Allele-specific analysis of transcription factors binding to promoter regions.
In vivo footprinting techniques are useful for the identification of regulatory elements mediating transcriptional control of a gene. However, regulation of a gene can differ between the two alleles, and further steps must be taken to distinguish between the regulatory elements occupied on one allele and those used on the second allele. Many hematologic malignancies result from chromosomal translocations, which, in some cases, relocate a gene to a transcriptionally active region leading to the deregulated expression of that gene. This situation provides an example of differential expression between two alleles. In studying the t(14; 18) and t(8; 14) translocations, which involve the bcl-2 and c-myc proto-oncogenes, respectively, we have been able to identify regulatory elements important in mediating the activation of the translocated alleles and the silencing of the normal alleles. Following in vivo methylation and isolation of genomic DNA, we were able to separate the translocated and normal alleles by electrophoresis. Using the ligation-mediated polymerase chain reaction (LMPCR) technique, we could then assess protein interactions on the two different alleles. A detailed description of this methodology with examples from our studies are provided with a discussion of how these techniques may be applied to the study of other genes.
Heckman
Caroline A
CA
Center for Molecular Biology in Medicine, Veterans Affairs Palo Alto Health Care System and the Department of Medicine, Stanford University School of Medicine, CA 94305, USA.
Boxer
Linda M
LM
D000483:Alleles; D015139:Blotting, Southern / Q000379:methods; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D019254:Genes, bcl-2 / Q000502:physiology; D016259:Genes, myc / Q000502:physiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA