Structure and promoter activity of the mouse CDC25A gene
Paul M. Epstein,
BASF Bioresearch Corp. 100 Research Drive, Worcester, Massachusetts 01605-4314, USA
Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA
Department Molecular Genetics and Microbiology, UMMC, Worcester, Massachusetts, USA
Department of Pharmacology and Toxicology, UMMC, Worcester, Massachusetts, USA
Received: 26 April 2000 / Accepted: 28 July 2000
Abstract. CDC25A is a member of a group of highly related,
dual-specificity phosphatases that promote cell cycle phase tran-
sitions by regulating the activity of cyclin-dependent kinases. Here
we report the cloning and genomic sequence of 21,067 nucleotides
encompassing the mouse CDC25A gene. The coding sequence is
expressed from 17,904 bp of genomic DNA comprising 15 exons.
We also mapped the transcription initiation site to a consensus
initiator element proximal to an SP1 site. Approximately 1 kb of
sequence upstream of the transcription initiation site confers pro-
moter activity and cell type specificity to a reporter gene construct.
Surprisingly, transcription from this promoter was repressed by
over-expression of catalytically active but not catalytically inactive
CDC25A protein. We also show, using NIH 3T3 cells, that murine
CDC25A mRNA levels fluctuate only modestly over the cell
cycle. Our findings provide insights into the regulation of
CDC25A expression and have facilitated construction of gene
Key transition points in the eukaryotic cell cycle are regulated by
the activity of cyclin-dependent kinases (cdks) (for review, see
Nurse 1994, 1997; Sherr 1994; King et al. 1994; Elledge 1996).
Activity of these molecules is regulated by many mechanisms
including phosphorylation and protein-protein interactions (re-
viewed in Lew and Kornbluth 1996). Cdks are activated by phos-
phorylation on Thr-160 by Cdk7, whereas phosphorylation on Thr-
14 and Tyr-15 by Myt1 and Wee1 is inhibitory (Russell and Nurse
1987; Gould and Nurse 1989; Krek and Nigg 1991). These two
consecutive residues are dephosphorylated by the CDC25 dual-
specificity protein phosphatases (Galaktionov and Beach 1991;
Millar et al. 1991). Separate but perhaps overlapping functions
have been assigned to each form of CDC25. CDC25A has been
proposed to mediate transition from G1 to S phase (Jinno et al.
1994). The role of CDC25B has been cast both at the G1/S and
G2/M transitions (Sebastian et al. 1993; Galaktionov et al. 1995a;
Gabrielli et al. 1997; Garner-Hamrick and Fisher 1998). CDC25C
has been proposed to be required for the onset of mitosis (Sadhu et
al. 1990; Nagata et al. 1991; Galaktionov and Beach 1991).
CDC25 phosphatase is expressed in all eukaryotes. A single
form of CDC25 is expressed in yeast (Russell and Nurse 1986) and
two forms in Drosophila (Alphey et al. 1992; Edgar and O’Farrell
1989). In mammals the three forms of CDC25 (A, B, and C) are
encoded by three highly related but distinct genes (reviewed by
Draetta and Eckstein 1997). Recently, C. elegans was reported to
harbor four forms of CDC25, raising the possibility that other
forms may exist in mammalian cells (Ashcrot et al. 1998). Crystal
structures were published recently for human CDC25A (Fauman et
al. 1998) and CDC25B (Reynolds et al. 1999).
Based on gene disruption studies, CDC25 phosphatases are
necessary for cell cycle progression in yeast and Drosophila (Rus-
sell and Nurse 1986, Edgar and O’Farrell 1989; Alphey et al.
1992). In mammalian cells at least one report implicates CDC25A
in regulating G1 arrest owing to DNA damage (Terada et al. 1995).
CDC25A has been reported to play a role in regulating cell cycle
progress in response to serum factors. For example, TGF␤ was
proposed to cause G1 arrest in certain cell types by inhibiting
CDC25A expression (Terada et al. 1995; Iavarone and Massague
1997). CDC25A has also been proposed to lie at the end of signal
transduction pathways activated by mitogens (Galaktionov et al.
1995a; Galaktionov et al. 1996a).
Consistent with their proposed function as cell cycle regula-
tors, over-expression of CDC25A and B, but not C, has been
reported in breast cancer (Galaktionov et al. 1995a), non-Hodgkin
lymphomas (Hernandez et al. 1998), in head and neck cancers
(Gasparotto et al. 1997), and in human gastric carcinomas (Kudo
et al. 1997). Moreover, it has been reported that CDC25A and B,
but not C, can cooperate with activated ras to immortalize mouse
embryo fibroblasts (Galaktionov 1995b).
In the mouse, CDC25A and B are expressed in overlapping but
distinct patterns in adults and embryos (Wickramasinghe et al.
1995; Kakizuka et al. 1992). In adult mice, CDC25A is expressed
most abundantly in testes, with little or no expression in lung and
spleen (Wickramasinghe et al. 1995). In the adult rat, CDC25B is
expressed most abundantly in spleen and lung, but is not detectable
in kidney (Kakizuka et al. 1992). In adult mice CDC25C is ex-
pressed most abundantly in thymus, but was not detected in lung
or kidney (Nargi and Woodford-Thomas 1994). The mechanism
by which this tissue-specific expression is achieved has not been
Here we report the complete genomic sequence of the murine
CDC25A gene and details of the CDC25A transcription unit ob-
tained from cDNA and RNA mapping. We also describe 5Ј regu-
latory sequences conferring cell type-specific expression and a
potentially autoregulatory loop. Finally, we report on the construc-
tion and recombination frequencies of gene-targeting vectors con-
structed with this sequence information.
Materials and methods
Cell synchronization and northern blotting.
3T3 cells were
synchronized at the G
/S boundary by double thymidine block protocol
(Pagano 1995). Cells were harvested at 0, 3, 6, 9, and 12 h post release. A
fraction of the cells were fixed, propidium iodide stained, and FACS ana-
lyzed to determine cell cycle distribution. Total RNA from the remaining
cells was isolated from 175 cm
flasks of synchronized 3T3 cells by uti-Correspondence to: J. Voss at BASF. E-mail: Vossj@BASF.com
Mammalian Genome 11, 1063–1069 (2000).
© Springer-Verlag New York Inc. 2000