Top CUT&RUN and CUT&Tag chromatin mapping papers of 2023

2023 was a remarkable year for science. The first CRISPR-based gene therapy was approved in the U.K. and the U.S. for sickle-cell anemia, a landmark in precision medicine. Also this year, Drs. Katalin Karikó and Drew Weissman won the Nobel Prize in Physiology or Medicine for their groundbreaking work on mRNA vaccine technology. And for better or worse, artificial intelligence tools like ChatGPT are being used across biomedical research, data analysis, and healthcare. What advancements were made in epigenetics and chromatin profiling this year?

CUT&RUN and CUT&Tag chromatin mapping assays are continuously improving, enabling exciting applications across increasingly diverse research areas. Indeed, the improved sensitivity of these strategies is allowing consideration of chromatin structure in cell and gene therapy, drug development, and biomarker discovery. EpiCypher now offers user-friendly kits for CUTANA™ CUT&RUN and CUT&Tag, a collection of validated antibodies, and CUTANA™ CUT&RUN Services! Here, we highlight six ways our customers leveraged CUTANA CUT&RUN and CUT&Tag assays in 2023.

Aberrant gene activation in synovial sarcoma relies on SSX specificity and increased PRC1.1 stability

Benabdallah et al. Nature Structural Biology, 2023. PMID: 37735617

Chromatin mapping assay: CUT&RUN

EpiCypher Products: CUTANA ChIC/CUT&RUN Kit (14-1048) and CUTANA DNA Purification Kit (14-0050)

Cell types: Human synovial sarcoma HS-SY-II and SYO-1 cells, human osteosarcoma KHOS-240S cells

Targets: H2AK119ub1, endogenous SS18-SSX1/2 fusion proteins, HA-tagged fusion proteins

Background & Description: Gene fusions involving chromatin modifying/remodeling complexes are common drivers of pediatric cancers. In synovial sarcoma, a soft tissue cancer, the SS18 subunit of the SWI/SNF BAF chromatin remodeling complex is fused with SSX1, SSX2, or SSX4 protein, forming a dominant transcriptional activator. Integration of SS18-SSX fusion proteins in BAF remodeling complexes disrupts normal function and results in re-localization of BAF to regions containing H2AK119ub1, a marker of Polycomb-repressed genes. BAF recruitment leads to aberrant activation of Polycomb genes, including developmental regulators, which increases cell proliferation and supports cancer development.

Understanding how these fusions alter transcription and promote oncogenesis is an important topic of biomedical and pharmaceutical research. Although the recruitment of SS18-SSX to H2AK119ub1 nucleosomes is well defined, the precise role of BAF remodeling is unknown. In this study, Benabdallah et al. use CRISPR/Cas9 technology to dissect the function of SS18-SSX fusion proteins, including the requirement for BAF remodeling complexes. They used CUT&RUN to analyze changes in H2AK119ub1 and SS18-SSX enrichment and studied alternative fusions expressed with HA tags. Surprisingly, their results indicate minimal roles for BAF, with C-terminal SSX being the most important region for SS18-SSX recruitment and oncogenic activity.

Why it makes the list: CRISPR/Cas9 technology is enabling major breakthroughs in gene therapy and biomedical research. It is critical to pair CRISPR experiments with appropriate strategies to investigate potential impacts on chromatin structure and gene expression, especially when targeting chromatin modifying complexes. In this study, the authors combine CRISPR/Cas9 deletion of Polycomb and SWI/SNF subunits with CUT&RUN chromatin mapping analyses, providing a genome-wide overview of chromatin alterations as well as key mechanistic insights. Keep an eye out for an upcoming blog, where we will discuss further applications of CUT&RUN/CUT&Tag in gene therapy applications, including CRISPR and CAR T-cell therapy!

Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag

Henikoff et al. Nature Communications, 2023. PMID: 37739938

Chromatin mapping assay: CUTAC (CUT&Tag modified for chromatin accessibility)

EpiCypher Products: CUTANA pAG-Tn5 for CUT&Tag (15-1117) and SNAP-Certified™ H3K4me2 antibody (13-0027)

Cell types: Formalin-fixed paraffin-embedded (FFPE) mouse tissues, including brain tumors, intrahepatic cholangiocarcinoma tumors, healthy brain, and healthy liver

Targets: H3K27ac, RNA Polymerase II Serine 5 phosphorylation (RNAPII-Ser5p; paused RNA Pol II), RNA Pol II Serine 2 and Serine 5 phosphorylation (RNAPII-Ser2,5p; elongating and paused RNA Pol II)

Background & Description: FFPE is the global standard for tissue preservation. Thousands of samples are stored in FFPE blocks, providing a massive resource to the biomedical research community. However, chromatin mapping approaches are historically incompatible with FFPE tissues, due to chromatin damage and high background inherent to heavy cross-linking. Previous strategies, such as ChIP-seq, also lacked the sensitivity needed to resolve target signal and struggled to generate reliable data from small inputs. Available chromatin accessibility assays, such as ATAC-seq, have high sensitivity, but are designed for native samples. Although advances have been made in recent years (e.g. FFPE-ATAC, FACT-seq), profiling from FFPE tissues remains a major challenge.

