Innovative applications of CUT&RUN in five recent papers

In a previous blog post we discussed key papers describing the development of Cleavage Under Targets and Release Using Nuclease (CUT&RUN) , a breakthrough chromatin mapping technology that is rapidly replacing ChIP-seq as the leading approach in the field. CUT&RUN has the ability to profile diverse biological targets with exquisite sensitivity and low background.

EpiCypher’s leading CUTANA™ CUT&RUN Kit and user-friendly CUTANA CUT&RUN protocol have been validated for mapping histone post-translational modifications (PTMs), histone variants, and chromatin associated proteins – including transcription factors and regulatory complexes – from both cells and nuclei. Furthermore, CUTANA CUT&RUN assays are compatible with low inputs (down to 5,000 cells), various sample processing methods (fresh, fixed, frozen), and only require 3-8 milion reads per sample, representing major advances over ChIP-seq. Here, we highlight several recent, high-profile publications that used CUT&RUN to generate key findings in developmental biology and oncology research.

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Exciting Applications of CUT&RUN

Elevated NSD3 histone methylation activity drives squamous cell lung cancer

Gang Yuan et al. Nature, 2021. PMID: 33536620

CUT&RUN Targets: H3K36me2 and H3K27me3

Sample Type: Mouse-derived lung squamous cell carcinoma (LUSC) cell line

Significance: Lung squamous cell carcinoma (LUSC) is an aggressive form of lung cancer and a leading cause of cancer-related deaths worldwide. The genomic amplicon indicated in LUSC development (8p11-12) contains multiple candidate oncogenes, most notably FGFR1. However, drugs targeting FGFR1 have not been clinically viable, suggesting that other genes within the LUSC amplicon are promoting cancer development. Here, Yuan et al. study another candidate gene in this amplified region, the H3K36 methyltransferase NSD3. They use a series of mouse models, patient xenograft studies and biochemical assays to demonstrate NSD3 as a key oncogenic driver in LUSC. By identifying a direct NSD3-BRD4 interaction, this work also opens the possibility of using BET inhibitors to treat this cancer.

How was CUT&RUN used? The authors generated a mouse model of LUSC containing the 8p11-12 amplicon, with and without inducible expression of hyperactive NSD3. CUT&RUN was performed using EpiCypher CUTANA pAG-MNase to examine the genomic distribution of H3K36me2 and H3K27me3 as a complement to other in vitro and in vivo studies. Importantly, NSD3-catalyzed H3K36me2 is associated with actively transcribed gene bodies, while H3K27me3 is localized to repressed gene bodies. Thus, these PTMs were used to link hyperactive NSD3 with changes in chromatin states and gene expression patterns contributing to tumorigenesis.

BONUS: The authors also used EpiCypher recombinant nucleosomes to identify and characterize the LUSC-associated, hyperactive NSD3 mutant used in this study. Similar to NSD2, they observed NSD3 is only active on nucleosome substrates (vs. free histone H3).

Histone H1 loss drives lymphoma by disrupting 3D chromatin architecture

Nevin Yusufova et al. Nature, 2020. PMID: 33299181

CUT&RUN Targets: H3K9me2 and H3K9me3

Sample Type: Mouse germinal center B cells

Significance: Linker histone H1 proteins are transcriptional repressors that bind nucleosomal linker DNA to condense chromatin and restrict access to transcriptional activators. H1 mutations are highly associated with B-cell lymphomas, but their contribution to malignancy remains unclear. Yusufova et al. found that disruption of H1 led to genome-wide chromatin relaxation and re-expression of silenced developmental and/or stem cell genes, supporting increased cell proliferation and subsequent malignant transformation. These results demonstrate a novel tumor suppressor mechanism, in which H1 acts to silence stem cell-related gene programs by establishing and maintaining repressive chromatin compartments.

How was CUT&RUN used? The authors generated a histone H1c/H1e dual knockout mouse model and investigated the effect of H1 depletion on chromatin accessibility, PTM dynamics, and transcriptional regulation in germinal center B cells. As part of this work, EpiCypher scientists performed CUTANA CUT&RUN assays to map the distribution of H3K9me2 and me3, markers of dense, constitutive heterochromatin. In H1c/H1e knockout cells, H3K9 methylation was significantly reduced in areas that displayed increased chromatin accessibility, correlating with cancer development and supporting a tumor suppressor function for H1. Importantly, this study demonstrates the capability of pAG-MNase to access highly compact heterochromatin in a native conformation, including ENCODE-defined “blacklist” regions containing highly repetitive DNA.

Video Summary of Yusufova et al. 2020

DNA double-strand breaks induce H2AX phosphorylation domains in a contact-dependent manner

Patrick L. Collins et al. Nature Communications, 2020. PMID: 32572033

CUT&RUN Targets: γH2AX

Sample Type: Mouse thymocytes

Significance: The DNA damage response describes the various mechanisms by which DNA damage is detected and repaired to ensure genome integrity. One of the earliest markers of double strand break (DSB) formation and DNA damage response activation in cells is phosphorylation of H2AX (H2AXS139ph, or γH2AX), which forms larges domains of modified chromatin near the DSB site. γH2AX has numerous roles in recruiting DNA repair machinery and initiating DSB repair. As Collins et al. note, γH2AX domains can span 1-2 Mb and have varying levels of this important PTM – yet how γH2AX domain size and intensity are regulated, and how this impacts the DNA damage response are unknown. Here, the authors report that γH2AX domain breadth and intensity are largely regulated by intra-chromosomal interactions, or topologically associated domains (TADs).

How was CUT&RUN used? The authors used CUT&RUN to characterize γH2AX domains in a mouse model with defined, persistent DSBs. As part of their analysis, they mapped the distribution of γH2AX to physical chromosomal contacts determined by Hi-C to examine how γH2AX profiles correlate with DSB interactions. The authors found that when DSBs disrupt TAD borders, γH2AX regions extend bidirectionally into both regions, whereas breaks that occur adjacent to a TAD boundary result in high levels of γH2AX asymmetry. The resulting instability explains why lesions at TAD-proximal locations are particularly harmful and often associate with malignant chromosomal rearrangements. In addition, this paper reflects the power of CUT&RUN when integrated with other sequencing modalities (i.e. Hi-C) to achieve deeper insights into biological mechanisms.

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

Jay F. Sarthy et al. eLife, 2020. PMID: 32902381

CUT&RUN Targets: Oncogenic variant histones (H3.3K27M, H3.1K27M), H3K4me2, H3K27me3, Polycomb proteins (SUZ12, MTF2)

Sample Type: Patient-derived glioma cell lines

Significance: Pediatric diffuse midline glioma (DMG) is linked with oncogenic H3K27M mutations in both histone H3.1 (canonical H3) and H3.3. In each case, the K27M mutation inhibits generation of H3K27me3, a repressive chromatin mark catalyzed by the Polycomb Repressive Complex 2 (PRC2). This loss of H3K27me3 has been hypothesized to support tumor development via re-expression of silenced oncogenes. Interestingly, previous work has shown that H3.3 and H3.1 K27M mutations associate with unique sets of secondary mutations and differences in disease severity, with H3.1K27M correlated with early onset DMG. However, the precise mechanisms underlying the divergent outputs of H3.3 vs H3.1 K27M oncohistones has remained unclear.

Here, Sarthy and co-authors discover that only K27M oncohistones deposited in actively proliferating cells prevented H3K27 methylation. This replication-dependent pathway may help explain the accelerated development of H3.1K27M gliomas, as H3.1 is incorporated genome-wide during S phase, while H3.3 is deposited at select sites independent of DNA replication. Furthermore, due to the different mechanisms of H3.1 and H3.3 deposition, H3K27me3 depletion occurs differently across DMG patients, likely contributing to their varying phenotypes and disease progression.

