3D Gene Expression Research Articles

Integration of scHi-C and scRNA-seq data defines distinct 3D-regulated and biological-context dependent cell subpopulations

An integration of 3D chromatin structure and gene expression at single-cell resolution has yet been demonstrated. Here, we develop a computational method, a multiomic data integration (MUDI) algorithm, which integrates scHi-C and scRNA-seq data to precisely define the 3D-regulated and biological-context dependent cell subpopulations or topologically integrated subpopulations (TISPs). We demonstrate its algorithmic utility on the publicly available and newly generated scHi-C and scRNA-seq data. We then test and apply MUDI in a breast cancer cell model system to demonstrate its biological-context dependent utility. We find the newly defined topologically conserved associating domain (CAD) is the characteristic single-cell 3D chromatin structure and better characterizes chromatin domains in single-cell resolution. We further identify 20 TISPs uniquely characterizing 3D-regulated breast cancer cellular states. We reveal two of TISPs are remarkably resemble to high cycling breast cancer persister cells and chromatin modifying enzymes might be functional regulators to drive the alteration of the 3D chromatin structures. Our comprehensive integration of scHi-C and scRNA-seq data in cancer cells at single-cell resolution provides mechanistic insights into 3D-regulated heterogeneity of developing drug-tolerant cancer cells.

Unsupervised pattern identification in spatial gene expression atlas reveals mouse brain regions beyond established ontology

The rapid growth of large-scale spatial gene expression data demands efficient and reliable computational tools to extract major trends of gene expression in their native spatial context. Here, we used stability-driven unsupervised learning (i.e., staNMF) to identify principal patterns (PPs) of 3D gene expression profiles and understand spatial gene distribution and anatomical localization at the whole mouse brain level. Our subsequent spatial correlation analysis systematically compared the PPs to known anatomical regions and ontology from the Allen Mouse Brain Atlas using spatial neighborhoods. We demonstrate that our stable and spatially coherent PPs, whose linear combinations accurately approximate the spatial gene data, are highly correlated with combinations of expert-annotated brain regions. These PPs yield a brain ontology based purely on spatial gene expression. Our PP identification approach outperforms principal component analysis and typical clustering algorithms on the same task. Moreover, we show that the stable PPs reveal marked regional imbalance of brainwide genetic architecture, leading to region-specific marker genes and gene coexpression networks. Our findings highlight the advantages of stability-driven machine learning for plausible biological discovery from dense spatial gene expression data, streamlining tasks that are infeasible by conventional manual approaches.

GAGE-seq concurrently profiles multiscale 3D genome organization and gene expression in single cells.

The organization of mammalian genomes features a complex, multiscale three-dimensional (3D) architecture, whose functional significance remains elusive because of limited single-cell technologies that can concurrently profile genome organization and transcriptional activities. Here, we introduce genome architecture and gene expression by sequencing (GAGE-seq), a scalable, robust single-cell co-assay measuring 3D genome structure and transcriptome simultaneously within the same cell. Applied to mouse brain cortex and human bone marrow CD34+ cells, GAGE-seq characterized the intricate relationships between 3D genome and gene expression, showing that multiscale 3D genome features inform cell-type-specific gene expression and link regulatory elements to target genes. Integration with spatial transcriptomic data revealed in situ 3D genome variations in mouse cortex. Observations in human hematopoiesis unveiled discordant changes between 3D genome organization and gene expression, underscoring a complex, temporal interplay at the single-cell level. GAGE-seq provides a powerful, cost-effective approach for exploring genome structure and gene expression relationships at the single-cell level across diverse biological contexts.

Simultaneous single-cell three-dimensional genome and gene expression profiling uncovers dynamic enhancer connectivity underlying olfactory receptor choice

The simultaneous measurement of three-dimensional (3D) genome structure and gene expression of individual cells is critical for understanding a genome’s structure–function relationship, yet this is challenging for existing methods. Here we present ‘Linking mRNA to Chromatin Architecture (LiMCA)’, which jointly profiles the 3D genome and transcriptome with exceptional sensitivity and from low-input materials. Combining LiMCA and our high-resolution scATAC-seq assay, METATAC, we successfully characterized chromatin accessibility, as well as paired 3D genome structures and gene expression information, of individual developing olfactory sensory neurons. We expanded the repertoire of known olfactory receptor (OR) enhancers and discovered unexpected rules of their dynamics: OR genes and their enhancers are most accessible during early differentiation. Furthermore, we revealed the dynamic spatial relationship between ORs and enhancers behind stepwise OR expression. These findings offer valuable insights into how 3D connectivity of ORs and enhancers dynamically orchestrate the ‘one neuron–one receptor’ selection process.

Mesenchymal Stem Cell-Laden In Situ-Forming Hydrogel for Preventing Corneal Stromal Opacity.

