Human Dbf4/ASK promoter is activated through the Sp1 and MluI cell-cycle box (MCB) transcription elements

Abstract

Dbf4 is the regulatory subunit of Cdc7 kinase, which is essential for entry into and traversing through S phase. The level of Dbf4, which is critical for the activation of Cdc7, is regulated by transcription and protein degradation. To gain a better understanding as to how the transcription of human Dbf4 (HuDbf4) is regulated, we have cloned and characterized its promoter. We found that HuDbf4 core promoter is localized within 7 211 to 7285 of the translation start-codon. This 75 bp DNA segment contains, among others, a putative MluI Cellcycle Box (MCB). A point mutation within the MCB dramatically reduced the promoter activity. This is the first example that an MCB element plays an essential role in the activation of a core promoter in mammalian cells. The auxiliary elements required for the full promoter activity are present within 162-bp upstream from the core promoter (i.e., 7286/7447). A point mutation within the Sp1 element at 7353/7361 resulted in a decrease of promoter activity to the basal level, while the deletion of the putative HES-1 at 7326/7331 dramatically increased the promoter activity. Taken together, our data suggests that the MCB element is essential for the core promoter activation, while the Sp1 positive regulator and the HES-1 repressor coordinately determine the efficiency of the HuDbf4 promoter. We have also found: (i) that the major transcription initiations occur at 7220, 7235 and 7245; (ii) that HuDbf4 gene consists of 12 exons, which spread over a 33-kb region.

ATR–Chk1–APC/CCdh1-dependent stabilization of Cdc7–ASK (Dbf4) kinase is required for DNA lesion bypass under replication stress

“…In budding yeast, protein stability of Dbf4 is regulated by the cell cycle regulatory ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) (Weinreich and Stillman 1999;Ferreira et al 2000). The level of human ASK protein fluctuates during the cell cycle, partly due to transcriptional regulation (Wu and Lee 2002;Yamada et al 2002). In contrast, post-transcriptional mechanisms of Cdc7 and ASK protein abundance control are unknown in mammals.…”

Transcription Factor Binding Profiles Reveal Cyclic Expression of Human Protein-coding Genes and Non-coding RNAs

“…The first cluster contain 6 TSSs, which are all predicted to be cell cycle regulated with a probability score >0.7; whereas the second cluster (11 kb away from the first cluster) contains 2 TSSs with probability score of 0.296 and 0.190 respectively. The DBF4 protein is known to be essential for initiation of DNA replication [32] and the transcription of its promoter is activated through cell-cycle box (MCB) transcription elements [33]. Assuming the TSS annotation is correct, our analysis imply that only the first cluster of transcript isoforms are regulated in a cell cycle dependent manner; and that the two isoforms in the second cluster may not be periodically expressed during the cell cycle, either not being involved in cell cycle regulation or impacting cell cycle in a different way from the first cluster of isoforms.…”

A High Through-Put Screen for Small Molecules Modulating MCM2 Phosphorylation Identifies Ryuvidine as an Inducer of the DNA Damage Response

“…Furthermore, studies using siRNA-mediated downregulation of CDC7, as well as CDC7 kinase inhibition with a wide variety of small molecule inhibitors, have demonstrated that Ser40 MCM2 phosphorylation is a robust and reliable indicator/biomarker of cellular CDC7 activity [10], [11]. Intracellular CDC7 activity is regulated at multiple levels: by the binding of a regulatory subunit, either DBF4A or DBF4B [12]–[14], by cell cycle dependent transcription of the catalytic and regulatory subunits [15], by APC dependent proteolysis [16] and by miRNA's [17]. CDC7-dependent phosphorylation of MCM proteins is then antagonized by cellular protein phosphatases, with PP1 having a major role in this process in both budding yeast and Xenopus [18], [19].…”