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The tumor suppressor interferon regulatory factor 1 interferes with SP1 activation to repress the human CDK2 promoter
Rong-Lin Xie1, Sunita Gupta, Angela Miele
1Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Abstract:
Cell growth control by interferons (IFNs) involves up-regulation of the tumor suppressor interferon regulatory factor 1 (IRF1). To exert its anti-proliferative effects, this factor must ultimately control transcription of several key genes that regulate cell cycle progression. Here we show that the G1/S phase-related cyclin-dependent kinase 2 (CDK2) gene is a novel proliferation-related downstream target of IRF1. We find that IRF1, but not IRF2, IRF3, or IRF7, selectively represses CDK2 gene transcription in a dose- and time-dependent manner. We delineate the IRF1-responsive repressor element between nt -68 to -31 of the CDK2 promoter. For comparison, the tumor suppressor p53 represses CDK2 promoter activity independently of IRF1 through sequences upstream of nt -68, and the CDP/cut/Cux1 homeodomain protein represses transcription down-stream of -31. Thus, IRF1 repression represents one of three distinct mechanisms to attenuate CDK2 levels. The -68/-31 segment lacks a canonical IRF responsive element but contains a single SP1 binding site. Mutation of this element abrogates SP1-dependent enhancement of CDK2 promoter activity as expected but also abolishes IRF1-mediated repression. Forced elevation of SP1 levels increases endogenous CDK2 levels, whereas IRF1 reduces both endogenous SP1 and CDK2 protein levels. Hence, IRF1 represses CDK2 gene expression by interfering with SP1-dependent transcriptional activation. Our findings establish a causal series of events that functionally connect the anti-proliferative effects of interferons with the IRF1-dependent suppression of the CDK2 gene, which encodes a key regulator of the G1/S phase transition.
Insights
Interferon regulatory factor 1 (IRF1) suppresses cyclin-dependent kinase 2 (CDK2) gene transcription, inhibiting cell growth. This IRF1-mediated repression of CDK2 occurs by interfering with SP1-dependent activation, linking interferons to cell cycle control.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Interferons (IFNs) control cell growth via up-regulation of the tumor suppressor interferon regulatory factor 1 (IRF1).
- IRF1's anti-proliferative effects depend on controlling key cell cycle progression genes.
- The cyclin-dependent kinase 2 (CDK2) gene regulates the G1/S phase transition.
Purpose of the Study:
- To identify novel proliferation-related downstream targets of IRF1.
- To investigate the mechanism by which IRF1 regulates CDK2 gene transcription.
- To elucidate the connection between interferon signaling and cell cycle control.
Main Methods:
- Reporter gene assays to assess promoter activity.
- Site-directed mutagenesis to identify regulatory elements.
- Western blotting to analyze protein levels of SP1 and CDK2.
- Analysis of IRF1, IRF2, IRF3, and IRF7 effects on CDK2 transcription.
Main Results:
- IRF1 selectively represses CDK2 gene transcription in a dose- and time-dependent manner.
- IRF1-mediated repression involves an element between nt -68 to -31 of the CDK2 promoter, containing an SP1 binding site.
- IRF1 represses CDK2 expression by interfering with SP1-dependent transcriptional activation, reducing both SP1 and CDK2 protein levels.
Conclusions:
- CDK2 is a novel proliferation-related downstream target of IRF1.
- IRF1 represses CDK2 gene expression through interference with SP1 transcriptional activation.
- This study establishes a functional link between interferon's anti-proliferative effects and IRF1-dependent suppression of CDK2.