Inhibition of Cyclin D1 Kinase Activity Is Associated with E2F-Mediated Inhibition of Cyclin D1 Promoter Activity through E2F and Sp1

Abstract

Coordinated interactions between cyclin-dependent kinases (Cdks), their target "pocket proteins" (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk inhibitors regulate orderly cell cycle progression. The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes, which phosphorylate the tumor suppressor pRB, leading to the release of free E2F-1. Overexpression of E2F-1 can induce apoptosis and may either promote or inhibit cellular proliferation, depending upon the cell type. In these studies overexpression of E2F-1 inhibited cyclin D1-dependent kinase activity, cyclin D1 protein levels, and promoter activity. The DNA binding domain, the pRB pocket binding region, and the amino-terminal Sp1 binding domain of E2F-1 were required for full repression of cyclin D1. Overexpression of pRB activated the cyclin D1 promoter, and a dominant interfering pRB mutant was defective in cyclin D1 promoter activation. Two regions of the cyclin D1 promoter were required for full E2F-1-dependent repression. The region proximal to the transcription initiation site at ؊127 bound Sp1, Sp3, and Sp4, and the distal region at ؊143 bound E2F-4-DP-1-p107. In contrast with E2F-1, E2F-4 induced cyclin D1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable functions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce pRB phosphorylation and E2F-1 levels in the cell.

Cyclin D1 Is Transcriptionally Down-Regulated by ZO-2 via an E Box and the Transcription Factor c-Myc

“…Because ChIP assays (Cicatiello et al, 2004) have revealed that E2F factors bind to DNA in a cell cycle progression-related manner, we next performed deletions of the E2F region, and we designed functional assays to test the particular relevance of these transcription factors in controlling the cyclin D1 promoter activity under ZO-2 effect. In accordance with the results found in other cell lines (Watanabe et al, 1998), the deleted distal "E2F" region seemed to contain a silencer element for the cyclin D1 promoter ( Figure 4B). The generation of different constructs where the region surrounding E2F was deleted allowed the detection of a fragment located between nt Ϫ694 and Ϫ550 that is required for attaining the negative response triggered by ZO-2 ( Figure 4B).…”

“…Inside the region located between nucleotides (nt) Ϫ737/ Ϫ730 (distal) or between nt Ϫ143/Ϫ133 (proximal), there are elements recognized by the transcription factors of the E2F family (Fukami-Kobayashi and Mitsui, 1998;Watanabe et al, 1998;Cicatiello et al, 2004). Because ChIP assays (Cicatiello et al, 2004) have revealed that E2F factors bind to DNA in a cell cycle progression-related manner, we next performed deletions of the E2F region, and we designed functional assays to test the particular relevance of these transcription factors in controlling the cyclin D1 promoter activity under ZO-2 effect.…”

“…The pXP2-CD1 reporter vector contains the cyclin D1 regulatory region here shown. TRE(AP-1), TRE, Ϫ924 to Ϫ938 (Herber et al, 1994); dE2F, distal E2F, Ϫ730 to Ϫ737 (Herber et al, 1994); E box, bHLH, and Zn finger transcription factors binding site, Ϫ547 to Ϫ553 (Herber et al, 1994); ZONAB, ZONAB binding site and inverted CCAAT box, Ϫ527 to Ϫ532 (Sourisseau et al, 2006); Oct, Oct binding site, Ϫ646 to Ϫ652 (Cicatiello et al, 2004); pE2F, proximal E2F, Ϫ133 to Ϫ143 (Watanabe et al, 1998); Tcf/Lef, Tcf/Lef binding site, Ϫ67 to Ϫ77 (Shtutman et al, 1999); CRE, Ϫ46 to Ϫ53, (Herber et al, 1994); and Start, site of transcription initiation, Ϫ1/ϩ1 (Herber et al, 1994). Broken arrow stands for the transcription start point.…”

Gastrin-mediated activation of cyclin D1 transcription involves β-catenin and CREB pathways in gastric cancer cells

“…The antibodies used were: cyclin D1 from Neomarkers (Freemont, CA, USA); b-catenin from BD Transduction Laboratories (Franklin Lakes, NJ, USA); ABC b-catenin from Upstate Biotechnology (Lake Placid, NY, USA); HA.11 antibody from Babco (Richmond, CA, USA); and CREB (total and phospho) from Cell Signaling Technology (Beverly, MA, USA). The À1745 human cyclin D1 promoter reporter construct containing 1745 bp upstream of the transcription initiation site, À1745 CD1-luc (TCF mutant), À1745 CD1-luc (CRE mutant), À66 CD1-luc (without the TCF sites) and the empty luciferase vector pA3-luc have been described elsewhere (Joyce et al, 1999;Watanabe et al, 1998). The pGL3OT and pGL3OF reporter constructs were from Dr Bert Vogelstein (He et al, 1998), the pCRE-luc reporter construct was from Stratagene, HAepitope-tagged wild-type b-catenin construct from Dr Stephen Byers (Orford et al, 1997), the wild-type CREB construct from Dr Marc Montminy (Du et al, 2000), the KCREB construct from Dr Richard Goodman (Walton et al, 1992), the pHYTCAxin-myc clone containing the mouse axin cDNA was from Dr Roel Nusse (Willert et al, 1999) and the dnTCF4 cDNA clone was from Dr Hans C Clevers (Korinek et al, 1997).…”

Immunohistochemical Expression of Cell Cycle Proteins E2F-1, Cdk-2, Cyclin E, p27kip1, and Ki-67 in Normal Placenta and Gestational Trophoblastic Disease

“…Cell cycle progression is regulated by the coordinated interactions of a variety of proteins, which include E2F-1, cyclin-dependent kinases (Cdks), cyclin E, and p27 kip1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The retinoblastoma protein (pRb) in its active, hypophosphorylated state is an inhibitor of cell cycle progression from G 1 to S phase (4,6).…”