Transcriptional regulation of the cystathionine-β-synthase gene in Down syndrome and non–Down syndrome megakaryocytic leukemia cell lines

Abstract

Children with Down syndrome (DS) with acute myeloid leukemia (AML) have significantly higher event-free survival rates compared to those with non-DS AML, linked to greater cytosine arabinoside (ara-C) sensitivity and higher transcript levels of the chromosome 21–localized gene, cystathionine-β-synthase(CBS), in DS myeloblasts. In this study, we examined the transcriptional regulation of the CBS gene in the DS megakaryocytic leukemia (AMkL) cell line, CMK, characterized by significantly higher CBS transcripts compared with the non-DS AMkL cell line, CMS. Rapid amplification of 5′-cDNA ends (5′-RACE) analysis demonstrated exclusive use of the CBS−1b promoter in the cell lines, and transient transfections with the full-length CBS −1b luciferase reporter gene construct showed 40-fold greater promoter activity in the CMK than CMS cells. Electrophoretic mobility shift assays showed enhanced binding of the transcription factors Sp1/Sp3 to 2 GC/GT-box elements (GC-f and GT-d) in the upstream regions of the CBS −1b promoter in CMK nuclear extracts and undetectable binding in CMS cells. Mutation of the GC-f– or GT-d–binding site resulted in an approximately 90% decrease of theCBS −1b promoter activity in transient transfections of CMK cells. Chromatin immunoprecipitation assays confirmed in vivo binding of Sp3, USF-1, and nuclear factor YA (NF-YA) to theCBS −1b promoter region in chromatin extracts of CMK and CMS cells. Decreased binding of Sp1/Sp3 in CMK nuclear extracts following treatment with calf alkaline phosphatase suggested a role for phosphorylation of Sp1/Sp3 in regulating CBS promoter activity and in the differential CBS expression between CMK and CMS cells. The results of this study with clinically relevant cell line models suggest potential mechanisms for disparate patterns ofCBS gene expression in DS and non-DS myeloblasts and may, in part, explain the greater sensitivity to chemotherapy shown by patients with DS AML.