How was CUT&RUN used? The authors sourced two H3.1K27M and two H3.3K27M DMG patient-derived cell lines, as well as two human glioma cell lines (with wild-type H3.1 and H3.3) as controls. CUT&RUN was used to profile H3.1K27M and H3.3K27M, active (H3K4me2) and repressive (H3K27me3) PTM signatures, and PRC2 occupancy. They found that H3K27me3 was more reduced in H3.1 compared to H3.3 mutants, reflecting the genome-wide deposition of H3.1 during cell replication. Surprisingly, neither mutant allele inhibited PRC2 binding in DMG cell lines, with H3.3K27M cells displaying normal H3K27me3 at a subset of domains. Combined, these data suggest a model of PRC2 “poisoning” by the K27M mutation, wherein H3K27me3 loss occurs progressively in replicating cells and drives cancer development.

Distinct dynamics and functions of H2AK119ub1 and H3K27me3 in mouse preimplantation embryos

Zhiyuan Chen, Mohamed Nadhir Djekidel, and Yi Zhang. Nature Genetics, 2021. PMID: 33821005

CUT&RUN Targets: H2AK119ub1, H3K27me3

Sample Type: Mouse germ cells and early stage embryos (zygote through blastocyst)

Significance: Polycomb Group proteins are critical epigenetic regulators that direct proper development. The two multi-subunit complexes PRC1 and PRC2 respectively coordinate deposition of H2AK119ub1 (H2Aub) and H3K27me3 to establish and maintain repressed chromatin states following differentiation. Furthermore, studies in embryonic stem cells and mouse models have demonstrated that these complexes have important roles in early preimplantation developmental processes, including “non-canonical” or H3K27me3-mediated imprinting (see Inoue et al. 2017, Genes Dev. and Nature). However, genomic investigation of PRC1/2 and their associated chromatin marks during these stages in vivo has been restricted due to the massive input requirements for traditional genomic analysis, such as ChIP-seq.

Here, Chen and colleagues leveraged recent advances in ultra-sensitive chromatin mapping methods to investigate H3K27me3 and H2Aub during early mouse embryonic development, from single-cell oocytes and zygotes through the blastocyst stage. Their results yield new insights into the distinct functions of PRC1- and PRC2-mediated marks during mammalian early development to establish proper chromatin architecture and gene expression programs.

How was CUT&RUN used? The authors applied CUT&RUN to profile H2Aub and H3K27me3 in individual mouse germ cells and early embryos, to better understand how repressive chromatin marks are established throughout embryonic development. They discovered that H2Aub and H3K27me3 are surprisingly dynamic following fertilization. Although the two PRC marks co-localize in oocytes, they display unique patterns of loss and reestablishment following fertilization and during early development. Additional CUT&RUN profiling of cells from PRC2 and PRC1 mutant mouse embryos suggest a model in which H3K27me3 is an essential mediator of noncanonical imprinting, while H2Aub is necessary to silence developmental genes prior to implantation.

Shop CUTANA CUT&RUN Products

The above examples showcase the ability of CUT&RUN to profile diverse targets in a wide variety of biological contexts, including primary tissue and patient-derived samples. If you are interested in setting up CUT&RUN in your laboratory, we recommend trying our user-friendly CUTANA CUT&RUN Kit which includes the reagents and protocols necessary to perform CUT&RUN assays and purify DNA for sequencing. We also provide a CUT&RUN protocol and comprehensive video walk-through highlighting key experimental considerations and adaptations for different types of cell inputs, mapping targets, and more.

To support CUTANA CUT&RUN assays, EpiCypher offers a collection of CUTANA-compatible antibodies to histone PTMs, reader proteins, chromatin remodelers, and more, as well as ConA beads, E. coli spike-in DNA, a DNA purification kit, and other reagents to help generate accurate, high-quality results. Each reagent is lot-validated and meticulously tested by EpiCypher scientists using our CUTANA CUT&RUN workflow, meaning that only best-in-class products are provided to end users.

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