2022 was a big year for CUTANA™ CUT&RUN. EpiCypher now offers a complete workflow with our CUTANA CUT&RUN Assay Kit and Library Prep Kit, as well as an expanding collection of CUT&RUN validated antibodies. The SNAP-CUTANA™ K-MetStat Panel provides an essential spike-in assay control, and can be used for antibody validation, workflow optimization, and as a direct metric of assay success. Together these advances have contributed to multiple publications worthy of recognition. Below we highlight a few studies showing how CUTANA CUT&RUN can be used across diverse fields of study. We hope these papers provide ideas for how CUT&RUN can be applied for your projects, and are excited for 2023!
Wang et al. Nucleic Acids Research, 2022. PMID: 35849129
Cell Types: Transfected HeLa cells and HEK293T cells
Description: Targeted modification of the epigenome represents a promising strategy for precision medicine, since it allows activation of genes without disrupting DNA sequence. CRISPR/Cas9 has been cleverly modified for this purpose by fusing a nuclease-defective Cas9 (dCas9) with transcriptional activation domains, such as histone lysine acetyltransferases CBP or p300. Here, Wang et al. used CUT&RUN to help analyze local and genome-wide changes in chromatin structure when using different dCas9 activation systems. In agreement with previous studies, they found that dCas9 activators were highly variable, with results varying by the type of dCas9 fusion protein, cell type, and genomic target. They also discovered that targeting dCas9 activators to enhancers can induce reciprocal epigenomic changes at target promoters, leading to increased gene expression.
Why it makes the list: Although dCas9 activation systems provide specific gene activation, broader changes to the epigenome are possible – and could have inadvertent consequences in therapeutic applications. As a fast and reliable chromatin mapping assay, CUT&RUN is easily integrated into dCas9 screening pipelines.
Liu et al. Nature Genetics, 2022. PMID: 36347944
Cell Types: MDA-MB-231 cells (hypoxia-sensitive breast cancer cells) and 293T cells; includes experiments with and without knockdown of EGLN2 using the inducible CRISPR v2 system
Targets: H3P16oh, EGLN2, KDM5A, H3K4me3
Description: Low oxygen (hypoxia) induces transcriptional changes that drive tumor growth and increase cancer severity. Although multiple studies show that chromatin is responsive to hypoxia and plays a role in these processes, additional information is needed to provide a cohesive mechanism. Here, Liu et al. defined a novel histone PTM directly linked to hypoxia: prolyl (proline) hydroxylation on histone H3, proline 16. As part of this work, the authors used CUTANA CUT&RUN to validate H3P16oh antibodies, characterize its enrichment, and determine its function during hypoxia. Their experiments revealed substantial crosstalk between H3P16oh and H3K4me3, establishing H3P16oh as a hypoxia-sensitive regulator of gene expression and cell proliferation in breast cancer cell lines.
Why it makes the list: This paper is an important example of how CUTANA CUT&RUN assays can be used to study new or emerging PTMs. Li et al. also show how the combinatorial input of histone PTMs regulates chromatin structure and gene expression in disease, supporting epigenetic-targeted drug development.
Sanchez-Priego et al. Cell Reports, 2022. PMID: 35649373
Cell Types: Excitatory glutamatergic neurons and inhibitory GABAergic neurons derived from human pluripotent stem cells, with and without stimulation (membrane depolarization)
Targets: H3K27ac (0, 30, 90 min), FOS (0, 2 hr)
Description: Genetic risk variants for psychiatric diseases are concentrated in cis-regulatory DNA, suggesting roles in cell-type specific gene expression. However, due to the inherent challenges of studying human brain tissues, these genomic regions remain largely unexplored. In this paper, Sanchez-Priego et al. generated large amounts of excitatory and inhibitory neurons using human pluripotent stem cells. They profiled cells using a variety of techniques, including CUT&RUN, ATAC-seq, and RNA-seq, to identify putative cis-regulatory elements (i.e. enhancers) associated with activity-dependent gene expression. To support the relevance of their results to human disease, the authors compared the list of candidate enhancers to a large database of psychiatric-disease risk variants, which revealed significant links to schizophrenia, ADHD, and bipolar disorder.
Why it makes the list: Instead of dissecting individual variants, Sanchez-Priego et al. took a global approach, defining epigenomic elements in disease-relevant cell types and cross-referencing with variant datasets. This method provides more resources for the field and can be applied to many areas of biomedical research.
Zhu et al. Cell Reports, 2022. PMID: 36323253
Cell Types: HCT116 cell line (all targets), HeLa cell line (NUP93 only)
Targets: NUP93, NUP35, NUP205, BRD4, SEC13
Description: The nuclear pore complex acts as the gateway to the nucleus in eukaryotic cells and is composed of ~30 different nucleoporin proteins (NUPs). There is ample evidence that the nuclear pore complex helps regulate 3D chromatin organization and transcription. However, the functions of NUP subunits in human cells are not defined. Here, Zhu et al. studied NUPs using multiple techniques, including CUTANA CUT&RUN, Hi-C, PRO-seq, and CRISPR/dCas9 tethering. CUT&RUN showed that NUP proteins were generally bound to active chromatin regions. NUP93 specifically associated with promoters and enhancers and directly regulated transcription (similar to Brown et al. and Ibarra et al.). Strikingly, the authors found that core NUP proteins were not required for 3D chromatin architecture, in contrast to leading hypotheses in the field.
Why it makes the list: The ability to map subunits of large molecular complexes under native conditions, without cross-linking, is a key advantage of CUTANA CUT&RUN. Of note, NUP proteins and the nuclear pore complex have been challenging to study in mammalian cells; accurate chromatin mapping will help accelerate this research.
For a full list of papers citing CUTANA CUT&RUN products see this page.