Regulation of CDK7-carboxyl-terminal domain kinase activity by the tumor suppressor p16(INK4A) contributes to cell cycle regulation.

The eukaryotic cell cycle is regulated by cyclin-dependent kinases (CDKs). CDK4 and CDK6, which are activated by D-type cyclins during the G(1) phase of the cell cycle, are thought to be responsible for phosphorylation of the retinoblastoma gene product (pRb). The tumor suppressor p16(INK4A) inhibits phosphorylation of pRb by CDK4 and CDK6 and can thereby block cell cycle progression at the G(1)/S boundary. Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II by general transcription factor TFIIH is believed to be an important regulatory event in transcription. TFIIH contains a CDK7 kinase subunit and phosphorylates the CTD. We have previously shown that p16(INK4A) inhibits phosphorylation of the CTD by TFIIH. Here we report that the ability of p16(INK4A) to inhibit CDK7-CTD kinase contributes to the capacity to induce cell cycle arrest. These results suggest that p16(INK4A) may regulate cell cycle progression by inhibiting not only CDK4-pRb kinase activity but also by modulating CDK7-CTD kinase activity. Regulation of CDK7-CTD kinase activity by p16(INK4A) thus may represent an alternative pathway for controlling cell cycle progression.

Structural organization of the human vitamin D receptor chromosomal gene and its promoter.

The vitamin D receptor (VDR) is known to mediate the pleiotropic biological actions of 1,25-dihydroxyvitamin D3 through its ability to modulate the expression of target genes. The regulation of this ligand-activated cellular transcription factor is reported to occur at both transcriptional and posttranslational levels. To begin to address the molecular basis by which the VDR gene is regulated transcriptionally, we report here an initial characterization of the human VDR gene and its promoter. We isolated several overlapping A-phage and cosmid clones that cover more than 100 kb of human DNA and contained the entire VDR gene. The gene is comprised of 11 exons that, together with intervening introns, span approximately 75 kb. The noncoding 5'-end of the gene includes exons 1A, 1B, and 1C. Eight additional exons (exons 2-9) encode the structural portion of the VDR gene product. While primer extension and S1 nuclease-mapping studies reveal several common transcriptional start sites, three unique mRNA species are produced as a result of the differential splicing of exons 1B and 1C. The DNA sequence lying upstream of exon 1A is GC rich and does not contain an apparent TATA box. Several potential binding sites for the transcription factor SP1 and other activators are evident. Fusion of DNA fragments containing putative promoter sequences upstream of the luciferase structural gene followed by transient transfection of these plasmids into several mammalian cell lines resulted in significant reporter activity. Due to the size and complexity of the 5'-end of the VDR gene, we examined the activity of a DNA fragment surrounding exon 1C. An intron fragment 3' of exon 1C conferred retinoic acid responsivity when fused to a reporter gene plasmid, suggesting a molecular mechanism for the previously observed ability of retinoic acid to induce the VDR. The recovery of the gene for the human VDR will enable further studies on the transcriptional regulation of this gene.