Single-cell CUT&Tag analysis of chromatin modifications in differentiation and tumor progression.

Methods for quantifying gene expression1 and chromatin accessibility2 in single cells are well established, but single-cell analysis of chromatin regions with specific histone modifications has been technically challenging. In this study, we adapted the CUT&Tag method3 to scalable nanowell and droplet-based single-cell platforms to profile chromatin landscapes in single cells (scCUT&Tag) from complex tissues and during the differentiation of human embryonic stem cells. We focused on profiling polycomb group (PcG) silenced regions marked by histone H3 Lys27 trimethylation (H3K27me3) in single cells as an orthogonal approach to chromatin accessibility for identifying cell states. We show that scCUT&Tag profiling of H3K27me3 distinguishes cell types in human blood and allows the generation of cell-type-specific PcG landscapes from heterogeneous tissues. Furthermore, we used scCUT&Tag to profile H3K27me3 in a patient with a brain tumor before and after treatment, identifying cell types in the tumor microenvironment and heterogeneity in PcG activity in the primary sample and after treatment.

Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation.

Chromatin accessibility mapping is a powerful approach to identify potential regulatory elements. A popular example is ATAC-seq, whereby Tn5 transposase inserts sequencing adapters into accessible DNA ('tagmentation'). CUT&Tag is a tagmentation-based epigenomic profiling method in which antibody tethering of Tn5 to a chromatin epitope of interest profiles specific chromatin features in small samples and single cells. Here, we show that by simply modifying the tagmentation conditions for histone H3K4me2 or H3K4me3 CUT&Tag, antibody-tethered tagmentation of accessible DNA sites is redirected to produce chromatin accessibility maps that are indistinguishable from the best ATAC-seq maps. Thus, chromatin accessibility maps can be produced in parallel with CUT&Tag maps of other epitopes with all steps from nuclei to amplified sequencing-ready libraries performed in single PCR tubes in the laboratory or on a home workbench. As H3K4 methylation is produced by transcription at promoters and enhancers, our method identifies transcription-coupled accessible regulatory sites.

Cells keep their DNA tidy by wrapping it into structures called nucleosomes. Each of these structures contains a short section of DNA wound around a cluster of proteins called histones. Not only do nucleosomes keep the genetic code organized, they also control whether the proteins that can switch genes on or off have access to the DNA. When genes turn on, the nucleosomes unwrap, exposing sections of genetic code called 'gene regulatory elements'. These elements attract the proteins that help read and copy nearby genes so the cell can make new proteins. Determining which regulatory elements are exposed at any given time can provide useful information about what is happening inside a cell, but the procedure can be expensive. The most popular way to map which regulatory elements are exposed is using a technique called Assay for Transposase-Accessible Chromatin using sequencing, or ATAC-seq for short. The 'transposase' in the acronym is an enzyme that cuts areas of DNA that are not wound around histones and prepares them for detection by DNA sequencing. Unfortunately, the data from ATAC-seq are often noisy (there are random factors that produce a signal that is detected but is not a 'real' result), so more sequencing is required to differentiate between real signal and noise, increasing the expense of ATAC-seq experiments. Furthermore, although ATAC-seq can identify unspooled sections of DNA, it cannot provide a direct connection between active genes and unwrapped DNA. To find the link between unspooled DNA and active genes, Henikoff et al. adapted a technique called CUT&Tag. Like ATAC-seq, it also uses transposases to cut the genome, but it allows more control over where the cuts occur. When genes are switched on, the proteins reading them leave chemical marks on the histones they pass. CUT&Tag attaches a transposase to a molecule that recognizes and binds to those marks. This allowed Henikoff et al. to guide the transposases to unspooled regions of DNA bordering active genes. The maps of gene regulatory elements produced using this method were the same as the best ATAC-seq maps. And, because the transposases could only access gaps near active genes, the data provided evidence that genes switching on leads to regulatory elements in the genome unwrapping. This new technique is simple enough that Henikoff et al. were able to perform it from home on the countertop of a laundry room. By tethering the transposases to histone marks it was possible to detect unspooled DNA that was active more efficiently than with ATAC-seq. This lowers laboratory costs by reducing the cost of DNA sequencing, and may also improve the detection of gaps between nucleosomes in single cells.

Efficient low-cost chromatin profiling with CUT&Tag.