The aims of this study were to construct a mesenchymal stem cell (MSC)-laden in situ-forming hydrogel and study its effects on preventing corneal stromal opacity. The native gellan gum was modified by high temperature and pressure, and the rabbit bone marrow MSCs were encapsulated before adding Ca 2+ to initiate cross-linking. The effects of the hydrogel on 3D culture and gene expression of the rabbit bone marrow MSCs were observed in vitro. Then, the MSC-hydrogel was used to repair corneal stromal injury in New Zealand white rabbits within 28 days postoperation. The short-chain gellan gum solution has a very low viscosity (<0.1 Pa·s) that is ideal for encapsulating cells. Moreover, mRNA expressions of 3D-cultured MSCs coding for corneal stromal components (decorin, lumican, and keratocan) were upregulated (by 127.8, 165.5, and 25.4 times, respectively) ( P < 0.05) on day 21 in vitro and were verified by Western blotting results. For the in vivo study, the corneal densitometry of the experimental group was (20.73 ± 1.85) grayscale units which was lower than the other groups ( P < 0.05). The MSC-hydrogel downregulated mRNA expression coding for fibrosis markers (α-smooth muscle actin, vimentin, collagen type 5-α1, and collagen type 1-α1) in the rabbit corneal stroma. Furthermore, some of the 5-ethynyl-2'-deoxyuridine (EdU)-labeled MSCs integrated into the upper corneal stroma and expressed keratocyte-specific antigens on day 28 postoperation. The short-chain gellan gum allows MSCs to slowly release to the corneal stromal defect and prevent corneal stromal opacity. Some of the implanted MSCs can integrate into the corneal stroma and differentiate into keratocytes.

Old school, new rules: floral meristem development revealed by 3D gene expression atlases and high-resolution transcription factor-chromatin dynamics.

The intricate morphology of the flower is primarily established within floral meristems in which floral organs will be defined and from where the developing flower will emerge. Floral meristem development involves multiscale-level regulation, including lineage and positional mechanisms for establishing cell-type identity, and transcriptional regulation mediated by changes in the chromatin environment. However, many key aspects of floral meristem development remain to be determined, such as: 1) the exact role of cellular location in connecting transcriptional inputs to morphological outcomes, and 2) the precise interactions between transcription factors and chromatin regulators underlying the transcriptional networks that regulate the transition from cell proliferation to differentiation during floral meristem development. Here, we highlight recent studies addressing these points through newly developed spatial reconstruction techniques and high-resolution transcription factor-chromatin environment interactions in the model plant Arabidopsis thaliana. Specifically, we feature studies that reconstructed 3D gene expression atlases of the floral meristem. We also discuss how the precise timing of floral meristem specification, floral organ patterning, and floral meristem termination is determined through temporally defined epigenetic dynamics for fine-tuning of gene expression. These studies offer fresh insights into the well-established principles of floral meristem development and outline the potential for further advances in this field in an age of integrated, powerful, multiscale resolution approaches.

A rapid and sensitive, multiplex, whole mount RNA fluorescence in situ hybridization and immunohistochemistry protocol

BackgroundIn the past few years, there has been an explosion in single-cell transcriptomics datasets, yet in vivo confirmation of these datasets is hampered in plants due to lack of robust validation methods. Likewise, modeling of plant development is hampered by paucity of spatial gene expression data. RNA fluorescence in situ hybridization (FISH) enables investigation of gene expression in the context of tissue type. Despite development of FISH methods for plants, easy and reliable whole mount FISH protocols have not yet been reported.ResultsWe adapt a 3-day whole mount RNA-FISH method for plant species based on a combination of prior protocols that employs hybridization chain reaction (HCR), which amplifies the probe signal in an antibody-free manner. Our whole mount HCR RNA-FISH method shows expected spatial signals with low background for gene transcripts with known spatial expression patterns in Arabidopsis inflorescences and monocot roots. It allows simultaneous detection of three transcripts in 3D. We also show that HCR RNA-FISH can be combined with endogenous fluorescent protein detection and with our improved immunohistochemistry (IHC) protocol.ConclusionsThe whole mount HCR RNA-FISH and IHC methods allow easy investigation of 3D spatial gene expression patterns in entire plant tissues.

Three-dimensional chromatin landscapes in hepatocellular carcinoma associated with hepatitis B virus.