In CUTAC (Cleavage Under Targeted Accessible Chromatin) assays, a low-salt pAG-Tn5 tagmentation step is used to release chromatin fragments marked by paused RNA Polymerase II (RNAPII-Ser5p), H3K27ac, or H3K4me2 antibodies. Here, Henikoff and colleagues optimized CUTAC for chromatin accessibility mapping in FFPE tissues, generating high-quality data from multiple FFPE samples.

Why it makes the list: The development of FFPE-CUTAC assays offers vast improvements to competing technologies. The simple protocol can be performed in two days and is compatible with direct-to-PCR CUT&Tag Kits, making it easy for others to try this strategy. Compared to FFPE-based FACT-seq assays, CUTAC shows improved signal to background and an increased fraction of reads in peaks (FRiP), indicative of high assay sensitivity. In addition, they were able to use CUTAC data to distinguish healthy tissue vs. tumor tissue on the same tissue section.

CUTAC makes profiling from FFPE samples a reality and opens the door to countless patient samples, mouse models, and more. The automation of related CUT&Tag assays will drive additional applications in clinical and pharmaceutical research for investigations of novel chromatin pathways.

Noncoding variants alter GATA2 expression in rhombomere 4 motor neurons and cause dominant hereditary congenital facial paresis

Tenney et al. Nature Genetics, 2023. PMID: 37386251

Chromatin mapping assay: Single-cell CUT&Tag

EpiCypher Product: CUTANA pAG-Tn5 (15-1117)

Cell type: FACS-isolated motor neurons from mouse embryos

Target: NR2F1

Background & Description: This paper identifies the genetic cause underlying hereditary congenital facial paresis type 1 (HCFP1), a rare autosomal dominant disorder that restricts facial movement. Previous work mapped HCFP1 to a specific region on chromosome 3, which includes GATA2, but no protein-coding variants were found. Here, Tenney et al. investigated non-coding regions in HCFP1 family cohorts, uncovering variants/duplications in a novel cis-regulatory element (cRE) of GATA2. A humanized HCFP1 mouse model shows that these variants alter neuronal differentiation during embryonic development, resulting in fewer facial branchial motor neurons – cell types critical for facial expression.

Why it makes the list: Many rare diseases are mapped to discrete genomic regions that lack variants in protein-coding genes, leading to a renewed focus on noncoding cREs, such as enhancers or silencers. However, the identification of cREs is far from straightforward, as many act in a cell-type specific or temporal manner to regulate gene expression and differentiation. Furthermore, in developmental diseases such as HCFP1, establishing cRE activity requires access to unique progenitor cells, which may be challenging to isolate and/or provide low cell numbers for analysis.

In this study, Tenney et al. used mouse model systems to fluorescently label motor neuron progenitor cells, enabling enrichment by FACS. Ultra-sensitive genomic technologies, including single-cell CUT&Tag, RNA-seq, and ATAC-seq assays, allowed functional analysis of the putative cRE. Taken together, this study provides an ideal workflow for studying cREs during development and disease.

Epigenetic dysregulation from chromosomal transit in micronuclei

Agustinus et al. Nature, 2023. PMID: 37286593

Chromatin mapping assay: CUT&RUN

EpiCypher Product: CUTANA CUT&RUN Services

Cell types: Telomerase-immortalized retinal pigment epithelial cells (RPE-1), DLD-1 colorectal cancer cells

Targets: H3K4me3, H3K27me3, H3K27ac

Background & Description: A hallmark of many cancers is chromosomal instability (CIN), which refers to large-scale genomic duplications, deletions, or rearrangements that occur during mitosis. CIN is often precipitated by abnormal chromosome separation in anaphase. These lagging chromosomes are sequestered in micronuclei, which are prone to rupture, resulting in genomic alterations characteristic of CIN. Damaged chromosomes can be reincorporated in the primary nucleus following cell division, leading to inheritance of new variants. Alternatively, micronuclei can be targeted by autophagy pathways and/or drive activation of inflammatory genes, all which support cancer progression.

However, the role of chromatin in this process is poorly understood. How does micronuclei development alter chromatin structure, and what is the long-term impact on cancer? Do micronuclei play a causative role in CIN? Here, the authors used multiple CIN/micronuclei model systems to study alterations in chromatin accessibility, histone PTMs, and gene expression. They found that chromosomes in micronuclei exhibit heritable changes in chromatin architecture that contribute to CIN. Interestingly, heterochromatin modifications, such as H3K27me3, remained stable. Active marks H3K4me3 and H3K27ac were redistributed, and aligned with changes in chromatin accessibility measured by ATAC-seq.

Why it makes the list: CIN contributes to tumor growth and is associated with poor prognosis as well as treatment resistance, making it a key area of biomedical research. Understanding the mechanisms that contribute to CIN will provide new drug targets and biomarkers, which are desperately needed.

