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Single-cell sequencing-based methods for profiling gene transcript levels have revealed substantial heterogeneity in expression levels among morphologically indistinguishable cells. This variability has important functional implications for tissue biology and disease states such as cancer. Mapping of epigenomic information such as chromatin accessibility, nucleosome positioning, histone tail modifications and enhancer–promoter interactions in both bulk-cell and single-cell samples has shown that these characteristics of chromatin state contribute to expression or repression of associated genes. Advances in single-cell epigenomic profiling methods are enabling high-resolution mapping of chromatin states in individual cells. Recent studies using these techniques provide evidence that variations in different aspects of chromatin organization collectively define gene expression heterogeneity among otherwise highly similar cells.In this Review, Carter and Zhao discuss how single-cell sequencing technologies are being applied to investigate epigenetic heterogeneity among seemingly homogeneous populations of cells and how this epigenetic variability relates to cell–cell differences in gene expression.

Although the function of DNA methylation in gene promoter regions is well established in transcriptional repression, the function of the evolutionarily conserved widespread distribution of DNA methylation in gene body regions remains incompletely understood. Here, we show that DNA methylation is enriched in included alternatively spliced exons （ASEs）, and that inhibition of DNA methylation results in aberrant splicing of ASEs. The methyl- CpG-binding protein MeCP2 is enriched in included ASEs, particularly those that are also highly methylated, and inhibition of DNA methylation disrupts specific targeting of MeCP2 to exons. Interestingly, ablation of MeCP2 results in increased histone acetylation and aberrant ASE-skipping events. We further show that inhibition of histone deacetylase （HDAC） activity leads to exon skipping that shows a highly significant degree of overlap with that caused by MeCP2 knockdown. Together, our data indicate that intragenic DNA methylation operates in exon definition to modulate alternative RNA splicing and can enhance exon recognition via recruitment of the multifunctional protein MeCP2, which thereby maintains local histone hypoacetylation through the subsequent recruitment of HDACs.

Our method for analyzing histone modifications, scChIC-seq (single-cell chromatin immunocleavage sequencing), involves targeting of the micrococcal nuclease (MNase) to a histone mark of choice by tethering to a specific antibody. Cleaved target sites are then selectively PCR amplified. We show that scChIC-seq reliably detects H3K4me3 and H3K27me3 target sites in single human white blood cells. The resulting data are used for clustering of blood cell types.Covalent linking of a histone-modification-specific antibody to MNase allows for the isolation of fragments with the desired histone mark, which can be amplified and sequenced. This approach is sensitive enough to profile histone modifications in single cells.

Modern next-generation sequencing-based methods have empowered researchers to assay the epigenetic states of individual cells. Existing techniques for profiling epigenetic marks in single cells often require the use and optimization of time-intensive procedures such as drop fluidics, chromatin fragmentation, and end repair. Here we describe ACT-seq, a streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. We conclude that ACT-seq present an attractive alternative to existing techniques for mapping epigenetic marks in single cells. The authors introduce ACT-seq: a Tn5-based method for rapidly profiling epigenetic marks in bulk-cell and single-cell samples. ACT-seq avoids many laborious or time-consuming steps required for similar techniques including chromatin fragmentation and end repair.

Innate lymphoid cells (ILCs) play important functions in immunity and tissue homeostasis, but their development is poorly understood. Through the use of single-cell approaches, we examined the transcriptional and functional heterogeneity of ILC progenitors, and studied the precursor–product relationships that link the subsets identified. This analysis identified two successive stages of ILC development within T cell factor 1-positive (TCF-1 + ) early innate lymphoid progenitors (EILPs), which we named ‘specified EILPs’ and ‘committed EILPs’. Specified EILPs generated dendritic cells, whereas this potential was greatly decreased in committed EILPs. TCF-1 was dispensable for the generation of specified EILPs, but required for the generation of committed EILPs. TCF-1 used a pre-existing regulatory landscape established in upstream lymphoid precursors to bind chromatin in EILPs. Our results provide insight into the mechanisms by which TCF-1 promotes developmental progression of ILC precursors, while constraining their dendritic cell lineage potential and enforcing commitment to ILC fate. Bhandoola and colleagues show that the transcription factor TCF-1 restrains the dendritic cell lineage potential in innate lymphoid cell progenitors.

