Effects of Overexpression of Human E2F1 on Gene Expression in Murine Keratinocytes
Introduction
Overexpression of the cell cycle-regulated transcription factor E2F1 can bypass growth arrest induced by a variety of methods. This is due to the ability of E2F1 to transactivate genes important for cell cycle progression, including genes for DNA biosynthetic enzymes, factors that control the initiation of DNA synthesis, cell cycle regulators, and several proto-oncogenes (1,2). In contrast to its positive effects on cell growth, E2F1 has also been shown to have a role in apoptosis in some systems. It has recently been reported that E2F1 mediates the induction of apoptosis by several oncoproteins, and this effect is mediated by stabilization of p53, in part by an induction of p19ARF (3-5). In other systems, however, p53 is not involved in the apoptotic response to E2F1, and in fact the transactivation domain of E2F1 may be dispensable (6,7). Thus, it is not clear to what extent activation, derepression or repression of downstream target genes is involved in E2F1-mediated apoptosis.
Dr. David Johnson's laboratory recently described the generation of transgenic mice expressing E2F1 under the control of a keratin 5 (K5) promoter (8,9). Deregulated expression of E2F1 in basal keratinocytes results in epidermal hyperplasia, hyperproliferation and aberrant p53-dependent apoptosis. In the absence of functional p53, E2F1-induced apoptosis is significantly reduced while E2F1-induced hyperproliferation is unchanged. Significantly, K5 E2F1 transgenic mice that are deficient (heterozygous or nullizygous) for p53 develop spontaneous skin carcinomas. We previously demonstrated that K5 E2F1 keratinocytes overexpress at least one endogenous E2F target, the cyclin E gene. To further explore the molecular mechanisms underlying the phenotype of these transgenic mice, we therefore wished to determine in more detail the changes in endogenous gene expression resulting from overexpression of the K5 E2F1 transgene.
To detect changes in gene expression due to overexpression of E2F1, paired reactions were performed with bitag preparations derived from short-term cultures of keratinocytes obtained from newborn wild type or K5 E2F1 transgenic mice. The relative concentrations of wild type and transgenic bitags were adjusted to give approximately equal expression ratios for a set of control genes (ribosomal proteins L5 and S17, GAPDH, beta-actin and gamma-actin) whose expression was not expected to change significantly with E2F1 overexpression. Duplicate RAGE reactions were performed using wild-type and transgenic bitags in an initial screen, and the assays were then repeated for those genes of interest.
Results
In all, we performed reactions for over 380 known murine genes, including genes previously shown to be regulated by E2F1 and other genes related to cell proliferation, apoptosis, transcriptional regulation and signal transduction. We were able to detect the expected amplimers in reactions with 223 pairs of primers. The remaining genes may not be expressed in keratinocytes, or their expression levels may be below the detection limit of the RAGE technique. A more complete description of the genes that we have assayed is given in Table 1 and Table 2. Each line of these Tables is linked to our GGEG Sequence Database, and the database entries are linked to Entrez and UniGene. The amplimers produced by RAGE primers for several representative genes are shown in Figure 1.
For genes that were of special interest, either intrinsically or due to expression changes detected in the first analyses, at least three independent determinations were carried out. Expression was increased by at least 2-fold for 23 genes out of 42 analyzed (Table 3). We first analyzed genes previously shown to be transcriptionally regulated by E2F (1,2,9). Six of these target genes exhibited 2-5-fold increases in steady-state expression in E2F1 over-expressing keratinocytes (Table 3), while three more targets changed less than 2-fold. The 5-fold increase in expression of cyclin E seen here agrees well with a previous determination (9) by Northern hybridization (approximately 6-fold). In addition, several other cell-cycle related genes, including cyclin B2, cyclin F, the cyclin activating kinase CDK7 and ornithine decarboxylase were upregulated 3-6-fold in the transgenic keratinocytes. Increased expression of several other transcription factors (HNF3, YY-1) not known to be E2F1-regulated was also seen.
To confirm the magnitude of changes in expression seen with the RAGE technique, we compared the expression of several selected genes between wild-type and transgenic keratinocytes by Northern analyses. An example of the Northern and RAGE analyses for Brca1 is shown in Figure 2, and expression ratios (transgenic divided by wild type) observed by the two techniques are given in Table 4. The overall agreement in expression ratios between the two techniques was quite good, although there were quantitative differences in some cases. There did not seem to be a consistent bias, either upwards or downwards, for either technique.
Among the 42 genes that we assayed, two of the genes upregulated by E2F1 overexpression were Brca1 and p19ARF (Table 3; Figure 1, reactions 8 and 9); in each of these cases the sequence of the amplimer was experimentally verified. Induction of p19ARF by E2F1 has recently been reported in other systems (3-5). The Brca1 gene has been shown to be cell cycle regulated (10,11), and potential E2F sites are found in the Brca1 promoter. Moreover, we have found that overexpression of E2F1 in transient transfection assays can transcriptionally activate the Brca1 promoter (12). Both of these genes have been reported to increase the transactivation activity of the tumor supressor protein p53 by post-transcriptional mechanisms (13-17). Interestingly, another gene that post-transcriptionally modulates p53, CDK7 (18),was among those genes showing modest increases in expression in transgenic keratinocytes. Evidence for cell cycle regulation of CDK7 in fibroblasts has recently been presented (19).
