A reliable method to display authentic DNase I hypersensitive sites at long-ranges in single-copy genes from large genomes

Nucleic Acids Research
Matthew E Pipkin, M G Lichtenheld

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.

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Citations

Jan 14, 2009·The Journal of Experimental Medicine·Fernando Cruz-GuillotyAnjana Rao
Feb 16, 2013·Nucleic Acids Research·Erbay YigitMatthew E Pipkin
Feb 6, 2009·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Sean A PintchovskiSteven Finkbeiner
Feb 27, 2008·Immunology·Matthew E Pipkin, Silvia Monticelli
Jun 12, 2010·Immunological Reviews·Matthew E PipkinMathias G Lichtenheld
Jan 16, 2007·Immunity·Matthew E PipkinMathias G Lichtenheld
Dec 17, 2008·Molecular BioSystems·Andreas Wagner
Aug 30, 2008·Molecular Genetics and Genomics : MGG·Andreas Wagner, Nicole de la Chaux
Sep 12, 2020·Proceedings of the National Academy of Sciences of the United States of America·Livia OdagiuNathalie Labrecque
Oct 22, 2020·Nature Communications·Giulia MazzuccoYlli Doksani

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Datasets Mentioned

BETA
GM11688
BC040163
ADAMTS-14

Methods Mentioned

BETA
PCR
electrophoresis
size exclusion chromatography
gel
transgenic
MDHA

Software Mentioned

FIGE
MDHA
RepeatMasker
OLIGO
ImageQuant
VISTA

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