Cloning of the cyclin A1 Genomic Structure and Characterization of the Promoter Region

Abstract

Cyclin A1 is a recently cloned cyclin with high level expression in meiotic cells in the testis. However, it is also frequently expressed at high levels in acute myeloid leukemia. To elucidate the regulation of cyclin A1 gene expression, we cloned and analyzed the genomic struc-ture of cyclin A1. It consists of 9 exons within 13 kilobase pairs. The TATA-less promoter initiates transcription from several start sites with the majority of transcripts beginning within a 4-base pair stretch. A construct con-taining a fragment from ؊190 to ؉145 showed the high-est transcriptional activity. Transfection of cyclin A1 promoter constructs into S2 Drosophila cells demon-strated that Sp1 is essential for the activity of the pro-moter. Sp1, as well as Sp3, bound to four GC boxes be-tween nucleotides ؊130 and ؊80 as observed by gel shift analysis. Mutations in two or more of the four GC boxes decreased promoter activity by >80%. The promoter was found to be cell cycle-regulated with highest activities found in late S and G 2 /M phase. Further analyses sug-gested that cell cycle regulation was accomplished by periodic repression of the GC boxes in G 1 phase. Taken together, our data show that cyclin A1 promoter activity critically depends on four GC boxes, and members of the Sp1 family appear to be involved in directing expression of cyclin A1 in both a tissue-and cell cycle-specific manner. A growing family of cyclin-dependent kinases (Cdk) 1 regu-lates a wide variety of cellular pathways (for review, see Ref. 1). Cdc 2 (Cdk 1) and Cdk 2 play a central role in the cell cycle of mammalian cells. Substrate specificity and activity of Cdks are controlled by their interaction with different cyclins that trig-ger the initiation of cell cycle events (2). Cdk 2 specifically interacts with cyclin E for G 1 /S progression and with cyclin A during S and G 2 /M phases. Cyclin B3 might be another partner for Cdc 2 and Cdk 2 during the G 2 /M phase (3). In accordance with their central role in mammalian cell cycle regulation, the levels of cyclins A and E (among other cyclins) oscillate in most if not all proliferating mammalian cells. Disruption of the mu-rine cyclin A2 (the homolog of human cyclin A) leads to early embryonic death suggesting an essential role for this gene in embryonic cell cycle in mammals (4). The human cyclin A2 (also known as cyclin A) was initially cloned because its gene locus was the site of integration by the hepatitis B virus in a case of hepatocellular carcinoma (5). It has also been implicated to be important in the recurrence of hepatocellular carcinoma (6). Recently, we cloned a second human cyclin A-like partner for Cdk 2, termed cyclin A1, that exhibits a highly restricted pat-tern of expression (7). The high level tissue-specific expression of the human and murine cyclin A1 in testis suggests a specific role in meiosis (7, 8). Very low levels are detected in other tissues by reverse transcriptase-PCR; however, high levels of human cyclin A1 were also found in acute myeloid leukemia cell lines (7) and myeloid leukemia samples from patients (9). This intriguing observation might suggest a possible role for cyclin A1 in proliferation and differentiation of hematopoietic progenitors and/or in promotion of growth of leukemic cells. Cyclin A1 shows homology to cyclin A2 and forms in vivo complexes with Rb as well as with E2F (10). Cyclin A1-Cdk 2 complexes phosphorylate these substrates in vitro (10). Our data showing that cyclin A1 is expressed in hematopoietic progenitors (9) and interacts with Rb family members and E2F, suggest that it may affect cell cycle progression in expressing cells. The pattern of cyclin A1 expression indicates that the regu-lation of its expression is different from that of cyclin A2. Furthermore, overexpression of cyclin A1 in myeloid leukemia originates at the transcriptional level. To elucidate the tran-scriptional mechanisms that underlie the tissue-specific pat-tern of expression, we cloned and analyzed the genomic orga-nization of the cyclin A1 gene and its promoter region. The highest transcriptional activity was assigned to a 335-bp frag-ment that required intact GC boxes located between Ϫ60 and Ϫ120 bp upstream of the main transcriptional start sites. These sites are also essential for cell cycle regulation of the promoter.