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CHF: a Novel Factor Binding to Cyclin a CHR Corepressor Element

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CHF: a Novel Factor Binding to Cyclin a CHR Corepressor Element Oncogene (1999) 18, 6222 ± 6232 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc CHF: a novel factor binding to cyclin A CHR corepressor element Alexandre Philips1,Se verine Chambeyron2, Ned Lamb2, Annick Vie 1 and Jean Marie Blanchard*,1 1Institut de GeÂneÂtique MoleÂculaire, CNRS, UMR 5535, 1919 route de Mende, F-34293 Montpellier cedex 5, France; 2Institut de GeÂneÂtique Humaine, CNRS, rue de la Cardonille, F-34396 Montpellier, France Cell cycle modulation of cyclin A expression is due to the cycle responsive element (CCRE) (Huet et al., 1996; periodic relief of a transcriptional repression mediated by Philips et al., 1998; Plet et al., 1997) or the cell cycle a bipartite negative DNA regulatory region. The 5' dependent element (CDE) (Zwicker et al., 1995), is element (Cell Cycle Responsive Element: CCRE; cell periodically occupied in G0/early G1 when transcrip- Cycle Dependent Element: CDE) is clearly occupied in a tion of cyclin A is o. Functional assays where this cyclic manner in vivo, whereas the 3' element, whose region directed the expression of a reporter gene, sequence is shared by B-myb, cdc25C and cdc2 genes revealed that mutation of the CCRE/CDE resulted in a (cell Cycle gene Homology Region: CHR), is involved in complete deregulation in G0/G1. This led thus to the more subtle interactions. Mutation of either element notion that a repressor was binding to this element, an results in complete deregulation of cyclin A promoter observation which was consistent with previous data activity. Whereas some reports claim that E2F/DP can obtained with human cyclin A promoter (Schulze et al., bind to the CCRE/CDE, the nature of the protein(s) 1995; Zwicker et al., 1995). 3' to the CCRE/CDE interacting with the CHR is unknown. In the present resides another element mutation of which also work we have characterized an activity present in abolished cyclin A repression in G0/G1 (Zwicker et quiescent cells and absent in cells blocked in S phase, al., 1995). Interestingly, this element, together with the which binds speci®cally to cyclin A CHR, but not to B- CCRE/CDE was found to be shared by other genes myb,ortocdc25C,ortocdc2 CHRs. A 90 kD protein, such as B-myb, cdc2 and cdc25C and was therefore named CHF (cyclin A CHR binding factor), has been termed cell cycle gene homology region (CHR). identi®ed through preparative electrophoresis and UV Transcription of genes involved in cell cycle control crosslinking experiments. In order to address in more requires coordinate activation and repression of speci®c functional terms the binding of CHF to cyclin A CHR, sets of transcription factors, among which members of we developed in vitro and in vivo oligonucleotide the E2F/DP family constitute a thoroughly studied competition assays. Both in vitro transcription and in archetype (for reviews, see: Dyson, 1998; Sardet et al., vivo microinjection experiments demonstrate that a 1997; Weinberg, 1995). Activity of E2F/DP transcrip- functional dierence exists between the composite tion factors is repressed by their association with CCRE/CDE-CHR repressor regions of cell cycle pocket proteins of the Rb family. The latter become regulated genes such as cyclin A and cdc25C. hyperphosphorylated as cells transit G1 and dissociate from the E2F/DP complex, leading to the activation of Keywords: cyclin A; transcription; cell cycle E2F-regulated genes. Cyclin A belongs to a group of genes whose transcription is repressed in G0/early G1 and derepressed prior to entry (B-myb, E2F-1) into or during S phase (cyclin A, cdc2), as well as in G2 Introduction (cdc25C). These genes are therefore potential target candidates for E2F/DP-mediated repression processes. Progression through the dierent phases of the cell Whereas E2F-containing complex have been reported division cycle is controlled by a family of cyclin- to interact with the CCRE/CDE (Liu et al., 1998; dependent protein kinases. Several types of cyclins Schulze et al., 1995; Zerfass-Thome et al., 1997) no have been identi®ed and cyclin A is synthesized at the CHR-binding protein has been puri®ed yet. However, onset of DNA replication as well as during the G2/M an in vitro binding activity termed CDF-1, which is not transition (for reviews, see Sardet et al., 1997; Sherr, related to E2F, has been characterized and proposed to 1996). We have previously analysed the expression of interact with both the CDE and the CHR of cdc25C cyclin A in primary as well as in human and rodent cell (Liu et al., 1997, 1998). Electromobility shift assays lines (Barlat et al., 1993; 1995; Philips et al., 1998; and oligonucleotide competition experiments suggested 1999; Plet et al., 1997), which led us to delineate the presence of a general factor which was not able to functional DNA sequences present in the mouse cyclin discriminate between the