title: A reliable method to display authentic DNase I hypersensitive sites at long-ranges in single-copy genes from large genomes.
abstract: The study of eukaryotic gene transcription depends on methods to discover distal cis-acting control sequences. Comparative bioinformatics is one powerful strategy to reveal these domains, but still requires conventional wet-bench techniques to elucidate their specificity and function. The DNase I hypersensitivity assay (DHA) is also a method to identify regulatory domains, but can also suggest their function. Technically however, the classical DHA is constrained to mapping gene loci in small increments of approximately 20 kb. This limitation hinders efficient and comprehensive analysis of distal gene regions. Here, we report an improved method termed mega-DHA that extends the range of existing DHAs to facilitate assaying intervals that approach 100 kb. We demonstrate its feasibility for efficient analysis of single-copy genes within a large and complex genome by assaying 230 kb of the human ADAMTS14-perforin-paladin gene cluster in four experiments. The results identify distinct networks of regulatory domains specific to expression of perforin and its two neighboring genes.
authors.affiliation: Department of Microbiology and Immunology, University of Miami, Miller School of Medicine, Miami, FL 33136, USA.
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Pipkin
authors.forename: Mathias G
authors.initials: MG
authors.lastname: Lichtenheld
mesh_terms: D000818:Animals; D002460:Cell Line; D002874:Chromosome Mapping; D002879:Chromosomes, Human, Pair 10; D003850:Deoxyribonuclease I; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D018628:Gene Dosage; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D050436:Regulatory Elements, Transcriptional; D014158:Transcription, Genetic
chemical_list: D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; D003850:Deoxyribonuclease I