Methylation of the asparagine synthetase promoter in human leukemic cell lines is associated with a specific methyl binding protein.
We have examined the methylation profiles of the asparagine synthetase (ASY) promoter in a number of human leukemic cell lines in relation to their asparagine (ASN) requirements in vitro. Cells in which the promoter is highly methylated are auxotrophs and express ASY at very low levels. Electromobility shift assays (EMSA) of nuclear extracts with oligomers from the promoting region show, in addition to recognized transcription factor binding, a novel methyl binding protein specific for a 12 base consensus sequence, which includes a single methylated CpG. This sequence overlaps that of the amino-acid response unit of the ASY promoter, which is activated byATF4 and C/EBP. Competition by the methyl binding protein could account for the observed failure of the methylated promoter to bind these transcription factors and consequently, although other mechanisms can also be operative, for the specific repression of the gene. The ASY methyl binding protein (ASMB) is present in leukemic lymphoid and myeloid cells irrespective of their methylation status, and in normal lymphocytes after phytohemagglutinin stimulation. It has been purified by affinity chromatography and has a molecular size of 40 kDa in 10% SDS-polyacrylamide gels.
Ren
Y
Y
Department of Pathology, North Shore University Hospital, Manhasset, NY 11030, USA.
Roy
S
S
Ding
Y
Y
Iqbal
J
J
Broome
J D
JD
D001217:Aspartate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007938:Leukemia / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014157:Transcription Factors; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C066141:MBD1 protein, human; D014157:Transcription Factors; D001217:Aspartate-Ammonia Ligase