We have applied epigenomic profiling to the classical salt fractionation method. Salt competes for interactions between the highly basic histone core and highly acidic DNA, and so salt-solubility measures a nucleosomal physical property. Chromatin fractions extracted with low salt after micrococcal nuclease digestion contain predominantly mononucleosomes and represent classical 'active' chromatin. We found that profiles of these low-salt soluble fractions displayed phased nucleosomes over transcriptionally active genes and correspond closely to profiles of the H2A replacement histone, H2A.Z
Another technology that we have introduced addresses the requirement for abundant cell-type-specific chromatin from tissues for epigenomic studies. A nuclear envelope protein is expressed under control of a cell-type-specific promoter, and in vivo biotin labeling is followed by affinity isolation of labeled nuclei to rapidly obtain large quantities of pure nuclei. We have applied this method to measure gene expression and chromatin features of the hair and non-hair cell types of the Arabidopsis root epidermis. We identified hundreds of genes that are preferentially expressed in each cell type and found that genes with the largest expression differences between hair and non-hair cells also show differences between cell types in H3K4me3 and H3K27me3. Our method should be applicable to any organism that is amenable to transformation.
We have also introduced a metabolic labeling strategy to obtain a more direct measure of nucleosome disruption genome-wide. Newly synthesized proteins are labeled with an amino acid analog, derivatized with a biotin moiety, nucleosome core particles are selectively extracted and affinity purified with streptavidin, and DNA is extracted for genome-wide profiling. We have successfully obtained genome-wide nucleosome turnover profiles for Drosophila cultured cells, and we have used these data to address the relationship between histone turnover and fundamental processes, including transcriptional initiation and elongation, epigenetic regulation and replication origin activity.
We have modified Chromatin Endogenous Cleavage (ChEC) for a DNA sequencing read-out (ChEC-seq). ChEC uses fusion of a protein of interest to MNase to target calcium-inducible cleavage in intact cells. Acquisition of ChEC-seq data on a seconds-to-minutes time-scale revealed two classes of sites for yeast TFs, one displaying rapid cleavage close to one side of consensus motifs and the second showing slow cleavage at non-motif sites. Remarkably, fast and slow sites showed nearly identical DNA shape (minor-groove width, helical twist, propeller twist and roll) profiles, which implies that time-resolved ChEC-seq detects both high-affinity interactions of TFs with consensus motifs and low-affinity sites preferentially sampled by TFs during scanning for DNA shape features. ChEC-seq is a simple, efficient method with high spatio-temporal resolution and orientation sensitivity that we anticipate will be broadly applicable for genome-wide profiling of protein-DNA dynamics.
We found that combining short read sequencing technology with native chromatin preparation for chromatin immune-precipitation could improve the resolution and dynamic range of epigenome mapping over protocols using formaldehyde cross-linking and sonication. Using this method, we have been able to map centromeric nucleosomes, chromatin remodelers and even transcription factors with greater sensitivity and accuracy.
To understand where RNA polymerase II is located on chromosomes, we took advantage of the fact that engaged RNAPII is insoluble and stable to develop a simple method for determining the position of the last nucleotide added to a growing nascent transcript, regardless of whether RNAPII is actively transcribing or stalled and backtracked. We used this method to determine that the first (+1) nucleosome at a transcription start site is a barrier to transcription in essentially all genes, while subsequent nucleosomes present less of a barrier, and H2A.Z incorporation appears to lower the barrier to transcription.
This method uses the intercalating agent trimethylpsoralen (TMP) to covalently crosslink both DNA strands at sites of supercoiling, then precisely identifies the cross-linked sites. Using this method, we have shown that torsion correlates with gene transcription, and inhibition of topoisomerases leads to rapid accumulation of torsional strain, which is accompanied by changes in RNAPII kinetics and chromatin properties.
Formaldehyde cross-linking ChIP protocols have typically used sonication to fragment the DNA, but this process is non-random and typically yields fragment of 200-500 bp, giving poor resolution for transcription factors and other chromatin proteins. We have achieved base-pair level resolution mapping of PolII and chromatin remodelers by using micrococcal nuclease with cross-linking.
The nucleosomal barrier to promoter escape by RNA polymerase II is overcome by the chromatin remodeler Chd1
Mapping In vivo Nascent Chromatin with EdU and sequencing (MINCE-seq) is a metabolic labeling method to characterize the genome-wide location of nucleosomes and other chromatin proteins behind replication forks at high temporal and spatial resolution. After a pulse of the thymidine analog ethynyl deoxyuridine (EdU), cells are cross-linked with formaldehyde, permeabilized and subjected to “Click” chemistry to attach a biotin tag to the incorporated base analog. Chromatin is solubilized, fragmented using micrococcal nuclease, and the newly replicated DNA fragments are extracted and separated from the bulk DNA using streptavidin beads. After paired-end sequencing, DNA fragments are mapped and classified as long fragments (nucleosomes) and short fragments (mostly transcription factors) to provide a high-resolution landscape of pulse-labeled chromatin.
Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an antibody-targeted chromatin profiling method in which micrococcal nuclease tethered to protein A binds to an antibody of choice and cuts immediately adjacent DNA, releasing DNA bound to the antibody target. The procedure is carried out in situ and produces precise transcription factor or histone modification profiles while avoiding crosslinking and solubilization issues. Extremely low backgrounds make profiling possible with typically one tenth of the sequencing depth required for ChIP, and permit profiling using low cell numbers without loss of quality. CUT&RUN can also be used to map long-range genomic contacts. Protocols and reagents are available on request.
We developed a chemical cleavage method that releases single nucleosome dyad-containing fragments, allowing us to precisely map both single nucleosomes and linkers with high accuracy genome-wide in yeast. Our single nucleosome positioning data reveal that nucleosomes occupy preferred positions that differ by integral multiples of the DNA helical repeat. By comparing nucleosome dyad positioning maps to existing genomic and transcriptomic data, we evaluated the contributions of sequence, transcription, and histones H1 and H2A.Z in defining the chromatin landscape. We present a biophysical model that neglects DNA sequence and shows that steric occlusion suffices to explain the salient features of nucleosome positioning.