CCRE/CDE-CHR modules A promoter (Barlat et al., 1995; Huet et al., 1996). In of cdc25C, cdc2 and cyclin A. No further characteriza- vivo genomic DMS footprinting has revealed the tion of the complex neither the determination of the presence of putative protein binding elements within number and molecular properties of its components a short region containing the major transcription were undertaken. The high sequence conservation of initiation sites. One of these elements, termed the cell the diverse CCRE/CDE-CHR modules together with the apparent lack of speci®city observed in vitro for the binding of CDF-1 are at odds with the observed in vivo sequential timing of promoter activity during the cell *Correspondence: JM Blanchard Received 17 February 1999; revised 14 June 1999; accepted 14 June cycle. Whereas the dierential binding of E2Fs and 1999 CDF could contribute to this timing (Lucibello et al., CHF: a novel factor binding to cyclin A CHR corepressor element A Philips et al 6223 1997), our recent data based on speci®c in vitro (pCycA-luc) were transfected into mouse primary electrophoretic mobility gel shift conditions prompted ®broblasts (MEF) which were then serum-starved for us to search for factors speci®c to each CHR. 24 h (Philips et al., 1998). Luciferase activity was In the present study, we present evidence that a 90 ± monitored 18 h after serum-restimulation. The CCRE- 95 kD polypeptide binds in vitro speci®cally to cyclin A CHR-dependent repression of cyclin A transcription CHR but not to cdc25C or to B-myb, or to cdc2 CHRs. was observed in quiescent cells and was relieved in Moreover, we also show that cyclin A and cdc25C CHRs serum-refed cells as expected (Figure 1). However, as are involved in functionally dierent interactions previously shown, a constitutive high activity was discriminating their two repressor elements. In vivo monitored whether cells were starved in low serum or microinjection and in vitro transcription experiments restimulated when the CCRE-mutated version of the using whole cell extracts were carried out in the presence cyclin A promoter was used (Figure 1), whereas, of competing excesses of cyclin A or cdc25C oligonucleo- mutations on the CRE or the NF-Y sites nearby led tides. When puri®ed plasmids were used as templates in to promoters only slightly impaired in their response to an in vitro transcription assay with whole cell extracts, serum (data not shown) (Huet et al., 1996; Philips et only a cyclin A oligonucleotide containing an intact al., 1999). In contrast, mutation of the sequence CHR was able to relieve cyclin A promoter from immediately 3' to the CCRE, shared by several other inhibitory factors. The inhibitory role of the CHR cell cycle-regulated genes and termed the `cell cycle binding polypeptide was further demonstrated by the gene homology region' (CHR), led to a dramatic use of depleted extracts which were reconstituted with upregulation of cyclin A promoter in quiescent cells the semi puri®ed CHF activity. Moreover, microinjec- and which was hardly stimulated by serum (Figure 1). tion in living ®broblasts of cyclin A wild type CCRE- Mutations on the CHR gave reproducibly rise to a CHR resulted in premature activation of cyclin A gene in stronger eect than on the CCRE, suggesting that a late G1 cells, while leaving unaected cdc25C.In hierarchy could exist between the two elements. This contrast, microinjection of oligonucleotides in which extends the observations previously made on the the CHR was mutated, was ineective in inducing cyclin human cdc25C, cdc2 and cyclin A promoters A. In addition, microinjection of an oligonucleotide (Zwicker et al., 1995) to the murine cyclin A promoter. containing cdc25C CDE-CHR did not result in cyclin A The repression of cyclin A transcription in G0/G1 premature expression. MEF is thus governed by a bipartite cell cycle- regulated repressor element consisting of the CCRE/ CDE and a directly adjacent CHR motif. Results Binding of factors to cyclin A CHR is cell cycle Mutations of either the CCRE or the CHR abolish regulated repression of murine cyclin A promoter in G0/G1 Electrophoretic mobility shift assays (EMSA) carried primary ®broblasts out with total cellular extracts from either MEF, HeLa, The wild type and the various mutated versions of the Daudi or Kit225 cells suggested the presence of factors cyclin A promoter linked to a luciferase reporter harbouring a speci®c interaction with cyclin A CHR. Figure 1 Cyclin A transcription is repressed through a bipartite repressor element. (a) Primary mouse ®broblasts (MEF) were transfected 18 h prior to serum starvation, serum-starved for 24 h, and released from mitogen deprivation by addition of complete medium. Fire¯y luciferase activity driven by the minimal cyclin A promoter, harbouring either a wild type structure (WT) or a mutation in respectively the CCRE (mCCRE), or the CHR (mCHR), is expressed relative to the activity of a co-transfected constitutive Renilla luciferase expression vector.
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