These results suggested the possibility that one or more aspects of p53 activity might be enhanced in the K5 E2F1 transgenic cells. Consistent with this possibility, expression of three known downstream targets for p53 transactivation, cyclin G, Bax-alpha and MDM2 (20), was increased about 2-fold in E2F1 transgenic keratinocytes (Table 3). A fourth target of p53, p21, was increased slightly in the transgenic cells and GADD45 was among the 10% of the genome that could not be assayed by the current technique.
Overexpression of E2F1 in mouse fibroblasts leads to p19ARF mediated apoptosis (3-5), and Brca1 overexpression has also been linked to p53-dependent apoptosis (13,21). The E2F1-mediated effects on the expression of genes that modulate p53 activity and on downstream targets of p53 seen in K5 E2F1 keratinocytes (Figure 3) may be manifested in vivo as an increased propensity of the transgenic keratinocytes to enter apoptosis in response to other stimuli. This would be consistent with the finding of increased apoptosis in the epidermis of the transgenic mice, and the fact that apoptosis is reduced in a p53 null background (9). Interestingly, while K5 E2F1 / p53 null mice have an increased incidence of spontaneous skin tumors, the single K5 E2F1 transgenics are resistant to chemical carcinogenesis in the skin (22). It will be of interest to determine whether cells damaged by a chemical carcinogen in K5 E2F1 mice are more susceptible to elimination by apoptosis than their wild type counterparts.
As noted above, each RAGE reaction has the potential to amplify several genes, and many non-targeted amplimer bands were noted in the course of these studies (see for example reaction 3 in Figure 1). We identified four non-targeted amplimer bands that exhibited changes in expression greater than three-fold in transgenic keratinocytes (Table 5), and sequenced these amplimers. One amplimer sequence (EIG-1) matched several ESTs, exemplified by AA24506, and a second (EIG-2) gave a partial match to the coding sequence of the gene for ALG-2-interacting protein X (AJ005073); ALG-2 is a gene required for apoptosis in murine T-cells (20). EIG-3 and EIG-4 had no matches in GenBank.
![[Figure 1. RAGE reactions for selected genes]](Images/RAGE_Proj_2_Fig_1.jpg)
Figure 1. RAGE reactions for selected genes.
PCR reactions were carried out using bitags prepared from wild-type (-) or K5-E2F1 transgenic (+) keratinocytes. The reaction products were electrophoresed on 8% polyacrylamide gels, stained with VistraGreen (Molecular Probes, Eugene, OR), and digital fluorescent images obtained with a FluorImager (Molecular Dynamics, Sunnyvale, CA). Reactions contained RAGE primers chosen to amplify specific genes and the expected amplimers are indicated in the Figure by black dots between the lanes. The selected genes and the size of the expected amplimers were : 1, gamma-actin, 98 bp; 2, ribosomal protein L5, 130 bp; 3, lamin A, 291 bp; 4, CDK7, 392 bp; 5, YY-1, 248 bp; 6, DHFR, 61 bp; 7, Genesis, 254 bp; 8, p19ARF, 508 bp; 9, Brca1, 291 bp.
![[Figure 2. Comparison of RAGE and Northern results]](Images/RAGE_Proj_2_Fig_2.jpg)
Figure 2. Comparison of RAGE and Northern results.
A) RAGE PCR reactions similar to those described in Figure 1 were carried out for the following genes: 1, RPS17; 2 HMG14; 3 and 4, Brca1 using two different preparations of bitags. B) 20 µg samples of total RNA from wild type(-) and K5-E2F1 transgenic (+) keratinocytes were used for Northern analyses with radioactive probes for the indicated genes. Similar amounts of mRNA for the control genes (GAPDH and gamma-actin) were detected in the two cell types, but Brca1 was over-expressed in the transgenic keratinocytes.
![[Figure 3. Model for E2F1 effects on p53 and apoptosis]](Images/RAGE_Proj_2_Fig_3.jpg)
Figure 3. Model for E2F1 effects on p53 and apoptosis.
Green and yellow ovals represent genes that exhibit increased expression in the transgenic keratinocytes. Overexpression of E2F1 in keratinocytes leads to increased expression of CDK7, Brca1 and p19ARF , which in turn increase p53 activity by the mechanisms indicated. This leads to selective increases in the expression of three downstream targets of p53, Mdm2, cyclin G and Bax-alpha, and this may predispose the cells to enter the apoptotic pathway under the influence of appropriate external stimuli, e.g. carcinogen-induced DNA damage.