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.
A simple method for gene expression and chromatin profiling of individual cell types within a tissue
The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana
Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling
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.
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.
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.
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.
Mapping regulatory factors by immunoprecipitation from native chromatin
High-resolution mapping of transcription factor binding sites on native chromatin
Mot1 redistributes TBP from TATA-containing to TATA-less promoters
ISWI and CHD chromatin remodelers bind promoters but act in gene bodies
Tripartite organization of centromeric chromatin in budding yeast
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
A simple method for generating high-resolution maps of genome-wide protein binding
Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono-nucleosomes, whereas cleavages within nucleosomes destroy them. Here, we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time course. As with human cells, we observe no overall differences in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.
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.
Methidiumpropyl-EDTA Fe(II) sequencing (MPE-seq) is a chemical cleavage method that intercalates into DNA and cleaves phosphodiester bonds which can be used similarly to MNase-seq but without exonuclease activity or AT bias. We used MPE-seq together with MNASE-seq to confirm that nucleosomes in Marseillevirus wrap 121 bp and are closely abutted without linkers or phasing over genes.
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.
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. CUT&RUN can be combined with salt fractionation (CUT&RUN.Salt) to profile different chromatin fractions based on solubility, or combined with immunoprecipitation to characterize protein components of chromatin complexes released by CUT&RUN (CUT&RUN.ChIP).
An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites
Targeted in situ genome-wide profiling with high efficiency for low cell numbers
Unexpected conformational variations of the human centromeric chromatin complex
RSC-Associated subnucleosomes define MNase-sensitive promoters in yeast
CUT&RUN with Drosophila
We have also constructed a 6xHis and HA-tagged protein A-protein G-MNase fusion (pAG-MNase) that allows direct binding of mouse antibodies that bind poorly to protein A, eliminating the need for a secondary antibody. The His tag allows purification of pAG-MNase with a commercial kit, while the HA tag can be used for pulling out pAG-MNase chromatin complexes for CUT&RUN.ChIP. We have also constructed a 6xHis and HA-tagged protein A-protein G-MNase fusion (pAG-MNase) that allows direct binding of mouse antibodies that bind poorly to protein A, eliminating the need for a secondary antibody. The His tag allows purification of pAG-MNase with a commercial kit, while the HA tag can be used for pulling out pAG-MNase chromatin complexes for CUT&RUN.ChIP. We developed low salt, high calcium conditions that prevent diffusion of released complexes into the supernatant, allowing for longer digestion times and increased yields without increased cleavage at non-specific accessible sites. E. coli DNA carried over from pA-MNase or pAG-MNase preparation is sufficient to provide internal calibration of samples without adding heterologous spike-in DNA. We also developed Sparse Enrichment Analysis for CUT&RUN (SEACR), a peak-calling algorithm that takes advantage of position and fragment spanning information in CUT&RUN data to improve peak-calling and domain detection.
SEACR is a peak-calling algorithm that takes advantage of position and fragment spanning information in CUT&RUN data to improve peak-calling and domain detection. SEACR is available as a web server.
CUT&RUN has been automated using a Beckman Biomek FX liquid-handling robot so that a 96 well format can be used to profile chromatin for high-throughput samples such as in a clinical setting. DNA end polishing and direct ligation of adaptors permits sample-to-Illumina library processing of 96 samples in two days. AutoCUT&RUN can be used for cell-type specific gene activity and enhancer profiling based on histone modifications and transcription factors, including in frozen tissue samples of tumor xenografts.
To distinguish presumed architectural roles of RNA from other functions, we applied a ribonuclease digestion strategy to our CUT&RUN in situ chromatin profiling method (CUT&RUN.RNase). We find that depletion of RNA compromises association of the murine nucleolar protein Nucleophosmin with pericentric heterochromatin and alters the chromatin environment of CCCTC-binding factor (CTCF) bound regions. Strikingly, we find that RNA maintains the integrity of both constitutive (H3K9me3 marked) and facultative (H3K27me3 marked) heterochromatic regions as compact domains, but only moderately stabilizes euchromatin.
Like CUT&RUN, CUT&Tag (Cleavage Under Targets and Tagmentation) is an enzyme tethering approach to profiling chromatin proteins, including histone marks, transcription factors and RNA Pol II. CUT&Tag generates sequence-ready libraries without the need for end polishing and adaptor ligation. It uses a proteinA-Tn5 fusion to tether Tn5 transposase near the site of an antibody to a chromatin protein of interest. A secondary antibody (such as guinea pig anti-rabbit) is used to increase the efficiency of tethering the pA-Tn5 to the target primary antibody. The pA-Tn5 complex is pre-loaded with sequencing adaptors that insert into adjacent DNA upon activation with magnesium. CUT&Tag has very low background and can be performed in a single tube in as little as a day, though more typically primary antibodies are incubated overnight. It can also be used with the ICELL8 nanodispensation system to profile single cells.
