Multiple DNA elements are required for the growth regulation of the mouse E2F1 promoter.

Abstract

To prepare for the DNA synthesis (S) phase of the cell cycle, transcription of many genes required for nucleotide biosynthesis increases. The promoters of several of these genes contain binding sites for the E2F family of transcription factors, and, in many cases, mutation of these sites abolishes growth-regulated transcription. The RNA levels of one family member, E2F1, increase about 15-fold at the G1/S-phase boundary and expression of E2F1 in quiescent cells activates transcription from some G1/S-phase-specific promoters, suggesting that E2F1 plays a critical role in preparing cells to enter S phase. To elucidate the signal transduction pathway leading to the activation of genes required for DNA synthesis, we are investigating the mechanism by which expression of E2F1 is regulated. To determine whether levels of E2F1 mRNA are controlled by changes in promoter activity, we have cloned and characterized the mouse E2F1 promoter. Sequence analysis revealed two sets of overlapping E2F-binding sites located between -12 and -40 relative to the transcription initiation site. We show that these sites bind cellular E2F and that an E2F1 promoter fragment can be activated up to 100-fold by coexpression of E2F proteins. We also show that the activity of this E2F1 promoter fragment increases -80-fold at the Gl/S-phase boundary and that this activation is, in part, regulated by Go-specific repression via the E2F sites. However, the E2F sites are not sufficient to mediate growth-regulated transcriptional activity; our results indicate that multiple DNA elements are required for transcription regulation of the E2F1 promoter at the G1/S-phase boundary.

Regulation of E2F-1 gene expression in avian cells

“…The deletion of E2F responsive elements on the E2F-1 promoter prevents the inactivation of the avian and mammalian promoter in G0/G1 phase, suggesting that these motifs are necessary for the down-regulation of E2F-1 gene expression in this particular phase of the cell cycle (Johnson et al, 1994;Neuman et al, 1996;Hsiao et al, 1994). This inhibition is probably mediated by p105 Rb or a related protein, as shown by we and others (Figure 8c; Johnson et al, 1994;Neuman et al, 1994;Hsiao et al, 1994;Johnson, 1995).…”

“…As shown in Figure 6, CAT transcript level is almost undetectable when the cells are quiescent (G0/G1 phase), whereas 12 h following the addition of serum, which corresponds to the G1/S transition, the CAT transcript level peaks. Therefore, the quail E2F-1 transcription is also strongly activated at the G1/S transition, as has been shown for the human and murine homologs of the E2F-1 gene (Johnson et al 1994;Hsiao 1994;Neuman et al, 1996). This result also indicates that the proE2F-CAT construct possesses most, if not all, regulatory elements allowing regulation of E2F-1 transcription in a cell cycle-dependent manner.…”

“…Analysis of the sequence of the promoter region also shows a good conservation between mammalian and avian species, especially near the two double E2F binding sites found as tandem repeats close to the transcription start site (Johnson et al, 1994;Hsiao et al, 1994;Neuman et al, 1996). Analysis of E2F-1 gene expression during the cell cycle has shown that this gene is only activated at the G1/S transition, like its mammalian homologs (Johnson et al 1994;Hsiao, 1994;Neuman et al, 1996).…”

“…Analysis of E2F-1 gene expression during the cell cycle has shown that this gene is only activated at the G1/S transition, like its mammalian homologs (Johnson et al 1994;Hsiao, 1994;Neuman et al, 1996). This suggests that regulatory mechanisms of E2F-1 expression have been conserved among these species at least in part.…”

“…Position of relevant restriction site is indicated. (c) Multiple alignment of promoter regions of E2F-1 genes from quail, mouse (Hsiao et al, 1994) and human (Johnson et al, 1994). Identical nucleotides are shaded.…”