Elsevier

Gene

Volume 228, Issues 1–2, 4 March 1999, Pages 93-100
Gene

Two short sequences have positive effects on the human p27Kip1 gene transcription

https://doi.org/10.1016/S0378-1119(99)00022-0Get rights and content

Abstract

The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 plays an important role in the progression from G1 to S phase in the cell cycle. To study the activities of its promoter and other regulatory elements, we have cloned and characterized the 5′-flanking region of the human p27Kip1 gene. This region, about 3 kb in length, is GC-rich and shares homology with that of the mouse p27Kip1 gene. Transcription start points (tsp) determined by the oligo-capping method are mapped in two regions, the cluster I (−479 to −403) and cluster II (−280 to −273). The cluster I was the primary functional site in transcription initiation. The luciferase activities of serial deletion mutants indicated that two short sequences (−581 to −557 and −556 to −526) had positive effects on transcription. The gel shift assay showed that factors in HeLa nuclear extract bound to these sequences. Sp1 was the major binding factor to the sequence of −556 to −526, wheres yet unidentified positive factors bound to the sequence of −581 to −557.

Introduction

Spatially and temporally precise activation of cyclin-dependent kinases (Cdks) is one of the key mechanisms by which mammalian cell cycle is controlled. Cdks are positively regulated by cyclins (Sherr, 1995). In progression from G1 to S phase, cyclin D binds to Cdk4/6 and cyclin E/A binds to Cdk2. These cyclin/Cdk complexes phosphorylate proteins such as the retinoblastoma protein (Rb). The phosphorylation of Rb inactivates its ability to inhibit transactivation of transcription factor E2F, which is essential for DNA replication (Weinberg, 1995). Cdks are also negatively regulated by Cdk inhibitors in progression from G1 to S phase (Sherr, 1995). Cdk inhibitors are classified into two families: Cip/Kip family and Ink family. The Cip/Kip family includes p21Cip1, p27Kip1 and p57Kip2 (Hengst and Reed, 1998), whereas the Ink family includes p15Ink4B, p16Ink4A, p18Ink4C and p19Ink4D (Carnero and Hannon, 1998). Additionally, the Ink family is specific for Cdk4/6, whereas the Cip/Kip family inhibits cyclin D-Cdk4/6 and cyclin E/A-Cdk2. Both families also block phosphorylation of Cdk by the Cdk-activating kinase (CAK).

The Cdk inhibitor p27Kip1 was cloned as a binding protein for the cyclin D1–Cdk4 complex (Toyoshima and Hunter, 1994). In addition, it was cloned independently as a binding protein for cyclin E–Cdk2 complex in transforming growth factorβ (TGFβ)-treated cells (Polyak et al., 1994a). The p27Kip1 has been suggested to mediate G1 arrest induced by TGFβ as well as rapamycin, cAMP, contact inhibition, or serum deprivation (Firpo et al., 1994, Kato et al., 1994, Nourse et al., 1994, Polyak et al., 1994a, Polyak et al., 1994b, Reynisdóttir et al., 1995). Recently there have been reports suggesting that p27Kip1 is involved in apoptosis and differentiation as well as cell proliferation (Ezhevsky et al., 1996, Kranenburg et al., 1995). Since p27Kip1 plays important roles as described above, its participation in tumorigenesis has also been investigated. Mice deficient in p27Kip1 developed multiorgan hyperplasia and pituitary tumors (Fero et al., 1996, Kiyokawa et al., 1996, Nakayama et al., 1996). Although the human p27Kip1 gene has been mapped to the chromosomal region 12p12–13.1 where deletions and rearrangements are often found in patients with leukemia, its mutations in human tumors are rare (Bullrich et al., 1995, Pietenpol et al., 1995, Ponce-Castañeda et al., 1995). However, the expression of the p27Kip1 protein is associated with the survival of patients with breast cancers and colorectal carcinomas, which suggests that the p27Kip1 protein may be important in tumor progression (Catzavelos et al., 1997, Loda et al., 1997, Porter et al., 1997).

The promoter region of the human p27Kip1 gene has not been studied in detail, but it is reported that the p27Kip1 mRNA remains unchanged during the cell cycle (Hengst and Reed, 1996). This paper reports the detailed characterization of the tsp and promoter of the human p27Kip1 gene. Our studies show that the major tsp is located in the region of −479 to −403 (the cluster I), which is different from that reported previously (Minami et al., 1997). Two short sequences had positive effects on transcription, and the factors binding to these sequences were studied.

Section snippets

Preparation of the adaptor-ligated DNA libraries

To isolate genomic clones containing the 5′-flanking region of the human p27Kip1 gene, we applied a PCR method reported by Siebert et al. (1995). Genomic DNA from Japanese male lymphocytes was digested with EcoRI, BglII or StuI. The digested DNA was ligated to an excess of oligonucleotide adaptors. The adaptors were formed with the oligonucleotide 5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3′ in combination with either one of the following oligonucleotides: 5′-PO4-ACCAGCCC-NH2-3′ for

Cloning of the 5′-flanking region

By using three libraries of adaptor-ligated genomic DNA fragments, we obtained three PCR products of about 2.5 kb in the EcoRI library, 2.6 kb in the BglII library and 3.0 kb in the StuI library. These fragments were cloned, and the 5′-flanking region of the human p27Kip1 gene was sequenced up to 3001 bp upstream from the adenine residue at the start codon (Accession No. AB005590, Fig. 1). A search of human nucleotide databases identified a short fragment of an unmethylated CpG island (Accession

Discussion

We found that the tsp of the human p27Kip1 gene were located in two regions: cluster I (−479 to −403) and cluster II (−280 to −273) (Fig. 1, Fig. 2). However, different tsp were reported previously (Minami et al., 1997). This discrepancy may be attributable to the different analysis methods employed. The oligo-capping method used in this study labels the 5′-end of mature mRNA directly (Maruyama and Sugano, 1994), whereas the primer extension method used by the other group depends on the

Acknowledgements

Part of the sequence analysis was performed using the computer system at the Human Genome Center, Institute of Medical Science, The University of Tokyo. This study was supported in part by the Ministry of Education, Science and Culture of Japan.

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    Present address: Medicinal Research Laboratories, Taisho Pharmaceutical Co. Ltd., Omiya, Japan.

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