We introduce a streamlined CUT&Tag protocol that suppresses DNA accessibility artefacts to ensure high-fidelity mapping of the antibody-targeted protein and improves the signal-to-noise ratio over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube, from cells to amplified libraries, providing low-cost genome-wide chromatin maps. By simplifying library preparation CUT&Tag-direct requires less than a day at the bench, from live cells to sequencing-ready barcoded libraries. As a result of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for as little as $25 per sample. This enables routine genome-wide profiling of chromatin proteins and modifications and requires no special skills or equipment.
We used CUT&Tag on Drosophila tissues to show that the oncohistone H3K27M, which dominantly inhibits H3K27 trimethylation, prevents developmental reprogramming by the wing-specific master regulator Vestigial (Vg) resulting in overgrowth of eye tissue, suggesting that growth dysregulation can result from the simple combination of crippled silencing and transcription factor mis-expression, an effect that may explain the origins of oncohistone-bearing cancers.
Single-cell analysis has become a powerful approach for the molecular characterization of complex tissues. Methods for quantifying gene expression and chromatin accessibility of single cells are now well-established, but analysis of chromatin regions with specific histone modifications has been technically challenging. We adapt CUT&Tag to scalable single-cell platforms to profile chromatin landscapes in single cells (scCUT&Tag) from complex tissues. We focus on profiling Polycomb Group (PcG) silenced regions marked by H3K27 trimethylation (H3K27me3) in single cells as an orthogonal approach to chromatin accessibility for identifying cell states. We show that scCUT&Tag profiling of H3K27me3 distinguishes cell types in human blood and allows the generation of cell-type-specific PcG landscapes from heterogeneous tissues. Furthermore, we use scCUT&Tag to profile H3K27me3 in a brain tumor patient before and after treatment, identifying cell types in the tumor microenvironment and heterogeneity in PcG activity in the primary sample and after treatment
Although the in vitro structural and in vivo spatial characteristics of transcription factor (TF) binding are well defined, TF interactions with chromatin and other companion TFs during development are poorly understood. To analyze such interactions in vivo, we profiled several TFs across a time course of human embryonic stem cell differentiation and studied their interactions with nucleosomes and co-occurring TFs by enhanced chromatin occupancy (EChO), a computational strategy for classifying TF interactions with chromatin. EChO shows that multiple individual TFs can employ either direct DNA binding or "pioneer" nucleosome binding at different enhancer targets.
For more than 40 years, chromatin hyperaccessibility mapping to identify regulatory elements has been performed using a variety of unrelated methods, with Tn5 transposase-mediated accessibility mapping emerging as the preferred method for most genome-wide applications. However, the mechanistic basis that underlies hyperaccessibility is uncertain. We describe a simple modification of our Tn5 transposase-mediated antibody-directed CUT&Tag method that provides high-quality accessibility mapping in parallel with mapping of specific components of the chromatin landscape. Our findings imply that regulatory sites detected by hyperaccessibility mapping are coupled to initiation of RNA Polymerase II transcription via H3K4 methylation. We expect that our Cleavage Under Targeted Accessible Chromatin (CUTAC) method will become highly popular, as it requires few resources and is sufficiently simple that it can be performed from nuclei to purified sequencing-ready libraries in single PCR tubes on a home workbench.
Technological progress in single-cell read-out technologies has fueled interest in “Multi-OMICs” where two different modalities, such as RNA-seq and ATAC-seq are performed in the same cells. Pol2S5p-CUTAC releases mostly subnucleosome-sized fragments corresponding to peaks of TF binding at promoters and enhancers, whereas H3K27me3-marked nucleosomes are in broad domains. We have taken advantage of these differences to use two antibodies for CUT&Tag simultaneously and then use a Bayesian deconvolution strategy to computationally separate active regulatory sites from developmentally silenced Polycomb domains based on fragment size and feature width, thereby profiling the active and repressive regulomes in the same single cells.
Multiple Targets Identified via Tagmentation (MulTI-Tag) is a CUT&Tag-based approach that uses identifying barcodes to profile multiple chromatin-associated proteins in the same individual cells. MulTI-Tag is as efficient as single-antibody CUT&Tag both in bulk and in single cells and represents a landmark advance in single cell chromatin profiling.
CUT&Tag has been automated using a Beckman Coulter Biomek FX liquid handling robot so that a 96 well format can be used to profile chromatin for high-throughput samples such as in a clinical setting. We used AutoCUT&Tag to profile the gene targets of fusions of the KMT2A lysine methyltransferase to other chromatin proteins, which characterize lymphoid, myeloid, and mixed lineage leukemias, uncovering heterogeneities that may underlie lineage plasticity.
We adapted CUT&Tag to profile RNA associated with chromatin epitopes by using a secondary antibody conjugated to streptavidin with a biotinylated oligo-dT fused to an adapter and protein A-Tn5 loaded with a second adapter, then performing reverse transcription and tagmentation of RNA-DNA hybrids simultaneously. Reverse transcribe and tagment (RT&Tag) is suitable for a variety of applications including detecting the Drosophila roX2 RNA associated with the male dosage compensation complex, RNAs associated with Polycomb complex (H3K27me3) domains, and N6-methyladenine-modifed mRNAs.