We recently introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei. These antibodies are then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds adapters ('tagmentation') for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses DNA accessibility artefacts to ensure high-fidelity mapping of the antibody-targeted protein and improves the signal-to-noise ratio over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube, from cells to amplified libraries, providing low-cost genome-wide chromatin maps. By simplifying library preparation CUT&Tag requires less than a day at the bench, from live cells to sequencing-ready barcoded libraries. As a result of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for as little as $25 per sample. This enables routine genome-wide profiling of chromatin proteins and modifications and requires no special skills or equipment.

CUT&Tag for efficient epigenomic profiling of small samples and single cells.

Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.

Msx Homeobox Genes Act Downstream of BMP2 to Regulate Endometrial Decidualization in Mice and in Humans.

Endometrial stromal cells differentiate to form decidual cells in a process known as decidualization, which is critical for embryo implantation and successful establishment of pregnancy. We previously reported that bone morphogenetic protein 2 (BMP2) mediates uterine stromal cell differentiation in mice and in humans. To identify the downstream target(s) of BMP2 signaling during decidualization, we performed gene-expression profiling of mouse uterine stromal cells, treated or not treated with recombinant BMP2. Our studies revealed that expression of Msx2, a member of the mammalian Msx homeobox gene family, was markedly upregulated in response to exogenous BMP2. Interestingly, conditional ablation of Msx2 in the uterus failed to prevent a decidual phenotype, presumably because of functional compensation of Msx2 by Msx1, a closely related member of the Msx family. Indeed, in Msx2-null uteri, the level of Msx1 expression in the stromal cells was markedly elevated. When conditional, tissue-specific ablation of both Msx1 and Msx2 was accomplished in the mouse uterus, a dramatically impaired decidual response was observed. In the absence of both Msx1 and Msx2, uterine stromal cells were able to proliferate, but they failed to undergo terminal differentiation. In parallel experiments, addition of BMP2 to human endometrial stromal cell cultures led to a robust enhancement of MSX1 and MSX2 expression and stimulated the differentiation process. Attenuation of MSX1 and MSX2 expression by small interfering RNAs greatly reduced human stromal differentiation in vitro, indicating a conservation of their roles as key mediators of BMP2-induced decidualization in mice and women.

Roles of Estrogen Receptor-α and the Coactivator MED1 During Human Endometrial Decidualization.

The steroid hormones 17ß-estradiol and progesterone are critical regulators of endometrial stromal cell differentiation, known as decidualization, which is a prerequisite for successful establishment of pregnancy. The present study using primary human endometrial stromal cells (HESCs) addressed the role of estrogen receptor-α (ESR1) in decidualization. Knockdown of ESR1 transcripts by RNA interference led to a marked reduction in decidualization of HESCs. Gene expression profiling at an early stage of decidualization indicated that ESR1 negatively regulates several cell cycle regulatory factors, thereby suppressing the proliferation of HESCs as these cells enter the differentiation program. ESR1 also controls the expression of WNT4, FOXO1, and progesterone receptor (PGR), well-known mediators of decidualization. Whereas ESR1 knockdown strongly inhibited the expression of FOXO1 and WNT4 transcripts within 24 hours of the initiation of decidualization, PGR expression remained unaffected at this early time point. Our study also revealed a major role of cAMP signaling in influencing the function of ESR1 during decidualization. Using a proteomic approach, we discovered that the cAMP-dependent protein kinase A (PKA) phosphorylates Mediator 1 (MED1), a subunit of the mediator coactivator complex, during HESC differentiation. Using immunoprecipitation, we demonstrated that PKA-phosphorylated MED1 interacts with ESR1. The PKA-dependent phosphorylation of MED1 was also correlated with its enhanced recruitment to estrogen-responsive elements in the WNT4 gene. Knockdown of MED1 transcripts impaired the expression of ESR1-induced WNT4 and FOXO1 transcripts and blocked decidualization. Based on these findings, we conclude that modulation of ESR1-MED1 interactions by cAMP signaling plays a critical role in human decidualization.

CRISPR EATING on a Low Budget.

In this issue of Developmental Cell, Lane et al. (2015) describe a simplified, inexpensive technique named "CRISPR EATING" for generating complex guide RNA libraries suitable for CRISPR/Cas9-based applications ranging from genome visualization and manipulation to genetic screens.