Recent epigenomic studies have predicted thousands of potential enhancers in the human genome. However, there has not been systematic characterization of target promoters for these potential enhancers. Using H3K4me2 as a mark for active enhancers, we identified genome-wide EP interactions in human CD4＋ T cells. Among the 6 520 longdistance chromatin interactions, we identify 2 067 enhancers that interact with 1 619 promoters and enhance their expression. These enhancers exist in accessible chromatin regions and are associated with various histone modifications and polymerase II binding. The promoters with interacting enhancers are expressed at higher levels than those without interacting enhancers, and their expression levels are positively correlated with the number of interacting enhancers. Interestingly, interacting promoters are co-expressed in a tissue-specific manner. We also find that chro- mosomes are organized into multiple levels of interacting domains. Our results define a global view of EP interactions and provide a data set to further understand mechanisms of enhancer targeting and long-range chromatin orga nization. The Gene Expression Omnibus accession number for the raw and analyzed chromatin interaction data is GSE32677.

The molecular pathways underlying the development of innate lymphoid cells (ILCs) are mostly unknown. Here we show that TGF-β signaling programs the development of ILC2s from their progenitors. Specifically, the deficiency of TGF-β receptor II in bone marrow progenitors results in inefficient development of ILC2s, but not ILC1s or ILC3s. Mechanistically, TGF-β signaling is required for the generation and maintenance of ILC2 progenitors (ILC2p). In addition, TGF-β upregulates the expression of the IL-33 receptor gene Il1rl1 (encoding IL-1 receptor-like 1, also known as ST2) in ILC2p and common helper-like innate lymphoid progenitors (CHILP), at least partially through the MEK-dependent pathway. These findings identify a function of TGF-β in the development of ILC2s from their progenitors. TGF-β is thought to be important for group 2 innate lymphoid cell (ILC2) function. Here the authors show that TGF-β drives expression of ST2 specifically in ILC2 progenitors and thereby is also important for the development of ILC2s in the bone marrow.

Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum ; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown. Results We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized. Conclusions It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.

Topoisomerase 3β (Top3β) is the only dual-activity topoisomerase in animals that can change topology for both DNA and RNA, and facilitate transcription on DNA and translation on mRNAs. Top3β mutations have been linked to schizophrenia, autism, epilepsy, and cognitive impairment. Here we show that Top3β knockout mice exhibit behavioural phenotypes related to psychiatric disorders and cognitive impairment. The mice also display impairments in hippocampal neurogenesis and synaptic plasticity. Notably, the brains of the mutant mice exhibit impaired global neuronal activity-dependent transcription in response to fear conditioning stress, and the affected genes include many with known neuronal functions. Our data suggest that Top3β is essential for normal brain function, and that defective neuronal activity-dependent transcription may be a mechanism by which Top3β deletion causes cognitive impairment and psychiatric disorders. Topoisomerase 3β (Top3β) solves topological stress in DNA or RNA metabolism and its mutations are linked to mental disorders. Here, the authors describe transcriptional and behavioural impairments in Top3β-knockout mice and show that these are linked to impaired neurogenesis and synaptic plasticity.

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. Genomic studies have revealed that HCC is a heterogeneous disease with multiple subtypes. BRG1, encoded by the SMARCA4 gene, is a key component of SWI/SNF chromatin-remodeling complexes. Based on TCGA studies, somatic mutations of SMARCA4 occur in ~3% of human HCC samples. Additional studies suggest that BRG1 is overexpressed in human HCC specimens and may promote HCC growth and invasion. However, the precise functional roles of BRG1 in HCC remain poorly delineated. Here, we analyzed BRG1 in human HCC samples as well as in mouse models. We found that BRG1 is overexpressed in most of human HCC samples, especially in those associated with poorer prognosis. BRG1 expression levels positively correlate with cell cycle and negatively with metabolic pathways in the Cancer Genome Atlas (TCGA) human HCC data set. In a murine HCC model induced by c-MYC overexpression, ablation of the Brg1 gene completely repressed HCC formation. In striking contrast, however, we discovered that concomitant deletion of Brg1 and overexpression of c-Met or mutant NRas (NRAS V12 ) triggered HCC formation in mice. Altogether, the present data indicate that BRG1 possesses both oncogenic and tumor-suppressing roles depending on the oncogenic stimuli during hepatocarcinogenesis.