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.
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.