Understanding the dynamic nature of chromatin is crucial for investigating the details of gene regulation and cellular function. CUT&RUN has emerged as a powerful tool in epigenetic research for probing chromatin dynamics, affording scientists unique insights into the spatial and temporal organization of chromatin. Here, we discuss how CUT&RUN can be applied to explore chromatin’s variable terrain and elucidate the regulatory mechanisms governing gene expression.
Addressing the Shortcomings of ChIP
Chromatin immunoprecipitation (ChIP) has been a gold standard for studying epigenetic modifications and protein-DNA interactions in cells. However, traditional ChIP has its limitations, including its requirement for large amounts of starting material, long processing times, and low resolution. The recently developed Cleavage Under Targets and Release Using Nuclease, or CUT&RUN, represents a significant advancement in the study of chromatin. Unlike traditional ChIP methods, CUT&RUN simplifies the process by means of an inventive fusion protein comprising a cleavage domain (such as micrococcal nuclease or MNase) and a protein A/G (pAG) domain that binds to an antibody against the protein of interest. The general procedure of CUT&RUN involves permeabilization of the nuclear membrane, followed by the targeted binding of antibody-conjugated pAG-MNase. Subsequent cleavage and release of DNA fragments result in a highly enriched chromatin sample ready for sequencing. This streamlined process reduces background noise and significantly enhances the signal-to-noise ratio, making CUT&RUN a preferred choice for chromatin analysis.
Chromatin Profiling
CUT&RUN's innovative approach provides high-resolution information on transcription factor binding sites, histone modifications, and chromatin accessibility:
Profiling Transcription Factor Binding Sites. CUT&RUN can identify the genomic locations where transcription factors (TFs) are bound to DNA. By selectively cleaving DNA around TF binding sites, CUT&RUN allows for the precise mapping of these regulatory elements. Researchers can examine how transcription factors dynamically interact with chromatin during different cellular states, uncovering the regulatory networks controlling gene expression.
Mapping Histone Modifications. Post-translational modifications of histone proteins play a crucial role in chromatin dynamics, influencing chromatin accessibility and transcription. CUT&RUN excels in profiling histone modifications with high precision. Investigators can study the dynamic changes of specific modifications in response to external stimuli or developmental cues, offering a comprehensive view of epigenetic regulation.
Chromatin Accessibility Studies. Chromatin accessibility is a key determinant of gene activity and refers to the ease with which specific regions of DNA are accessible or open to various cellular components like transcription factors and regulatory proteins. It plays a crucial role in gene regulation, as the accessibility of DNA determines the ability of the cellular machinery to read and transcribe genes. CUT&RUN can be used to profile regions of open chromatin, shedding light on the accessibility landscape during cellular processes such as differentiation. Dynamic changes in chromatin accessibility can be correlated with alterations in gene expression.
Future Innovations
As CUT&RUN continues to be optimized and improved, it is likely to become even more widely used in the field of epigenetics. To this end, EpigenTek has developed two improved methods, EpiNext™ CUT&RUN Fast and EpiNext™ CUT&Tag In-Place Sequencing, for enriching protein-bound DNA and mapping genome-wide protein-DNA interactions. These novel approaches combine the advantages of CUT&RUN with the fastest procedures in convenient and affordable all-in-one kits for reliably identifying true target protein-enriched regions and achieving high-resolution mapping. Both techniques utilize a unique nucleic acid cleavage enzyme mix to simultaneously fragment chromatin and cleave/remove unbound DNA sequences at both ends of the target protein-DNA complex in situ without affecting DNA occupied by the target protein, thereby minimizing immunocapture and sequencing background. DNA enrichment and library generation can be achieved in just a few hours, starting from as little as 500 cells or 50 ng of chromatin.
