Disruption of mutually negative regulatory feedback loop between interferon-inducible p202 protein and the E2F family of transcription factors in lupus-prone mice

Abstract

Studies have identified IFN-inducible Ifi202 gene as a lupus susceptibility gene (encoding p202 protein) in mouse models of lupus disease. However, signaling pathways that regulate the Ifi202 expression in cells remain to be elucidated. We found that steady-state levels of Ifi202 mRNA and protein were high in mouse embryonic fibroblasts (MEFs) from E2F1 knockout (E2F1(-/-)) and E2F1 and E2F2 double knockout (E2F1(-/-)E2F2(-/-)) mice than isogenic wild-type MEFs. Moreover, overexpression of E2F1 in mouse fibroblasts decreased expression of p202. Furthermore, expression of E2F1, but not E2F4, transcription factor in mouse fibroblasts repressed the activity of 202-luc-reporter in promoter-reporter assays. Interestingly, the E2F1-mediated transcriptional repression of the 202-luc-reporter was independent of p53 and pRb expression. However, the repression was dependent on the ability of E2F1 to bind DNA. We have identified a potential E2F DNA-binding site in the 5'-regulatory region of the Ifi202 gene, and mutations in this E2F DNA-binding site reduced the E2F1-mediated transcriptional repression of 202-luc-reporter. Because p202 inhibits the E2F1-mediated transcriptional activation of genes, we compared the expression of E2F1 and its target genes in splenic cells from lupus-prone B6.Nba2 congenic mice, which express increased levels of p202, with age-matched C57BL/6 mice. We found that increased expression of Ifi202 in the congenic mice was associated with inhibition of E2F1-mediated transcription and decreased expression of E2F1 and its target genes that encode proapoptotic proteins. Our observations support the idea that increased Ifi202 expression in certain strains of mice contributes to lupus susceptibility in part by inhibiting E2F1-mediated functions.

FIGURE 1. E2F1 negatively regulates expression of Ifi202

(A) Total RNA was isolated from sub-confluent cultures of the wild type (E2F1^+/+, lane 1) or E2F1-null (E2F1^-/-, lane 2) MEFs and steady state levels Ifi202 and actin mRNA were analyzed by semi-quantitative RT-PCR. (B) Total cell extracts were prepared from sub-confluent cultures of E2F1-null (lane 1) or wild type (lane 2) MEFs and steady state levels p202 and p68 protein (a protein detected by antiserum to p202 and serves as an internal control for protein amounts, ref. 19) were analyzed by immunoblotting. (C) Total cell extracts were prepared from NIH3T3 cells stably transfected with control vector (lane 1) or plasmid expressing E2F1 (lane 2). The extracts were analyzed by immunoblotting using antibodies specific to the indicated proteins.

FIGURE 2. E2F1-mediated transcriptional repression of Ifi202 is independent of p53 and pRb expression, but dependent on DNA-binding domain of E2F1

(A) Sub-confluent cultures of wild type or p53-null MEFs were transfected with 202-luc-reporter plasmid (2.5 μg) and pRL-TK plasmid (0.5 μg) along with pCMV (2.5 μg, column 1) or increasing amounts (1 μg or 2 μg, columns 2 and 3, respectively) of pCMV-mE2F1 plasmid using a calcium phosphate transfection kit as described in methods. Cells were harvested after 44 h to assays for the firefly and Renilla luciferase activities as described in methods. Normalized relative luciferase activity is shown. Results from a representative experiment are shown. (B) Sub-confluent cultures of p53-null MEFs were transfected with 202-luc-reporter plasmid (2.5 μg) and pRL-TK plasmid (0.5 μg) along with pCMV (2.5 μg, column 1) or increasing amounts (1 μg, 2 μg, or 3 μg, columns 2, 3, and 4, respectively) of pCMV-mE2F1 plasmid using a calcium phosphate transfection kit. Cells were harvested after 44 h to assays dual luciferase activities as described in (A) above. Normalized relative luciferase activity is shown. Results from a representative experiment are shown. (C) Sub-confluent cultures of Rb-null MEFs were transfected with 202-luc-reporter plasmid (2.5 μg) and pRL-TK plasmid (0.5 μg) along with pCMV (2.5 μg, column 1) or increasing amounts (1 μg or 2 μg columns 2 and 3, respectively) of pCMV-mE2F1 plasmid using a calcium phosphate transfection kit. Cells were harvested after 44 h to assays dual luciferase activities as described in (A) above. Normalized relative luciferase activity is shown. Results from a representative experiment are shown. (D) Sub-confluent cultures of p53-null MEFs were transfected with 202-luc-reporter plasmid (2.5 μg) and pRL-TK plasmid (0.5 μg) along with pCMV plasmid (2.5 μg, column 1) or plasmid (pCMV-mE2F1; 2.5 μg, column 2) encoding wild type mE2F1, or plasmid (2.5 μg) encoding various mutants of E2F1 [E2F1 (E132), column 3; E2F1 Δ206-220, column 4; E2F1 Δ1-88, column 5; and E2F1 (411/421) column 6] using a calcium phosphate transfection kit. Cells were harvested after 46 h to assays dual luciferase activities as described in (A) above. Normalized relative luciferase activity is shown. Results from a representative experiment are shown.

