Identification of a novel E2F3 product suggests a mechanism for determining specificity of repression by Rb proteins.
(9/148)
The tumor suppressor function of Rb is intimately related to its ability to interact with E2F and repress the transcription of E2F target genes. Here we describe a novel E2F product that specifically interacts with Rb in quiescent cells. This novel E2F, which we term E2F3b, is encoded by a unique mRNA transcribed from an intronic promoter within the E2F3 locus. The E2F3b RNA differs from the previously characterized E2F3 RNA, which we now term E2F3a, by the utilization of a unique coding exon. In contrast to the E2F3a product that is tightly regulated by cell growth, the E2F3b product is expressed equivalently in quiescent and proliferating cells. But, unlike the E2F4 and E2F5 proteins, which are also expressed in quiescent cells and form complexes with the p130 protein, the E2F3b protein associates with Rb and represents the predominant E2F-Rb complex in quiescent cells. Thus, the previously described specificity of Rb function as a transcriptional repressor in quiescent cells coincides with the association of Rb with this novel E2F product. (+info)
Complex transcriptional regulatory mechanisms control expression of the E2F3 locus.
(10/148)
E2F transcription activity has been shown to play a critical role in cell growth control, regulating the expression of a variety of genes that encode proteins important for the initiation of DNA replication and cell cycle regulation. We have shown that the E2F3 locus encodes two protein products: the E2F3a product, which is tightly regulated by cell growth, and the E2F3b product, which is constitutively expressed throughout the cell cycle. To further explore the mechanism controlling the expression of the two E2F3 gene products, we analyzed the genomic sequences flanking the 5' region of E2F3a and E2F3b. We find that a series of E2F binding sites confer negative control on the E2F3a promoter in quiescent cells, similar to the control of the E2F1 and E2F2 promoters. In addition, a group of E-box elements, which are Myc binding sites, confer responsiveness to Myc and are necessary for full activation of the E2F3a promoter in response to growth stimulation. Based on these results and past experiments, it appears that the E2F1, E2F2, and E2F3a genes are similarly regulated by growth stimulation, involving a combination of E2F-dependent negative control and Myc-mediated positive control. In contrast, the constitutive expression of the E2F3b gene more closely reflects the control of expression of the E2F4 and E2F5 genes. (+info)
CpG methylation as a mechanism for the regulation of E2F activity.
(11/148)
Regulation of gene expression in mammals through methylation of cytosine residues at CpG dinucleotides is involved in the development and progression of tumors. Because many genes that are involved in the control of cell proliferation are regulated by members of the E2F family of transcription factors and because some E2F DNA-binding sites are methylated in vivo, we have investigated whether CpG methylation can regulate E2F functions. We show here that methylation of E2F elements derived from the dihydrofolate reductase, E2F1, and cdc2 promoters prevents the binding of all E2F family members tested (E2F1 through E2F5). In contrast, methylation of the E2F elements derived from the c-myc and c-myb promoters minimally affects the binding of E2F2, E2F3, E2F4, and E2F5 but significantly inhibits the binding of E2F1. Consistent with these studies, E2F3, but not E2F1, activates transcription through methylated E2F sites derived from the c-myb and c-myc genes whereas both E2F1 and E2F3 fail to transactivate a reporter gene that is under the control of a methylated dihydrofolate reductase E2F site. Together, these data illustrate a means through which E2F activity can be controlled. (+info)
Expression of the E2F family of transcription factors during murine development.
