Single-cell analysis of chromatin silencing programs in development and tumor progression

Abstract

Single-cell analysis has become a powerful approach for the molecular characterization of complex tissues. Methods for quantifying gene expression and DNA accessibility of single cells are now well-established, but analysis of chromatin regions with specific histone modifications has been technically challenging. Here, we adapt the recently published CUT&Tag method to scalable single-cell platforms to profile chromatin landscapes in single cells (scCUT&Tag) from complex tissues. We focus on profiling Polycomb Group (PcG) silenced regions marked by H3K27 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 use scCUT&Tag to profile H3K27me3 in a brain tumor patient before and after treatment, identifying cell types in the tumor microenvironment and heterogeneity in PcG activity in the primary sample and after treatment.

Deep learning-based enhancement of epigenomics data with AtacWorks

“…We also demonstrate that AtacWorks can be adapted for crossmodality prediction of transcription factor footprints and ChIP-seq peaks from low-input ATAC-seq. As such, we anticipate this framework may be broadly useful for other deep learning applications in genomics, such as DNase, MNase, ChIP-seq, and the recently-developed method CUT&RUN 20 , which has comparable high-throughput single-cell adaptations 32,33 Finally, the robustness and speed of AtacWorks enable its application to high-throughput single-cell ATAC-seq datasets of heterogeneous tissues. We show that our method can be used on small subsets of rare lineage-priming cells to denoise signal and identify accessible regulatory regions at previously-unattainable genomic resolution.…”

Hierarchical chromatin regulation during blood formation uncovered by single-cell sortChIC

“…pA-Tn5-based strategies profile histone modifications with high sensitivity and throughput, but requires optimizing experimental conditions to reduce the intrinsic affinity of Tn5 to open chromatin regions, especially when studying repressive chromatin states (Bartosovic et al, 2021;Harada et al, 2019;Janssens et al, 2020;Kaya-Okur et al, 2019;Wang et al, 2019;Wu et al, 2021). pA-MNase-based methods profile histone modifications with high sensitivity, and have robust detection of modifications associated with euchromatic regions as well as heterochromatic regions, but has generally less throughput compared with Tn5-based methods (Hainer et al, 2019;Ku et al, 2019).…”

“…Alternatives to ChIP (Schmid et al, 2004) circumvent this pulldown by using antibody tethering of either protein-Amicrococcal nuclease (pA-MNase) (Ku et al, 2019;Skene and Henikoff, 2017) or protein-A-Tn5 transposase (Harada et al, 2019;Kaya-Okur et al, 2019), improving signal-to-noise by cutting only at specific sites of the genome. Although these tethering-based strategies have enabled large-scale profiling of histone modifications in single cells (Bartosovic et al, 2021;Janssens et al, 2020;Wu et al, 2021), they generally do not enrich for different cell types, making it difficult to dissect chromatin regulation in rare cell types, such as hematopoietic stem and progenitor cells in the bone marrow.…”

Deconvolving multiplexed histone modifications in single cells

“…Large-scale efforts characterizing different histone modifications in a variety of cell populations commonly use chromatin immunoprecipitation followed by sequencing (ChIP-seq) 10,11,[13][14][15][16] . Alternative strategies to ChIP-seq based on enzyme tethering (chromatin immunocleavage, ChIC) have reduced the background signal in profiling the epigenome 17 , and have enabled single-cell profiling of histone modifications [1][2][3][4][5][6][7][8][9]12 . Tethering strategies involve incubating cells with an antibody against a histone modification of interest, which then tethers either protein A-MNase 1,3,9,12 or protein A-Tn5 2, 4-8 fusion protein to generate targeted fragments in single cells.…”

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Recent advances have enabled mapping of histone modifications in single cells [1][2][3][4][5][6][7][8][9][10][11][12] , but current methods are constrained to profile only one histone modification per cell. Here we present an integrated experimental and computational framework, scChIX (single-cell chromatin immunocleavage and unmixing), to map multiple histone modifications in single cells.

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