Transcriptional regulation of the mouse deoxycytidine kinase: identification and functional analysis of nuclear protein binding sites at the proximal promoter

Abstract

Deoxycytidine kinase (EC 2.7.1.74, dCK) catalyzes the phosphorylation of deoxynucleosides and several nucleoside analogues that are important in antiviral and cancer chemotherapy. The enzyme is predominantly expressed in lymphoid tissue by as yet poorly defined mechanisms. In this work, we have studied the mouse dCK regulatory region to understand the molecular details of the tissue specific expression of the enzyme. DNase I footprinting and electrophoretic mobility shift assays using nuclear extracts from mouse lymphocytes (EL-4, T cells; J558, B cells) and non-lymphoid cells (L929, fibroblasts) demonstrated the existence of at least six cis-acting elements (FP-1–FP-6) within the proximal promoter region. Functional analysis revealed that all the elements necessary to promote high level transcription of the mdCK gene are located downstream the transcription start site. 5′-Deletion and site-directed mutagenesis assays demonstrated the importance of four GC-rich regions, which bind Sp-1 and Sp-3 transcription factors. In addition, we identified a site (FP-3) located at the −282 to −310 nucleotide region of the promoter, which binds NF-1, only in B cells. Analysis of point mutations introduced at the different regions revealed functional differences in their role in mdCK transcription in the cell lines used.