RNA Polymerase II hypertranscription at histone genes in cancer FFPE samples.

Genome-wide hypertranscription is common in human cancer and predicts poor prognosis. To understand how hypertranscription might drive cancer, we applied our FFPE-CUTAC method for mapping RNA Polymerase II (RNAPII) genome-wide in formalin-fixed paraffin-embedded (FFPE) sections. We demonstrate global RNAPII elevations in mouse gliomas and assorted human tumors in small clinical samples and discover regional elevations corresponding to HER2 amplifications punctuated by likely selective sweeps.

A giant virus genome is densely packaged by stable nucleosomes within virions.

The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 base pairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage, and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA-wrapped cores without linker DNA or phasing along genes.

Simplified Epigenome Profiling Using Antibody-tethered Tagmentation.

We previously introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling method in which antibody tethering of the Tn5 transposase to a chromatin epitope of interest maps specific chromatin features in small samples and single cells. With CUT&Tag, intact cells or nuclei are permeabilized, followed by successive addition of a primary antibody, a secondary antibody, and a chimeric Protein A-Transposase fusion protein that binds to the antibody. Addition of Mg activates the transposase and inserts sequencing adapters into adjacent DNA .

Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation.

Chromatin accessibility mapping is a powerful approach to identify potential regulatory elements. A popular example is ATAC-seq, whereby Tn5 transposase inserts sequencing adapters into accessible DNA ('tagmentation'). CUT&Tag is a tagmentation-based epigenomic profiling method in which antibody tethering of Tn5 to a chromatin epitope of interest profiles specific chromatin features in small samples and single cells.

Efficient low-cost chromatin profiling with CUT&Tag.

We recently introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei. These antibodies are then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds adapters ('tagmentation') for paired-end DNA sequencing.

Histone deposition pathways determine the chromatin landscapes of H3.1 and H3.3 K27M oncohistones.

Lysine 27-to-methionine (K27M) mutations in the H3.1 or H3.3 histone genes are characteristic of pediatric diffuse midline gliomas (DMGs).

Improved CUT&RUN chromatin profiling tools.

Previously, we described a novel alternative to chromatin immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a protein A-Micrococcal Nuclease (pA/MNase) fusion protein (Skene and Henikoff, 2017). Here we introduce three enhancements to CUT&RUN: A hybrid protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification, a modified digestion protocol that inhibits premature release of the nuclease-bound complex, and a calibration strategy based on carry-over of DNA introduced with the fusion protein. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

CUT&Tag for efficient epigenomic profiling of small samples and single cells.

Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components.

Targeted in situ genome-wide profiling with high efficiency for low cell numbers.

Cleavage under targets and release using nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely used chromatin immunoprecipitation (ChIP) protocols in resolution, signal-to-noise ratio and depth of sequencing required.

A unique chromatin complex occupies young α-satellite arrays of human centromeres.

The intractability of homogeneous α-satellite arrays has impeded understanding of human centromeres. Artificial centromeres are produced from higher-order repeats (HORs) present at centromere edges, although the exact sequences and chromatin conformations of centromere cores remain unknown. We use high-resolution chromatin immunoprecipitation (ChIP) of centromere components followed by clustering of sequence data as an unbiased approach to identify functional centromere sequences.

The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo.

In budding yeast, a single cenH3 (Cse4) nucleosome occupies the ∼120-bp functional centromere, however conflicting structural models for the particle have been proposed. To resolve this controversy, we have applied H4S47C-anchored cleavage mapping, which reveals the precise position of histone H4 in every nucleosome in the genome. We find that cleavage patterns at centromeres are unique within the genome and are incompatible with symmetrical structures, including octameric nucleosomes and (Cse4/H4)2 tetrasomes.

The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres.

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core.

"Point" centromeres of Saccharomyces harbor single centromere-specific nucleosomes.

