Qingsong Tang

Expression and regulation of intergenic long noncoding RNAs during T cell development and differentiation

Although intergenic long noncoding RNAs (lincRNAs) have been linked to gene regulation in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identified 1,524 lincRNA clusters in 42 T cell samples, from early T cell progenitors to terminally differentiated helper T cell subsets. Our analysis revealed highly dynamic and cell-specific expression patterns for lincRNAs during T cell differentiation. These lincRNAs were located in genomic regions enriched for genes that encode proteins with immunoregulatory functions. Many were bound and regulated by the key transcription factors T-bet, GATA-3, STAT4 and STAT6. We found that the lincRNA LincR-Ccr2-5′AS, together with GATA-3, was an essential component of a regulatory circuit in gene expression specific to the TH2 subset of helper T cells and was important for the migration of TH2 cells.

H2A.Z Facilitates Access of Active and Repressive Complexes to Chromatin in Embryonic Stem Cell Self-Renewal and Differentiation

Chromatin modifications have been implicated in the self-renewal and differentiation of embryonic stem cells (ESCs). However, the function of histone variant H2A.Z in ESCs remains unclear. We show that H2A.Z is highly enriched at promoters and enhancers and is required for both efficient self-renewal and differentiation of murine ESCs. H2A.Z deposition leads to an abnormal nucleosome structure, decreased nucleosome occupancy, and increased chromatin accessibility. In self-renewing ESCs, knockdown of H2A.Z compromises OCT4 binding to its target genes and leads to decreased binding of MLL complexes to active genes and of PRC2 complex to repressed genes. During differentiation of ESCs, inhibition of H2A.Z also compromises RA-induced RARα binding, activation of differentiation markers, and the repression of pluripotency genes. We propose that H2A.Z mediates such contrasting activities by acting as a general facilitator that generates access for a variety of complexes, both activating and repressive.

Detection of single nucleotide variations in expressed exons of the human genome using RNA-Seq

Whole-genome resequencing is still a costly method to detect genetic mutations that lead to altered forms of proteins and may be associated with disease development. Since the majority of disease-related single nucleotide variations (SNVs) are found in protein-coding regions, we propose to identify SNVs in expressed exons of the human genome using the recently developed RNA-Seq technique. We identify 12 176 and 10 621 SNVs, respectively, in Jurkat T cells and CD4+ T cells from a healthy donor. Interestingly, our data show that one copy of the TAL-1 proto-oncogene has a point mutation in 3′ UTR and only the mutant allele is expressed in Jurkat cells. We provide a comprehensive dataset for further understanding the cancer biology of Jurkat cells. Our results indicate that this is a cost-effective and efficient strategy to systematically identify SNVs in the expressed regions of the human genome.

Characterization of genome-wide enhancer-promoter interactions reveals co-expression of interacting genes and modes of higher order chromatin organization

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 long-distance 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 chromosomes 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 organization. The Gene Expression Omnibus accession number for the raw and analyzed chromatin interaction data is GSE32677.

Regulation of nucleosome landscape and transcription factor targeting at tissue-specific enhancers by BRG1

Enhancers of transcription activate transcription via binding of sequence-specific transcription factors to their target sites in chromatin. In this report, we identify GATA1-bound distal sites genome-wide and find a global reorganization of the nucleosomes at these potential enhancers during differentiation of hematopoietic stem cells (HSCs) to erythrocytes. We show that the catalytic subunit BRG1 of BAF complexes localizes to these distal sites during differentiation and generates a longer nucleosome linker region surrounding the GATA1 sites by shifting the flanking nucleosomes away. Intriguingly, we find that the nucleosome shifting specifically facilitates binding of TAL1 but not GATA1 and is linked to subsequent transcriptional regulation of target genes.

Genome-wide detection of DNase I hypersensitive sites in single cells and FFPE tissue samples

DNase I hypersensitive sites (DHSs) provide important information on the presence of transcriptional regulatory elements and the state of chromatin in mammalian cells. Conventional DNase sequencing (DNase-seq) for genome-wide DHSs profiling is limited by the requirement of millions of cells. Here we report an ultrasensitive strategy, called single-cell DNase sequencing (scDNase-seq) for detection of genome-wide DHSs in single cells. We show that DHS patterns at the single-cell level are highly reproducible among individual cells. Among different single cells, highly expressed gene promoters and enhancers associated with multiple active histone modifications display constitutive DHS whereas chromatin regions with fewer histone modifications exhibit high variation of DHS. Furthermore, the single-cell DHSs predict enhancers that regulate cell-specific gene expression programs and the cell-to-cell variations of DHS are predictive of gene expression. Finally, we apply scDNase-seq to pools of tumour cells and pools of normal cells, dissected from formalin-fixed paraffin-embedded tissue slides from patients with thyroid cancer, and detect thousands of tumour-specific DHSs. Many of these DHSs are associated with promoters and enhancers critically involved in cancer development. Analysis of the DHS sequences uncovers one mutation (chr18: 52417839G>C) in the tumour cells of a patient with follicular thyroid carcinoma, which affects the binding of the tumour suppressor protein p53 and correlates with decreased expression of its target gene TXNL1. In conclusion, scDNase-seq can reliably detect DHSs in single cells, greatly extending the range of applications of DHS analysis both for basic and for translational research, and may provide critical information for personalized medicine.

