Analysis of protein binding in the MLL translocation breakpoint region.
Much sequence and structure analysis has been done to characterize the MLL bcr to elucidate the mechanism of MLL translocation. Various studies have shown evidence for potential recombination mediating features, including a DNA topoisomerase II cleavage site, a DNase I hypersensitivity site and recombination signal sequences for V(D)J gene segment joining. In addition, the region is rich in repetitive DNA with several Alu repeats which could facilitate homologous recombination with loci throughout the genome.
However, little is known about the protein-binding characteristics of this region. We have begun analyzing protein binding in the MLL bcr using electrophoretic mobility shift assay (EMSA). Detection of differential protein binding potential within the bcr may provide insight into the regions of the MLL bcr which are necessary for recombination with other loci. More important, eventual identification of specific MLL bcr binding proteins would provide essential clues to the mechanism of aberrant recombination involving MLL.
Methods: Nuclear extracts from hematopoietic and non-hematopoietic cells are incubated with labeled double stranded oligonucleotides from 50-200 bp in length in EMSA binding buffer. Binding of proteins to specific MLL bcr oligonucleotides is indicated by retarded migration in 6% polyacrylamide gels relative to oligonucleotide migration in the absence of proteins. Specific binding is shown by competition with unlabeled oligonucleotides.
Results: Binding of nuclear protein(s) has been detected in the extreme 5' end of the 8 kb MLL bcr. The binding is sequence specific as shown by competition with unlabeled oligonucleotide of the same sequence. Binding was seen with nuclear extracts from two hematopoietic cell lines, REH and Jurkat, but not with a purified control protein (OCT2 transcription factor). The degree of retardation of migration indicates a possible protein complex binding the 5' oligonucleotide. Other regions of the MLL bcr, including the extreme 3' boundary of the MLL bcr and a region described as an in vivo DNA topoisomerase II cleavage site were did not show retarded migration in EMSA with REH or Jurkat nuclear extracts.
Conclusions: We have identified one apparent region of protein binding
within the MLL bcr. The region marks the extreme 5' boundary of the bcr and may be relevant in the formation of MLL translocations. Experiments are underway to narrow the 200-bp region of binding by using small overlapping oligonucleotides from the region and to test binding with nuclear extracts from non-hematopoietic cell lines. Ultimately protein binding will be compared throughout the MLL bcr with the final goal of identifying potential recombination-mediating nuclear proteins.
Heidi J. Super, Alysa L. Anderson and Aileen Aldrich
Department of Biology, Minot State University, Minot ND 58707
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|Author:||Super, Heidi J.; Anderson, Alysa L.; Aldrich, Aileen|
|Publication:||Proceedings of the North Dakota Academy of Science|
|Date:||Apr 1, 2007|
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