The Tumor Suppressor Interferon Regulatory Factor 1 Interferes with SP1 Activation to Repress the Human CDK2 Promoter

Abstract

Cell growth control by interferons (IFNs) involves upregulation 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 G 1 /S phase-related cyclindependent kinase 2 (CDK2) gene is a novel proliferationrelated 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 downstream 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 G 1 /S phase transition.Interferon regulatory factors (IRFs) 1 are activated by the anti-proliferative actions of interferons through JAK/STATmediated signaling mechanisms to inhibit cell growth (1-3).Genetic evidence suggests IRF1, the prototypical member of the IRF class of transcription factors, functions as a tumor suppressor (4, 5) presumably by regulating cell growth-related target genes (6). There are few experimentally validated IRF1 target genes, and only a subset of these may contribute to the cell growth inhibitory potential of IRF1 (1, 6 -10). To clarify the biological functions of IRF1 as a tumor suppressor, it is necessary to define additional cell growth regulatory genes that are IRF1-responsive.Our laboratory has shown that IRF1, as well as the closely related protein IRF2 (also known as histone nuclear factor-M (HiNF-M)), can function in the activation of histone H4 gene transcription at the G 1 /S phase transition through a phylogenetically conserved cell cycle regulatory element (9 -16), which encompasses a canonical IRF consensus sequence (17). A characteristic N-terminal DNA binding domain spanning a winged helix-turn-helix motif (18 -20) mediates the interaction of IRF factors with their cognate sites. The C-terminal region supports transcriptional enhancement or repression (21-23). The transcriptional activity of IRF factors is influenced by posttranslational modifications, ...

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VRK1 Signaling Pathway in the Context of the Proliferation Phenotype in Head and Neck Squamous Cell Carcinoma

“…In Cos1 cells, the same response region has been detected, but in the presence of VRK1, there is a significant increase in transcriptional activity. The human CDK2 gene promoter has a response element located between À440 and À68, which is important for basal expression in NIH3T3 cells (58). This element is also functional in Cos1 cells, but this region also increases its transcriptional activity in the presence of VRK1.…”

“…pX-p16, pCMV-E2F1, and pCMV-Rb were from M. Malumbres (Centro Nacional de Investigaciones Oncológicas, Madrid, Spain). Plasmid p-68-CDK2-Luc, p-440-CDK2-Luc, p-683-CDK2-Luc, and p-2400-CDK2-Luc have been previously described (58). The survivin gene promoter constructs pLuc1430c, pLuc-649c, pLuc-441c, pLuc-230c, and pLuc-42c were previously reported (74).…”

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