Dynamic regulation of epigenomic landscapes during hematopoiesis

BackgroundHuman blood develops from self-renewing hematopoietic stem cells to terminal lineages and necessitates regulator and effector gene expression changes; each cell type specifically expresses a subset of genes to carry out a specific function. Gene expression changes coincide with histone modification, histone variant deposition, and recruitment of transcription-related enzymes to specific genetic loci. Transcriptional regulation has been mostly studied using in vitro systems while epigenetic changes occurring during in vivo development remain poorly understood.ResultsBy integrating previously published and novel global expression profiles from human CD34+/CD133+ hematopoietic stem and progenitor cells (HSPCs), in vivo differentiated human CD4+ T-cells and CD19+ B-cells, and in vitro differentiated CD36+ erythrocyte precursors, we identified hundreds of transcripts specifically expressed in each cell type. To relate concurrent epigenomic changes to expression, we examined genome-wide distributions of H3K4me1, H3K4me3, H3K27me1, H3K27me3, histone variant H2A.Z, ATP-dependent chromatin remodeler BRG1, and RNA Polymerase II in these cell types, as well as embryonic stem cells. These datasets revealed that numerous differentiation genes are primed for subsequent downstream expression by BRG1 and PolII binding in HSPCs, as well as the bivalent H3K4me3 and H3K27me3 modifications in the HSPCs prior to their expression in downstream, differentiated cell types; much HSPC bivalency is retained from embryonic stem cells. After differentiation, bivalency resolves to active chromatin configuration in the specific lineage, while it remains in parallel differentiated lineages. PolII and BRG1 are lost in closer lineages; bivalency resolves to silent monovalency in more distant lineages. Correlation of expression with epigenomic changes predicts tens of thousands of potential common and tissue-specific enhancers, which may contribute to expression patterns and differentiation pathways.ConclusionsSeveral crucial lineage factors are bivalently prepared for their eventual expression or repression. Bivalency is not only resolved during differentiation but is also established in a step-wise manner in differentiated cell types. We note a progressive, specific silencing of alternate lineage genes in certain cell types coinciding with H3K27me3 enrichment, though expression silencing is maintained in its absence. Globally, the expression of type-specific genes across many cell types correlates strongly with their epigenetic profiles. These epigenomic data appear useful for further understanding mechanisms of differentiation and function of human blood lineages.

Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq

H2A is a nucleosome core subunit involved in organizing DNA into a chromatin structure that is often inaccessible to regulatory enzymes. Replacement of H2A by its variant H2A.Z renders chromatin accessible at enhancers and promoters. However, it remains unclear how H2A.Z functions so differently from canonical H2A. Here we report the genome-wide identification of proteins that directly interact with H2A and H2A.Z in vivo using a novel strategy, bPPI-seq. We show that bPPI-seq is a sensitive and robust technique to identify protein-protein interactions in vivo. Our data indicate that H2A.Z-interacting proteins and H2A-interacting proteins participate in distinct biological processes. In contrast to H2A-interacting proteins, the H2A.Z-interacting proteins are involved in transcriptional regulation. We found that the transcription factor Osr1 interacts with H2A.Z both in vitro and in vivo. It also mediates H2A.Z incorporation to a large number of target sites and regulates gene expression. Our data indicate that bPPI-seq can be widely applied to identify genome-wide interacting proteins under physiological conditions.

Trac-looping measures genome structure and chromatin accessibility

Long-range chromatin interactions play critical roles in genome organization and regulation of transcription. We now report transposase-mediated analysis of chromatin looping (Trac-looping) for simultaneous detection of multiscale genome-wide chromatin interactions among regulatory elements and chromatin accessibility. With this technique, a bivalent oligonucleotide linker is inserted between two interacting regions such that the chromatin interactions are captured without prior chromatin fragmentation and proximity-based ligation. Application of Trac-looping to human CD4+ T cells revealed substantial reorganization of enhancer–promoter interactions associated with changes in gene expression after T cell receptor stimulation.Insertion of a bivalent linker between two regions of interest allows chromatin contacts to be probed without proximity ligation.

Principles of nucleosome organization revealed by single-cell micrococcal nuclease sequencing

Nucleosome positioning is critical to chromatin accessibility and is associated with gene expression programs in cells1–3. Previous nucleosome mapping methods assemble profiles from cell populations and reveal a cell-averaged pattern: nucleosomes are positioned and form a phased array that surrounds the transcription start sites of active genes3–6 and DNase I hypersensitive sites7. However, even in a homogenous population of cells, cells exhibit heterogeneity in expression in response to active signalling8,9 that may be related to heterogeneity in chromatin accessibility10–12. Here we report a technique, termed single-cell micrococcal nuclease sequencing (scMNase-seq), that can be used to simultaneously measure genome-wide nucleosome positioning and chromatin accessibility in single cells. Application of scMNase-seq to NIH3T3 cells, mouse primary naive CD4 T cells and mouse embryonic stem cells reveals two principles of nucleosome organization: first, nucleosomes in heterochromatin regions, or that surround the transcription start sites of silent genes, show large variation in positioning across different cells but are highly uniformly spaced along the nucleosome array; and second, nucleosomes that surround the transcription start sites of active genes and DNase I hypersensitive sites show little variation in positioning across different cells but are relatively heterogeneously spaced along the nucleosome array. We found a bimodal distribution of nucleosome spacing at DNase I hypersensitive sites, which corresponds to inaccessible and accessible states and is associated with nucleosome variation and variation in accessibility across cells. Nucleosome variation is smaller within single cells than across cells, and smaller within the same cell type than across cell types. A large fraction of naive CD4 T cells and mouse embryonic stem cells shows depleted nucleosome occupancy at the de novo enhancers detected in their respective differentiated lineages, revealing the existence of cells primed for differentiation to specific lineages in undifferentiated cell populations.Single-cell micrococcal nuclease sequencing simultaneously measures chromatin accessibility and genome-wide nucleosome positioning in single cells to reveal principles of nucleosome organization.