and inhibited significantly by the overexpression of a dominant-negative PKC beta 2 isoform but not by the expression of PKC alpha, delta, and zeta isoforms. Association of PKC beta 2 isoform with retinoblastoma protein was discovered in retinal endothelial cells, and PKC beta 2 isoform increased retinoblastoma phosphorylation under basal and VEGF-stimulated conditions. The potential functional consequences of PKC beta-induced retinoblastoma phosphorylation could include enhanced E2 promoter binding factor transcriptional activity and increased VEGF-induced endothelial cell proliferation.

COMPLEX REGULATION OF THE ADENOVIRUS E2 PROMOTER BY THE VIRAL ONCOPROTEINS E1A AND E7 AND TUMOR-SUPPRESSOR RB

The adenovirus E2 gene promoter contains two positive regulatory elements, the activating transcription factor (ATF) and E2 promoter binding factor (E2F) sites, in physical proximity which are important for adenovirus E1A-induced transcriptional activation of the E2 promoter. It is also known that E1A trans-activates through the E2F site by freeing E2F from inactive complexes containing E2F and tumor suppressor Rb or other proteins. In this study, we demonstrated that E1A, in addition to activating the E2F sites, could trans-activate the E2 promoter through enhancing the DNA binding ability of ATF; and enhanced ATF binding was phosphorylation-dependent. Besides, overexpression of Rb can block both the transcriptional effect of E1A and human papillomavirus type 16 (HPV-16) E7, and E1A-induced ATF binding. Moreover, Rb can overcome trans-activation through the E2F sites by E1A or E7. These data suggest that E1A can trans-activate the E2 promoter through two regulatory elements by different mechanisms, and Rb can antagonize the trans-activation effect of E1A or E7.

An adenovirus E4 gene product trans-activates E2 transcription and stimulates stable E2F binding through a direct association with E2F.

The adenovirus E4 gene encodes a trans-activating function that can stimulate the E2 promoter. E2 promoter sequences required for E4 trans-activation are identical to those required for E1A trans-activation, and these principally are the E2 promoter binding factor (E2F) binding sites. Furthermore, full activation of E2F DNA binding activity requires both E1A and E4 action. Analysis of a series of mutant E4 viruses identifies open reading frame (orf) 6/7 of the E4 transcription unit as that required for activation of E2F binding activity. In addition, the assay of various E4 cDNAs demonstrates that the E4 orf 6/7 also is responsible for the trans-activation of E2 transcription. Translation of the E4 orf 6/7 mRNA, but not a control mRNA, in a reticulocyte extract generates an activity that can stimulate cooperative binding of E2F in vitro, consistent with recent in vivo assays that demonstrate a role for the E4 gene in E2F stable complex formation. This stimulation is due to a direct interaction of the E4 protein with E2F since an antibody that recognizes the E4 orf 6/7 polypeptide detects this E4 protein in the E2F-DNA complex. We conclude that the E4 orf 6/7 product interacts with the E2F factor altering binding to allow formation of a stable complex that results in a stimulation of transcription.

Promoter interaction of the E1A-inducible factor E2F and its potential role in the formation of a multi-component complex.

The precise binding site in the adenovirus E2 promoter for the E1A-inducible factor E2F was determined. DNase footprinting revealed two distinct regions of protection which spanned sequences from -33 to -49 and from -53 to -71. Chemical modifications of DNA further delineated nucleotides involved in DNA-protein contacts in each binding region. The E2F binding sites are clearly distinct from the binding site for another E2 promoter binding factor, located at -68 to -80, previously described by SivaRaman et al. [(1986) Proc. Natl. Acad. Sci. USA, 83, 5914-5918]. As determined by DNase footprinting using crude nuclear extracts, both factors were present in extracts of Ad5-infected cells and were found to bind simultaneously to their respective sites on the promoter. In contrast, E2F was not evident in extracts of uninfected cells, whereas there was no difference in the -68 to -80 footprint as a function of the extract. Thus, although multiple factors interact with the E2 promoter, only the E2F factor is unique to the infected extract. The implications of the formation of a multi-factor promoter complex as a possible mechanism of transcriptional regulation are discussed.

Role of an adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control.

A product of the adenovirus gene E1A is responsible for the stimulation of transcription from six viral promoters as well as at least two cellular promoters. We have detected a HeLa cell factor, termed E2 promoter binding factor (E2F), that appears to mediate the transcriptional stimulation of the viral E2 promoter. Competition experiments revealed that E2F did not recognize and bind to the E1B, E3, E4, or major late promoter sequences. Furthermore, three additional promoters stimulated by E1A, heat shock protein 70, beta-globin, and early simian virus 40, do not bind E2F. In contrast, the factor does recognize sequences in the E1A enhancer, and within the E1A enhancer are duplicated binding sites for E2F. Finally, a single E2F binding site from the E1A enhancer can confer increased transcription to a mouse beta-globin promoter, dependent on the action of the E1A gene product. This stimulation requires binding of E2F since methylation of the binding site, which blocks binding in vitro, reduces transcription stimulation in vivo. We, therefore, conclude that E2F is likely to be responsible for the E1A-mediated stimulation of the E1A gene as well as the E2 gene but is not involved in the activation of the other E1A-inducible promoters.