Three-dimensional (3D) chromatin architecture frequently altered in cancer. However, its changes during the pathogenesis of hepatocellular carcinoma (HCC) remained elusive. Hi-C and RNA-seq were applied to study the 3D chromatin landscapes and gene expression of HCC and ANHT. Hi-C Pro was used to generate genome-wide raw interaction matrices, which were normalized via iterative correction (ICE). Moreover, the chromosomes were divided into different compartments according to the first principal component (E1). Furthermore, topologically associated domains (TADs) were visualized via WashU Epigenome Browser. Furthermore, differential expression analysis of ANHT and HCC was performed using the DESeq2 R package. Additionally, dysregulated genes associated with 3D genome architecture altered were confirmed using TCGA, qRT-PCR, immunohistochemistry (IHC), etc. RESULTS: First, the intrachromosomal interactions of chr1, chr2, chr5, and chr11 were significantly different, and the interchromosomal interactions of chr4-chr10, chr13-chr21, chr15-chr22, and chr16-chr19 are remarkably different between ANHT and HCC, which resulted in the up-regulation of TP53I3 and ZNF738 and the down-regulation of APOC3 and APOA5 in HCC. Second, 49 compartment regions on 18 chromosomes have significantly switched (A-B or B-A) during HCC tumorigenesis, contributing to up-regulation of RAP2A. Finally, a tumor-specific TAD boundary located on chr5: 6271000-6478000 and enhancer hijacking were identified in HCC tissues, potentially associated with the elevated expression of MED10, whose expression were associated with poor prognosis of HCC patients. This study demonstrates the crucial role of chromosomal structure variation in HCC oncogenesis and potential novel biomarkers of HCC, laying a foundation for cancer precision medicine development.

Remodeling of the 3D chromatin architecture in the marine microalga Nannochloropsis oceanica during lipid accumulation

BackgroundGenomic three-dimensional (3D) spatial organization plays a key role in shaping gene expression and associated chromatin modification, and it is highly sensitive to environmental stress conditions. In microalgae, exposure to nitrogen stress can drive lipid accumulation, yet the associated functional alterations in the spatial organization of the microalgal genome have yet to be effectively characterized.ResultsAccordingly, the present study employed RNA-seq, Hi-C, and ChIP-seq approaches to explore the relationship between 3D chromosomal architecture and gene expression during lipid accumulation in the marine microalga Nannochloropsis oceanica in response to nitrogen deprivation (ND). These analyses revealed that ND resulted in various changes in chromosomal organization, including A/B compartment transitions, topologically associating domain (TAD) shifts, and the disruption of short-range interactions. Significantly higher levels of gene expression were evident in A compartments and TAD boundary regions relative to B compartments and TAD interior regions, consistent with observed histone modification enrichment in these areas. ND-induced differentially expressed genes (DEGs) were notably enriched in altered TAD-associated regions and regions exhibiting differential genomic contact. These DEGs were subjected to Gene Ontology (GO) term analyses that indicated they were enriched in the ‘fatty acid metabolism’, ‘response to stress’, ‘carbon fixation’ and ‘photosynthesis’ functional categories, in line with the ND treatment conditions used to conduct this study. These data indicate that Nannochloropsis cells exhibit a clear association between chromatin organization and transcriptional activity under nitrogen stress conditions. Pronounced and extensive histone modifications were evident in response to ND. Observed changes in chromatin architecture were linked to shifts in histone modifications and gene expression.ConclusionsOverall, the reprogramming of many lipid metabolism-associated genes was evident under nitrogen stress conditions with respect to both histone modifications and chromosomal organization. Together these results revealed that higher-order chromatin architecture represents a new layer that can guide efforts to understand the transcriptional regulation of lipid metabolism in nitrogen-deprived microalgae.

Exogenous interleukin-1 beta stimulation regulates equine tenocyte function and gene expression in three-dimensional culture which can be rescued by pharmacological inhibition of interleukin 1 receptor, but not nuclear factor kappa B, signaling

We investigated how Interleukin 1 beta (IL-1β) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF-KB) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1β for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF-KB inhibitors on gel contraction and IL6 secretion were measured in 3D culture, with NF-KB-P65 nuclear translocation by immunofluorescence and gene expression by qPCR measured in two-dimensional (2D) monolayer culture. In addition, daily 3D gel contraction and transcriptomic analysis was performed on interleukin 1 receptor antagonist-treated 3D gels at day 14. IL-1β increased NF-KB-P65 nuclear translocation in 2D culture and IL6 secretion in 3D culture, but reduced daily tenocyte 3D gel contraction and impacted > 2500 genes at day 14, with enrichment for NF-KB signaling. Administering direct pharmacological inhibitors of NF-KB did reduce NF-KB-P65 nuclear translocation, but had no effect on 3D gel contraction or IL6 secretion in the presence of IL-1β. However, IL1Ra restored 3D gel contraction and partially rescued global gene expression. Tenocyte 3D gel contraction and gene expression is adversely impacted by IL-1β which can only be rescued by blockade of interleukin 1 receptor, but not NF-KB, signalling.