(12/148)
The E2F family of transcription factors plays a crucial role in the control of cell cycle progression and regulation of cellular proliferation, both processes fundamental to mammalian development. In the present study, we have examined the levels of expression of the six currently identified E2F proteins in murine embryos/fetuses as a function of gestational age, compared the expression of these six proteins in selected developing and adult tissues, and examined E2F expression in the embryonic murine palate, a tissue in which perturbation of proliferation is associated with induction of cleft palate. Our results indicate that: 1) multiple forms of individual E2F family members are present in embryonic, fetal and adult cells/tissues; 2) each of the six E2Fs is expressed in a tissue specific manner in both adult and embryonic/fetal organs; 3) certain forms of individual E2F family members are preferentially detected in adult tissues, whereas others are preferentially expressed in embryonic/fetal tissues; 4) expression of the various E2Fs and their isoforms follows distinct temporal patterns during murine gestation; and 5) individual E2F family members also exhibit differential patterns of temporal expression during murine palatogenesis. (+info)
A genetic screen to identify genes that rescue the slow growth phenotype of c-myc null fibroblasts.
(13/148)
The c-myc gene is frequently over-expressed in human cancers and is involved in regulation of proliferation, differentiation and apoptosis. c-Myc is a transcription factor that acts primarily by regulating the expression of other genes. However, it has been very difficult to identify bona fide c-Myc target genes that explain its diverse biological activities. The recent generation of c-myc deficient Rat1A fibroblasts with a profound and stable growth defect provides a new system to search for genes that can substitute for c-myc in proliferation. In this study, we have attempted to identify genes that rescue the slow growth phenotype of c-myc null cells through introduction of a series of potent cell cycle regulatory genes and several retroviral cDNA expression libraries. None of the candidate genes tested, including SV40 T-antigen and adenovirus E1A, caused reversal of the c-myc null growth defect. Furthermore, extensive screens with high-complexity retroviral cDNA libraries from three different tissue sources revealed that only c-myc and N-myc rescued the c-myc null slow-growth phenotype. Our data support the notion that there are no functional equivalents of the myc family of proto-oncogenes and also suggest that there are no c-Myc-activated genes that alone can substitute for c-Myc in control of cell proliferation. (+info)
Identification of E2F-3B, an alternative form of E2F-3 lacking a conserved N-terminal region.
(14/148)
We have identified a novel form of the full-length E2F-3 protein that we term E2F-3B. In contrast to full-length E2F-3, which is expressed only at the G1/S boundary, E2F-3B is detected throughout the cell cycle with peak levels in GO where it is associated with Rb. Transfection and in vitro translation experiments demonstrate that a protein identical to E2F-3B in size and iso-electric point is produced from the E2F-3 mRNA via the use of an alternative translational start site. This alternative initiation codon was mapped by mutagenesis to codon 102, an ACG codon. Mutation of the ACG codon at position 102 abolished E2F-3B expression, whereas the conversion of ACG 102 to a consensus ATG led to the expression of a protein indistinguishable from E2F-3B. Given these results, E2F-3B is missing 101 N-terminal amino acids relative to full-length E2F-3. This region includes a moderately conserved sequence of unknown function that is present only in the growth-promoting E2F family members, including E2F-1, 2 and full-length E2F-3. These observations make E2F-3B the first example of an E2F gene giving rise to two different protein species and also suggest that E2F-3 and E2F-3B may have opposing roles in cell cycle control. (+info)
E2F transcription factors are differentially expressed in murine gametes and early embryos.
(15/148)
We have examined the murine genes encoding transcription factors E2F1, -3, -5 and -6 in gametes and early embryos. All genes are expressed as maternal transcripts and all are efficiently transcribed after the blastocyst stage. Between those two stages, each E2F mRNA is transcribed with a distinctive and unique pattern. E2F proteins are also differentially expressed and compartmentalized in pre-implantation embryos. (+info)
E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis.
(16/148)
The retinoblastoma protein (pRB) and its two relatives, p107 and p130, regulate development and cell proliferation in part by inhibiting the activity of E2F-regulated promoters. We have used high-density oligonucleotide arrays to identify genes in which expression changed in response to activation of E2F1, E2F2, and E2F3. We show that the E2Fs control the expression of several genes that are involved in cell proliferation. We also show that the E2Fs regulate a number of genes involved in apoptosis, differentiation, and development. These results provide possible genetic explanations to the variety of phenotypes observed as a consequence of a deregulated pRB/E2F pathway. (+info)