*BONUS* This is one of the first publications supported by EpiCypher’s CUTANA CUT&RUN Services. Our CUT&RUN Services provide direct access to our genomics experts, automated workflows, and data analysis pipelines. Read more here.

A multi-organoid platform identifies CIART as a key factor for SARS-CoV-2 infection

Tang et al. Nature Cell Biology, 2023. PMID: 36918693

Chromatin mapping assay: CUT&RUN

EpiCypher Product: CUTANA ChIC/CUT&RUN Kit (14-1048)

Cell type: Human pluripotent stem cell (hPSC)-derived cardiomyocytes

Target: Flag-tagged CIART

Background & Description: COVID-19 has widespread impacts on the body, including long-term effects on respiratory, cardiac, and neurological function. Studying infected cells is crucial to developing effective therapeutics; however, isolating each of these cell types from individual human donors is difficult, if not impossible. To address these concerns, the authors previously developed lung organoid and cardiomyocyte cell models using hPSCs, and successfully leveraged these systems for SARS-CoV-2 infection and drug screening studies. Here, Tang et al. examined gene expression in these COVID-19 cell models, which uncovered a role for the circadian-controlled transcriptional repressor CIART in multi-organ pathogenesis. Using a combination of RNA-seq, ATAC-seq, and CUT&RUN assays, they show that knockout of CIART renders cells resistant to SARS-CoV-2 infection, through altered regulation of Retinoid X Receptor signaling.

Why it makes the list: Organoids and other cell-based model systems form an important branch of disease research and drug testing. The study of chromatin structure in these models may identify cell-type specific mechanisms, particularly in the case of COVID-19, which effects multiple organs. Many studies overlook mapping transcription factors or histone PTMs in favor of RNA-seq and ATAC-seq technologies, due to the low sensitivity and poor reliability of historical chromatin profiling assays (i.e. ChIP-seq). Here, the authors took an exhaustive multiomic approach, demonstrating the power of CUT&RUN for emerging biological platforms. Further, CUT&RUN helped define novel gene regulatory pathways in SARS-CoV-2 infection, which could lead to improved treatment strategies.

Canonical BAF complex activity shapes the enhancer landscape that licenses CD8+ T cell effector and memory fates

McDonald et al. Immunity, 2023. PMID: 37315534

Chromatin mapping assay: CUT&RUN

EpiCypher Product: CUTANA pAG-MNase for ChIC/CUT&RUN Workflows (15-1016)

Cell type: CD8+ T cells

Targets: ARID1A, H3K27ac, T-bet, BATF, ETS1

Background & Description: Cytotoxic CD8+ T cells form an essential part of adaptive immunity, playing critical roles in viral and bacterial infection as well as tumor surveillance. During infection, naïve CD8+ T cells are activated by their cognate antigen, leading to the production of short-lived effector cells and long-term memory cells. The cell surface markers, transcription factors (e.g. T-Bet), and corresponding gene expression programs involved in CD8+ T cell differentiation are well-defined. However, the mechanisms that govern transcription factor binding – particularly in the development of CD8+ memory T cells – are less understood.

Could chromatin remodeling play a role? Variants in chromatin remodeling enzymes are frequently linked to cancer, including T cell lymphomas. In addition, previous work found that the SWI/SNF canonical BAF (cBAF) complex subunits ARID1A and SMARCA4 are required for T cell lineages in mice. In this 2023 paper, McDonald and colleagues investigated cBAF functions during antiviral immune responses. They found that cBAF complexes regulate enhancer accessibility in a temporal manner, enabling CD8+ T cell effector and memory cell formation.

Why it makes the list: T cell differentiation pathways are a major topic in cell and gene therapy research, particularly in relation to T cell exhaustion, which occurs during prolonged infection and cancer. Exhausted CD8+ T cells are dysfunctional, lack cytotoxic activity, and are transcriptionally distinct from effector and memory cell types. As a result, exhausted T cells are a major barrier to cancer treatment, as they reduce the efficacy of immunotherapy. In the case of CAR T-cell therapy, T cell exhaustion prohibits application to solid tumors.

The ability to block or reverse T cell exhaustion would be a massive advance in cancer research. This work suggests that SWI/SNF chromatin remodelers regulate T cell fate, providing a feasible drug target. In fact, multiple SWI/SNF inhibitors are already in clinical trials (NCT04891757, NCT04879017).

Conclusions

Chromatin is one of the most rapidly expanding areas of scientific research. Mapping the genome-wide enrichment of chromatin features, such as transcription factors, chromatin remodeling enzymes, histone post-translational modifications, can provide powerful insights about pathogenesis and drug efficacy. Understanding chromatin regulation can also reveal novel pathways that impact disease development, resulting in new drug targets and potential biomarkers.

The next year is sure to bring additional innovations and discoveries. We are particularly interested in the application of epigenomics to liquid biopsies, which has potential uses in diagnosis, drug response tracking, and more. Single-cell and low-input epigenomic technologies are also gaining steam, along with integrative analysis tools to enable deeper insights. And the inclusion of CUT&RUN in CRISPR/Cas9 experiments highlights the flexibility of CUTANA technology for cell and gene therapy research.