EpiCypher’s H3K4me3 Antibody Provides a Useful Positive Control for CUT&RUN

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) assays are one of the latest advancements in chromatin profiling technologies, allowing researchers to map chromatin interacting proteins and histone post-translational modifications (PTMs) at a high resolution with fewer cells and sequencing reads compared to standard ChIP-seq experiments1-3 . To help scientists apply CUT&RUN to their own research projects, EpiCypher has released CUTANA™ CUT&RUN reagents, including a rigorously validated H3K4me3 antibody that can be used as a reliable positive control in CUT&RUN experiments.

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The quality of CUT&RUN sequencing data is truly remarkable. Unlike ChIP-seq, which requires 20-40 million reads per sample due to variable background and low signal-to-noise, CUT&RUN experiments only require 3-5 million reads per sample1. Even at this low sequencing depth, CUT&RUN consistently generates higher quality data vs. ChIP-seq, with low backgrounds and improved [signal : noise]. Furthermore, these advantages make it feasible to profile from low cell inputs, even down to single cell resolution in a recent paper4.

But how can you be sure it is working as intended? What controls are available for CUT&RUN?

Testing CUT&RUN: The Need for Controls

Controls are a fundamental part of every scientific experiment, including CUT&RUN. In fact, for new assays, controls are even more important, as they validate your technique and bolster confidence in the data from your experimental samples.

The most common controls for chromatin profiling experiments, such as ChIP-seq, are:

1) A positive control antibody - Generates robust chromatin profiles for a control histone PTM or chromatin-associated protein

2) A negative control antibody - Does not bind any chromatin associated factors, should generate little to no data, representing background signal in experiment (i.e. anti-IgG)

An H3K4me3 antibody is often used as a positive control for ChIP-seq, as it reliably denotes active transcription start sites, exhibiting sharp peaks that are easy to analyze. Moreover, H3K4me3 has been profiled in countless cell and tissue types. In fact, aberrant H3K4me3 methylation is a hallmark of many human cancers 5, including hepatocellular carcinoma6, breast7, 8 and colorectal cancer9, and enzymes that regulate H3K4me3 have been linked to leukemia10-12 and neurodegeneration13, 14.

But are there reliable H3K4me3 antibodies for CUT&RUN?

Antibody Validation for Chromatin Profiling Assays

It is generally assumed that “ChIP-grade” antibodies will also be successful in CUT&RUN. However, it remains unclear if antibodies that are validated for ChIP-seq will always work for CUT&RUN. Furthermore, many ChIP-grade antibodies display severe cross-reactivity, including highly cited H3K4me3 antibodies15 (see this blog post to learn more).

To address these concerns, EpiCypher developed SNAP-ChIP®, in which panels of DNA-barcoded modified designer nucleosomes (dNucs) are leveraged as physiological spike-in controls and used to quantify antibody specificity and enrichment within the context of a ChIP experiment15, 16. This method is essential to identify high-quality antibodies for ChIP-seq, as other methods (i.e. histone peptide arrays) do not replicate experimental conditions or the natural chromatin architecture, and thus fail to accurately define antibody binding activity15.

EpiCypher is developing similar spike-in technologies for our CUTANA CUT&RUN platform, which will allow unprecedented user control in these high-resolution chromatin profiling assays.

But what should scientists use as a positive control right now? EpiCypher turned to our existing line of SNAP-ChIP Certified Antibodies to identify an antibody that could be used for CUT&RUN.

Developing a CUTANA-Compatible Positive Control H3K4me3 Antibody

EpiCypher recently launched a new SNAP-ChIP certified H3K4me3 antibody, which shows best-in-class specificity and efficiency of target enrichment in ChIP-seq. In our efforts to deliver robust reagents for CUT&RUN assays, and to help scientists find a suitable positive control for their experiments, we decided to test this new H3K4me3 antibody using our optimized CUT&RUN protocol and standard ChIP-seq workflows15.

First, we examined ChIP-seq and CUT&RUN localization around annotated transcription start sites (TSS), as shown in Figure 1. The enrichment patterns are similar between both assays, and are typical of published H3K4me3 profiles. We also performed a Pearson correlation analysis, to examine the linear relationship between ChIP-seq and CUT&RUN data generated using this H3K4me3 antibody (Figure 2). The data from these assays were highly correlated (r = 0.811), providing additional support for the use of this antibody in CUT&RUN.

Importantly, we also compared our CUT&RUN and ChIP-seq tracks with highly cited H3K4me3 ChIP-seq data collected by the ENCODE consortium (using a different antibody). The ChIP tracks displayed in Figure 3A are in near-perfect agreement, confirming the reliability of our antibody across multiple assays and with published datasets. Additionally, we compared our ChIP-seq and CUT&RUN tracks to ENCODE data generated using an H3K4me3 antibody with documented cross-reactivity to H3K4me2 (see this blog posts for more info). As seen in Figure 3B, our H3K4me3 antibody displays no peaks in the cross-reactive region, indicating that our antibody has low levels of cross reactivity in both ChIP-seq and CUT&RUN.

The FIRST CUTANA CUT&RUN-Compatible Control Antibody

EpiCypher has provided convincing data supporting the use of our SNAP-ChIP certified H3K4me3 antibody as a positive control in CUT&RUN. This is the first commercial antibody shown to exhibit reliable performance in CUT&RUN assays, as it has been both:

1) Validated in a chromatin profiling platform using physiological nucleosome spike-in controls (i.e. SNAP-ChIP)

2) Thoroughly tested and compared in both ChIP-seq and CUT&RUN

Notably, the specificity of this antibody was defined in ChIP-seq experiments, using SNAP-ChIP spike-ins. Our previous work strongly emphasized the necessity of using defined nucleosome spike-ins to quantify antibody binding activity directly in the target application15. Thus, EpiCypher is currently developing similar controls for CUT&RUN, which will enable a direct assessment of antibody specificity and efficiency in this novel chromatin mapping assay.

Nevertheless, the development of a positive control is a critical innovation in the CUT&RUN field. Our H3K4me3 antibody will allow scientists to perform additional quality control analyses on their CUT&RUN experiments, which will add confidence to resulting sample data and expand the application of CUT&RUN.

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References

1. Meers MP, et al. Improved CUT&RUN chromatin profiling tools. Elife, 2019. 8: p. (PubMed PMID: 31232687) (PMC6598765)

2. Skene PJ, et al. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc, 2018. 13(5): p. 1006-19.(PubMed PMID: 29651053)

3. Skene PJ, Henikoff S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife, 2017. 6: p. (PubMed PMID: 28079019) (PMC5310842)

4. Hainer SJ, et al. Profiling of Pluripotency Factors in Single Cells and Early Embryos. Cell, 2019. 177(5): p. 1319-29 e11. (PubMed PMID: 30955888) (PMC6525046)

5. Rao RC, Dou Y. Hijacked in cancer: the KMT2 (MLL) family of methyltransferases. Nat Rev Cancer, 2015. 15(6): p. 334-46. (PubMed PMID: 25998713) (PMC4493861)

6. He C, et al. High expression of trimethylated histone H3 lysine 4 is associated with poor prognosis in hepatocellular carcinoma. Hum Pathol, 2012. 43(9): p. 1425-35. (PubMed PMID: 22406368)

7. Chen X, et al. A novel subtype classification and risk of breast cancer by histone modification profiling. Breast Cancer Res Treat, 2016. 157(2): p. 267-79. (PubMed PMID: 27178334)

8. Xi Y, et al. Histone modification profiling in breast cancer cell lines highlights commonalities and differences among subtypes. BMC Genomics, 2018. 19(1): p. 150. (PubMed PMID: 29458327) (PMC5819162)

9. Benard A, et al. Histone trimethylation at H3K4, H3K9 and H4K20 correlates with patient survival and tumor recurrence in early-stage colon cancer. BMC Cancer, 2014. 14: p. 531. (PubMed PMID: 25047223) (PMC4223547)

10. Winters AC, Bernt KM. MLL-Rearranged Leukemias-An Update on Science and Clinical Approaches. Front Pediatr, 2017. 5: p. 4. (PubMed PMID: 28232907) (PMC5299633)

11. Tkachuk DC, et al. Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell, 1992. 71(4): p. 691-700. (PubMed PMID: 1423624)

12. Gu Y, et al. The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene. Cell, 1992. 71(4): p. 701-8. (PubMed PMID: 1423625)

13. Vashishtha M, et al. Targeting H3K4 trimethylation in Huntington disease. Proc Natl Acad Sci U S A, 2013. 110(32): p. E3027-36. (PubMed PMID: 23872847)(PMC3740882)

14. Berson A, et al. Epigenetic Regulation in Neurodegenerative Diseases. Trends Neurosci, 2018. 41(9): p. 587-98. (PubMed PMID: 29885742) (PMC6174532)

15. Shah RN, et al. Examining the Roles of H3K4 Methylation States with Systematically Characterized Antibodies. Mol Cell, 2018. 72(1): p. 162-77 e7. (PubMed PMID: 30244833) (PMC6173622)

16. Grzybowski AT, et al. Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide. Mol Cell, 2015. 58(5): p. 886-99. (PubMed PMID: 26004229) (PMC4458216)