ABSTRACT
Combinatorial binding of transcription factors to regulatory DNA underpins gene regulation in all organisms. Genetic variation in regulatory regions has been connected with diseases and diverse phenotypic traits1, but it remains challenging to distinguish variants that affect regulatory function2. Genomic DNase I footprinting enables the quantitative, nucleotide-resolution delineation of sites of transcription factor occupancy within native chromatin3-6. However, only a small fraction of such sites have been precisely resolved on the human genome sequence6. Here, to enable comprehensive mapping of transcription factor footprints, we produced high-density DNase I cleavage maps from 243 human cell and tissue types and states and integrated these data to delineate about 4.5 million compact genomic elements that encode transcription factor occupancy at nucleotide resolution. We map the fine-scale structure within about 1.6 million DNase I-hypersensitive sites and show that the overwhelming majority are populated by well-spaced sites of single transcription factor-DNA interaction. Cell-context-dependent cis-regulation is chiefly executed by wholesale modulation of accessibility at regulatory DNA rather than by differential transcription factor occupancy within accessible elements. We also show that the enrichment of genetic variants associated with diseases or phenotypic traits in regulatory regions1,7 is almost entirely attributable to variants within footprints, and that functional variants that affect transcription factor occupancy are nearly evenly partitioned between loss- and gain-of-function alleles. Unexpectedly, we find increased density of human genetic variation within transcription factor footprints, revealing an unappreciated driver of cis-regulatory evolution. Our results provide a framework for both global and nucleotide-precision analyses of gene regulatory mechanisms and functional genetic variation.
Subject(s)
DNA Footprinting/standards , Genome, Human/genetics , Transcription Factors/metabolism , Consensus Sequence , DNA/genetics , DNA/metabolism , Deoxyribonuclease I/metabolism , Genetics, Population , Genome-Wide Association Study , Humans , Models, Molecular , Polymorphism, Single Nucleotide , Regulatory Sequences, Nucleic Acid/geneticsABSTRACT
We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how to measure the degree of chromatin assembly in the absence and presence of histone H1 using topological analysis and how to perform micrococcal nuclease digestion to confirm H1 incorporation and determine the quality of in vitro chromatin templates. Further, we describe the use of sucrose gradient ultracentrifugation to verify that no unincorporated H1 remains as a second means for deciding on the proper H1 to core histone ratio during assembly. Additionally, we discuss the use of both yeast and Drosophila NAP-1 (yNAP-1 and dNAP-1, respectively) in the assembly of H1-containing chromatin. Finally, we provide detailed description of functional assays for investigating the mechanism of transcriptional regulation in a chromatin context (transcription, histone acetyltransferase activity, and protein association with promoter-bound complexes using immobilized chromatin templates).
Subject(s)
Chromatin Assembly and Disassembly/genetics , Drosophila Proteins/genetics , Histones/genetics , Molecular Biology/methods , Transcription, Genetic/genetics , Animals , Baculoviridae , DNA Footprinting/methods , DNA Footprinting/standards , Drosophila Proteins/isolation & purification , Drosophila Proteins/metabolism , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Histones/isolation & purification , Histones/metabolism , Molecular Biology/standards , Molecular Chaperones/genetics , Plasmids/genetics , Quality Control , Ultracentrifugation/methods , Ultracentrifugation/standards , Viral Plaque Assay/standards , Yeasts/geneticsABSTRACT
Hydroxyl radical footprinting can probe the solvent accessibility of the ribose moiety of the individual nucleotides of DNA and RNA. Semi-automated analytical tools are presented for the quantitative analyses of nucleic acid footprint transitions in which processes such as folding or ligand binding are followed as a function of time or ligand concentration. Efficient quantitation of the intensities of the electrophoretic bands comprising the footprinting reaction products is achieved by fitting a series of Lorentzian curves to line profiles obtained from gels utilizing sequentially relaxed constraints consistent with electrophoretic mobility. An automated process of data 'standardization' has been developed that corrects for differences in the loading amounts in the electrophoresis. This process enhances the accuracy of the derived transitions and makes generating them easier. Together with visualization of the processed footprinting in false-color two-dimensional maps, DNA and RNA footprinting data can be accurately, precisely and efficiently processed allowing transitions to be objectively and comprehensively analyzed. The utility of this new analysis approach is illustrated by its application to the ion-meditated folding of a large RNA molecule.
Subject(s)
DNA Footprinting/methods , Hydroxyl Radical/chemistry , Molecular Probe Techniques , RNA/chemistry , Autoradiography , DNA Footprinting/standards , Molecular Probe Techniques/standards , Molecular Probes , Nucleic Acid Conformation , RNA, Catalytic/chemistryABSTRACT
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