FIGURE 3. E2Fs differentially regulate expression of Ifi202

Sub-confluent cultures of NIH3T3 cells were transfected with 202-luc-reporter plasmid (2.5 μg) and pRL-TK plasmid (0.5 μg) along with equal amounts (2.5 μg) of pCMV (column 1), pCMV-E2F1 (column 2), pCMV-E2F2 (column 3), pCMV-E2F3 (column 4), or pCMV-E2F4 (column 5) using a calcium phosphate transfection kit as described in methods. Cells were harvested after 44 h to assays for the firefly and Renilla luciferase activities as described in methods. Normalized relative luciferase activity is shown.

FIGURE 4. Identification of a potential E2F DNA-binding site in the 5′-regulatory region of Ifi202 gene in gel-mobility shift assays

(A) Schematic representation of a potential E2F DNA-binding site (202-E2F-BS) in the 5′-regulatory region of Ifi202 gene. (B) Nuclear extracts from NIH 3T3 cells were subjected to gel-mobility shift assays using radio-labeled oligonucleotide containing the 202-E2F DNA-binding sequence (probe) as described in methods. The nuclear extract without any treatment (lane 2), after treatment with deoxycholate (lane 3), or after competition with 50-fold excess of cold oligonucleotide containing the E2F DNA-binding consensus sequence (purchased from Santa Cruz Biotech.). As a control, we also analyzed the probe without incubation with the extracts (lane 1). (C) Nuclear extracts from NIH3T3 cells were subjected to gel-mobility shift assays using radio-labeled oligonucleotide containing the E2F DNA-binding consensus sequence from the DHFR gene (probe) as described in methods. The nuclear extract without any treatment (lane 2), after competition with 50-fold excess of cold oligonucleotide containing the E2F-CS (lane 3), after competition with 20-fold (lane 4), 50-fold (lane 5), or 100-fold (lane 6) excess of cold oligonucleotide containing the 202-E2F-BS. As a control, we also analyzed the probe without incubation with the extracts (lane 1). The arrow indicates the location of E2F transcription factors in complex with the pocket family of proteins and the star indicates the location of “free” E2F transcription factors.

FIGURE 5. E2F1 binds to potential E2F DNA-binding site in the 5′-regulatory region of Ifi202 gene in chromatin immunoprecipitation assays

(A) Schematic representation of the 5′-regulatory region of Ifi202 gene containing the 202-E2F-BS and relative location of PCR primers that were used to amplify the immunoprecipitated chromatin. (B) Soluble chromatin was prepared from NIH3T3 (lanes 3, 5, and 7) or NIH3T3-E2F1 (lanes 4, 6, and 8) cells. Chromatin was incubated with antibodies to E2F1 (lanes 7 and 8) or, as a negative control, with isotype IgG1 antibodies (lanes 5 and 6). DNA was extracted from immunoprecipitates and PCR amplified (upper panel, 32 cycles; lower panel, 36 cycles) using a pair of primers that covered the E2F1 DNA-binding site in the 5′-regulatory region of the Ifi202 gene. As a positive control for PCR, we also amplified the input chromatin DNA from NIH3T3 (lane 3) and NIH3T3-E2F1 (lane 4) cells. As a negative control for PCR, we did not include any template DNA in PCR reaction (lane 2).

FIGURE 6

The E2F1 transcriptionally represses Ifi202 expression through the E2F1 DNA-binding site. Sub-confluent cultures of p53-null MEFs were transfected with wild type or mutant 202-luc-reporter plasmid (2.5 μg) and pRL-TK plasmid (0.5 μg) along with pCMV (2.5 μg, column 1) or pCMV-mE2F1 (2.5 μg, column 2) plasmid using a calcium phosphate transfection kit as described in methods. Cells were harvested after 44 h to assays for the firefly and Renilla luciferase activities as described in methods. Normalized relative luciferase activity is shown. Results from a representative experiment are shown.

FIGURE 7

Increased expression of Ifi202 in B6.Nba2 mice is associated with reduced expression levels of E2F1 and inhibition of E2F1-mediated transcription of target genes. (A) Sub-confluent cultures of B6 or B6.Nba2 MEFs were transfected with E2F-luc-reporter plasmid (2.5 μg) and pRL-TK plasmid (0.5 μg) using a calcium phosphate transfection kit as described in methods. Cells were harvested after 44 h to assays for the firefly and Renilla luciferase activities as described in methods. Normalized relative luciferase activity is shown. (B) Total RNA was isolated from splenocytes from age-matched (10 weeks) B6 (lane 1) or B6.Nba2 (lane 2) female mice and steady state levels of mRNAs were analyzed by semi-quantitative RT-PCR using a pair of primers specific for the indicated individual genes. (C) Total cell extracts were prepared from splenocytes isolated from age-matched (10 weeks) B6 (lane 1) or B6.Nba2 (lane 2) female mice and steady state levels of proteins were analyzed by immunoblotting using antibodies specific for the indicated proteins. (D) Total cell extracts were prepared from splenocytes isolated from age-matched (10 weeks) B6 (lanes 1 and 2) or B6.Nba2 (lanes 3 and 4) female mice and steady state levels p73 and actin proteins were analyzed by immunoblotting using specific antibodies.

FIGURE 8

Regulation of Ifi202gene by E2F1 transcription factor and the role of p202 in lupus susceptibility through inhibition of E2F-mediated transcription of target genes.