MulTI-Tag: your ticket to profiling chromatin

From the Henikoff Lab, Cancer Basic Biology Program, Cancer Consortium.

“Sydney Brenner is widely quoted as saying the following regarding technology development: ‘Progress in science depends on new techniques, new discoveries and new ideas, probably in that order’,” Dr. Michael Meers told me. The Henikoff Lab, where Dr. Meers completed his postdoc before taking a faculty job in the Department of Genetics at the Washington University School of Medicine, is no stranger to developing new techniques. Under the guidance of Dr. Steven Henikoff, Professor in the Basic Sciences Division, the Henikoff Lab has developed several techniques for investigating chromatin biology including CUT&Tag, CUT&RUN, MINCE-seq, ChEC-seq – the list goes on and on. Now Dr. Meers has added yet another technique, MulTI-Tag, to that list. He describes the single-cell technology in an article published in Nature Biotechnology on Halloween.

MulTI-Tag, or Multiple Target Identification by Tagmentation, builds upon the CUT&Tag technology developed by Dr. Hatice Kaya-Okur. Whereas CUT&Tag lets researchers profile single cells for a given epigenetic feature, such as a histone modification or a transcription factor, MulTI-Tag aims to map multiple targets simultaneously. This method could be applied to mixed cell populations to understand gene regulation in individual cell types, or to the same population over time to track lineage progression during development. “I have a long-standing interest in how pioneer transcription factors contend with chromatin barriers that occlude their binding sites, which often reside in essential developmental enhancers,” Dr. Meers confided. Pioneer transcription factors are thought to initiate transcription at otherwise silenced genes in multiple ways, including binding their recognition motifs directly and binding partially exposed motifs on the surface of nucleosomes and triggering eviction of said nucleosomes. Dr. Meers continued, “this is a fiendishly difficult problem because to understand it we need to be able to visualize the moment of encounter between a pioneer factor and the chromatin landscape at specific loci we care about, which is impossible with current technology. I knew [that solving this problem] was going to be a long, multi-step technology development process that would first involve figuring out how to map any chromatin proteins in tandem…in the same cells.” So, Dr. Meers started thinking about ways to modify the CUT&Tag protocol. The genius of CUT&Tag lies in “a specific detail of how [Dr. Kaya-Okur] engineered the technique: a Tn5 transposase fusion protein that delivered a payload of DNA sequencing adapters directly at sites where a protein of interest resided, guided by an antibody,” said Dr. Meers. “In talking with Hatice (Kaya-Okur), it was immediately obvious that the adapters could serve a dual purpose—marking not just the protein’s location, but also its identity, by smuggling in an extra DNA barcode identifier that would be read out in the same sequencing reaction”. Using MulTI-Tag this is possible through sequential antibody incubation and tagmentation, wherein each primary antibody introduces a specific barcoded forward adapter allowing for downstream identification. Dr. Meers continued, “That would enable us to theoretically map a pioneer factor and the other proteins that compose the chromatin landscape at the same time, tagged with identifiers. So the lightbulb moment really came before any of the work had even begun as its potential became clear.”

A graphic showing the workflow of MulTI-Tag, from sequential binding of barcoded antibodies to sequencing of resulting DNA fragments.
The new technique from the Henikoff Lab, “MulTI-Tag”, uses individually barcoded Tn5 transposases to profile up to three chromatin factors simultaneously within single cells. Figure adapted from article.

“After the initial excitement at the prospects for a new method, we hit an immediate stumbling block,” Dr. Meers sighed. “The barcodes identifying specific proteins had a habit of ‘swapping’ with each other so that they lost their specificity. We went to great pains to reengineer the protocol so that barcodes were physically separated in situ in a way that prevented swapping. It was a bit of a letdown at first since the new measures made the protocol more complicated than I had originally envisioned, but the eventual payoff was worth it.” And the data quickly revealed some exciting patterns. “Even within what amounted to a proof of principle for a more ambitious experimental goal, a major theme emerged: chromatin exerts network-level regulatory effects in very different ways depending on the developmental context,” explained Dr. Meers. “This became clear by mapping multiple histone modifications across fine-grain, single-cell trajectories of ESC [embryonic stem cell] differentiation, where specific modifications were prominently featured at crucial developmental genes in one trajectory, but nearly dispensed of entirely in another.” For instance, Dr. Meers was able to “watch” as human ESCs transitioned into primary germ layers using inferred pseudotime trajectories derived from the enrichment patterns of H3K27me3 and H3K4me1 in individual cells. In tracking chromatin changes at a single-cell resolution with MulTI-Tag, Dr. Meers was able to establish a pseudo-time “roadmap” predicting the trajectories from ESC to endoderm, mesoderm, and ectoderm, based solely on the dynamic localization of three histone marks.

And, now that Dr. Meers has invented the tool he needed, he can get back to his real goal: “map[ping] pioneer factors alongside the chromatin proteins they interact with in the same cellular context, and learn the details of how chromatin barriers are overcome to specify developmental lineage.” Dr. Meers continued, “Any chromatin characteristic for which an antibody is available can be added to the mix, so the future applications for comprehensive chromatin mapping are wide-ranging. With any luck, this technique will pull its scientific weight in discoveries and ideas down the road.”

Meers M, Llagas G, Janssens DH, Codomo CA, and Henikoff S. 2022. Multifactorial profiling of epigenetic landscapes at single-cell resolution using MulTI-Tag. Nature Biotechnology.

This work was funded by the Howard Hughes Medical Institute and the National Institutes of Health.

Cancer Consortium member Dr. Steven Henikoff contributed to this work.