Methylation of the asparagine synthetase promoter in human leukemic cell lines is associated with a specific methyl binding protein

Abstract

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.

Epigenetic changes in the repression and induction of asparagine synthetase in human leukemic cell lines

“…12 This was not derived from the supporting cells. Not only were these firmly attached to the glass and therefore rare in the suspensions used, but the measurement of ASY expression by RT-PCR employed primers specific for human cDNA.…”

“…Although in initial studies both wild-type 1929 and 1873 were fully methylated in the ASY promoting region, 12 after coculture for 30 or more days, methylation was largely lost and individual DNA clones showed only scanty and inconstant methylation sites (Figure 4). Importantly, these changes occurred in an indistinguishable way in 1929 and 1873 cocultures whether or not the medium in which they had been established was supplemented with ASN ( Figure 4c and d).…”

“…The cells used have been described earlier, 12,16 the first, designated 1873 has B-cell and myleoid characteristics and t(4;11) (q21;q23), the other, 1929, expresses B-cell markers and has an anomalous t(9;22) (q34;q11). These cells grow in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and ASN, 50 mg/l.…”

“…12 The promoting region of p16 was amplified as described by Coombes et al, 17 using the primers 5 0 -GCGCTAGATTCCAATTC-3 0 and 5 0 -GCAGCATGGAGCCTTCG GCTGAC-3 0 . ChIP grade antibodies to histone H3 diacetyl K9 and H3 dimethyl K4 were obtained from Upstate Biotechnology, Lake Placid, NY, USA and to H3 dimethyl K9 from Abcam Inc., Cambridge, MA, USA.…”

“…In these and in other lymphoid cells, a specific methyl-binding protein was identified whose binding motif overlapped the ASY response element so that transcription factor binding to oligomers containing this site was inhibited. 12 Further experiments, to be described in this paper, show that although methylation may have a role in inhibiting ASY in leukemic cells, and certainly expression is induced by 5-aza-2 0 deoxycytidine, 12 silencing occurs at multiple levels and methylation is not the sole, or necessarily the primary, component.…”