The "point" centromere of budding yeast is genetically defined by an ≈ 125-bp sequence. Recent fluorescence measurements of kinetochore clusters have suggested that this sequence specifies multiple centromere histone 3 (CenH3) nucleosomes. However, high-resolution mapping demonstrates that there is only one CenH3 nucleosome per centromere, providing biochemical confirmation of the point centromere model.

Tripartite organization of centromeric chromatin in budding yeast.

The centromere is the genetic locus that organizes the proteinaceous kinetochore and is responsible for attachment of the chromosome to the spindle at mitosis and meiosis. In most eukaryotes, the centromere consists of highly repetitive DNA sequences that are occupied by nucleosomes containing the CenH3 histone variant, whereas in budding yeast, a ∼120-bp centromere DNA element (CDE) that is sufficient for centromere function is occupied by a single right-handed histone variant CenH3 (Cse4) nucleosome. However, these in vivo observations are inconsistent with in vitro evidence for left-handed octameric CenH3 nucleosomes.

Epigenome characterization at single base-pair resolution.

We have combined standard micrococcal nuclease (MNase) digestion of nuclei with a modified protocol for constructing paired-end DNA sequencing libraries to map both nucleosomes and subnucleosome-sized particles at single base-pair resolution throughout the budding yeast genome. We found that partially unwrapped nucleosomes and subnucleosome-sized particles can occupy the same position within a cell population, suggesting dynamic behavior. By varying the time of MNase digestion, we have been able to observe changes that reflect differential sensitivity of particles, including the eviction of nucleosomes.

Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project.

We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor-binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs.

Identification of functional elements and regulatory circuits by Drosophila modENCODE.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction.

H2A.Z nucleosomes enriched over active genes are homotypic.

Nucleosomes that contain the histone variant H2A.Z are enriched around transcriptional start sites, but the mechanistic basis for this enrichment is unknown. A single octameric nucleosome can contain two H2A.

Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones.

Nucleosome disruption and replacement are crucial activities that maintain epigenomes, but these highly dynamic processes have been difficult to study. Here, we describe a direct method for measuring nucleosome turnover dynamics genome-wide. We found that nucleosome turnover is most rapid over active gene bodies, epigenetic regulatory elements, and replication origins in Drosophila cells.

A comprehensive map of insulator elements for the Drosophila genome.

Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP-chip the genome-wide binding sites of 6 insulator-associated proteins-dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF-to obtain the first comprehensive map of insulator elements in Drosophila embryos.

A native chromatin purification system for epigenomic profiling in Caenorhabditis elegans.

High-resolution mapping of chromatin features has emerged as an important strategy for understanding gene regulation and epigenetic inheritance. We describe an in vivo tagging system coupled to chromatin purification for genome-wide epigenetic profiling in Caenorhabditis elegans. In this system, we coexpressed the Escherichia coli biotin ligase enzyme (BirA), together with the C.

Genome-wide profiling of salt fractions maps physical properties of chromatin.

We applied genome-wide profiling to successive salt-extracted fractions of micrococcal nuclease-treated Drosophila chromatin. Chromatin fractions extracted with 80 mM or 150 mM NaCl after digestion contain predominantly mononucleosomes and represent classical "active" chromatin. Profiles of these low-salt soluble fractions display phased nucleosomes over transcriptionally active genes that are locally depleted of histone H3.

Using the blocks database to recognize functional domains.

Blocks are ungapped multiple alignments of related protein sequence segments that correspond to the most conserved regions of the proteins. The Blocks Database is a collection of blocks representing known protein families that can be used to compare a protein or DNA sequence with documented families of proteins. Protocols in this unit describe the analysis of proteins and families using Blocks-based tools, including searching, exploring relationships with trees, making new blocks, and designing PCR primers from blocks for isolating homologous sequences.

Histone replacement marks the boundaries of cis-regulatory domains.

Cellular memory is maintained at homeotic genes by cis-regulatory elements whose mechanism of action is unknown. We have examined chromatin at Drosophila homeotic gene clusters by measuring, at high resolution, levels of histone replacement and nucleosome occupancy. Homeotic gene clusters display conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement.