An important aspect of gene expression involves the action of transcription factors (TFs) at their binding sites on DNA. This process affects myriad functional pathways and biological processes, and is relevant in both normal physiology and disease pathobiology. Advances in deep sequencing in combination with other technologies has enabled the study of transcriptional regulation to shift from a gene-by-gene level to a genome-wide scale. Profiling of TFs is most commonly carried out using chromatin immunoprecipitation (ChIP), a protocol introduced over 30 years ago. Since then, there have been efforts to revolutionize and refine this method to allow for more precise and higher resolution TF profiling. The Henikoff laboratory in the Basic Sciences Division has been developing novel technologies to do just that, and show how one strategy developed in 2017 has come of age in two recently published papers.
Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike ChIP, which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. In CUT&RUN, DNA in the starting cells are initially intact, allowing protein-DNA interactions to be maintained in their natural state. When applied to yeast and human nuclei, CUT&RUN yields precise transcription factor profiles while avoiding crosslinking and solubilization issues.
In an example of the latest application of CUT&RUN, Dr. Henikoff and a postdoctoral fellow in his lab, Dr. Sandipan Brahma used CUT&RUN followed by ChIP to map chromatin-factor co-occupancies with nucleosomal histones. The landscape of nucleosomes at promoter regions can affect transcription regulation, however, ChIP experiments have fallen short of proving that the nucleosome-depleted region (NDR) are not nucleosome free, but instead, occupied by particles referred to as fragile nucleosomes. Published in a recent issue of Molecular Cell, Drs. Brahma and Henikoff found that fragile particles represent the occupancy of the remodeling the structure of chromatin (RSC) and RSC-bound, partially unwrapped nucleosomal intermediates. This suggests that nucleosomes unravel when there is RSC binding, which facilitates the subsequent binding of general regulatory factors that lead to a dynamic cycle of nucleosome deposition and clearance at certain NDRs. Dr. Brahma explained the significance of this work: “We have introduced a simple and efficient method for profiling the co-occupancy of chromatin-associated factors with nucleosomal histones, which is expected to be broadly applicable and easily adaptable. Previously, an efficient way to directly test co-occupancies was lacking, while some involved complicated barcoding and computational approaches, and most conclusions were based on correlations. Also, the concept of "fragile nucleosomes" has been out there for almost a decade, and their existence has been debated. Subnucleosomal intermediates at gene-regulatory regions such as the NDRs in yeast, as we show here, we think is a way to fine-tune gene expression patterns.” This study was done with Saccharomyces cerevisiae, but Dr. Brahma reveals his plans following this study to “address how subnucleosomal intermediates and the chromatin-remodeling complex BAF may be involved in regulating the pluripotency of embryonic stem cells or their differentiation into defined lineages.”
In addition, members of the Henikoff laboratory reported in a recent issue of Epigenetics & Chromatin that they have successfully automated the CUT&RUN platform. Postdoctoral fellow Dr. Derek Janssens led the effort, and explained the significance of automating CUT&RUN: “It expands the throughput potential and consistency of the CUT&RUN technique, allowing a single user to map protein footprints on the DNA in up to 96 samples in two days.” Making this an easy and cost-effective workflow was no easy feat. “The first major challenge was to modify the chemistry of the CUT&RUN reaction to streamline it for automation, and the second was to figure out how to properly ‘zero’ all the equipment and get the biomek to behave.” said Dr. Janssens. The ability to automate this technology so that it performs consistently and quickly will make many researchers cheer. Indeed, for us lucky ones at the Fred Hutch, it may well be soon that we get to use automated CUT&RUN. Dr. Janssens revealed: “The next step of this project will be working with the genomics shared resources core to establish either Auto CUT&RUN or a similar method we call CUT&TAG as a core facility that is open to all labs at the Hutch. We are still in the process of figuring out which method will be more suitable for this purpose, but hope to have it ready to go this spring.”
Brahma S, Henikoff S. (2018). RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast. Molecular Cell. 73:1-12
Janssens DH, Wu SJ, Sarthy JF, Meers MP, Myers CH, Olson JM, Ahmad K, Henikoff S. 2018. Automated in situ chromatin profiling efficiently resolves cell types and gene regulatory programs. Epigenetics Chromatin. 11(1), 74.
Funding was provided by the Chan-Zuckerberg Initiative, Howard Hughes Medical Institute, and the Damon Runyon-Sohn Foundation.