Spatial transcriptomics reveals a cnidarian segment polarity program in Nematostella vectensis

During early animal evolution, the emergence of axially polarized segments was central to the diversification of complex bilaterian body plans. Nevertheless, precisely how and when segment polarity pathways arose remains obscure. Here, we demonstrate the molecular basis for segment polarization in developing larvae of the sea anemone Nematostella vectensis. Utilizing spatial transcriptomics, we first constructed a 3D gene expression atlas of developing larval segments. Capitalizing on accurate in silico predictions, we identified Lbx and Uncx, conserved homeodomain-containing genes that occupy opposing subsegmental domains under the control of both bone morphogenetic protein (BMP) signaling and the Hox-Gbx cascade. Functionally, Lbx mutagenesis eliminated all molecular evidence of segment polarization at the larval stage and caused an aberrant mirror-symmetric pattern of retractor muscles (RMs) in primary polyps. These results demonstrate the molecular basis for segment polarity in a non-bilaterian animal, suggesting that polarized metameric structures were present in the Cnidaria-Bilateria common ancestor over 600 million years ago.

Simultaneous Profiling of Chromosome Conformation and Gene Expression in Single Cells.

Rapid development in single-cell chromosome conformation capture technologies has provided valuable insights into the importance of spatial genome architecture for gene regulation. However, a long-standing technical gap remains in the simultaneous characterization of three-dimensional genomes and transcriptomes in the same cell. We have described an assay named Hi-C and RNA-seq employed simultaneously (HiRES), which integrates in situ reverse transcription and chromosome conformation capture (3C) for the parallel analysis of chromatin organization and gene expression. Here, we provide a detailed implementation of the assay, using mouse embryos and cerebral cortices as examples. The versatility of this method extends beyond these two samples, with the potential to be used in various other cell types. Key features • A multi-omics sequencing approach to profile 3D genome structure and gene expression simultaneously in single cells. • Compatible with animal tissues. • One-tube amplification of both DNA and RNA components. • Requires three days to complete.

De novo discovery of traits co-occurring with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous group of chronic lung conditions. Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) associated with COPD and the co-occurring conditions, suggesting common biological mechanisms underlying COPD and these co-occurring conditions. To identify them, we have integrated information across different biological levels (i.e., genetic variants, lung-specific 3D genome structure, gene expression and protein-protein interactions) to build lung-specific gene regulatory and protein-protein interaction networks. We have queried these networks using disease-associated SNPs for COPD, unipolar depression and coronary artery disease. COPD-associated SNPs can control genes involved in the regulation of lung or pulmonary function, asthma, brain region volumes, cortical surface area, depressed affect, neuroticism, Parkinson's disease, white matter microstructure and smoking behaviour. We describe the regulatory connections, genes and biochemical pathways that underlay these co-occurring trait-SNP-gene associations. Collectively, our findings provide new avenues for the investigation of the underlying biology and diverse clinical presentations of COPD. In so doing, we identify a collection of genetic variants and genes that may aid COPD patient stratification and treatment.

Combined method of whole mount and block-face imaging: Acquisition of 3D data of gene expression pattern from conventional in situ hybridization.

Visualization of spatiotemporal expression of a gene of interest is a fundamental technique for analyzing the involvements of genes in organ development. In situ hybridization (ISH) is one of the most popular methods for visualizing gene expression. When conventional ISH is performed on sections or whole-mount specimens, the gene expression pattern is represented in 2-dimensional (2D) microscopic images or in the surface view of the specimen. To obtain 3-dimensional (3D) data of gene expression from conventional ISH, the "serial section method" has traditionally been employed. However, this method requires an extensive amount of time and labor because it requires researchers to collect a tremendous number of sections, label all sections by ISH, and image them before 3D reconstruction. Here, we proposed a rapid and low-cost 3D imaging method that can create 3D gene expression patterns from conventional ISH-labeled specimens. Our method consists of a combination of whole-mount ISH and Correlative Microscopy and Blockface imaging (CoMBI). The whole-mount ISH-labeled specimens were sliced using a microtome or cryostat, and all block-faces were imaged and used to reconstruct 3D images by CoMBI. The 3D data acquired using our method showed sufficient quality to analyze the morphology and gene expression patterns in the developing mouse heart. In addition, 2D microscopic images of the sections can be obtained when needed. Correlating 2D microscopic images and 3D data can help annotate gene expression patterns and understand the anatomy of developing organs. These results indicated that our method can be useful in the field of developmental biology.

Apoptotic effects of Phlomis armeniaca mediated biosynthesized silver nanoparticles in monolayer (2D) and spheroid (3D) cultures of human breast cancer cell lines.

The purpose of current research was to assess the apoptotic effects of biofabrication silver nanoparticles (AgNPs) mediated by the aqueous extract of Phlomis armeniaca on human breast cancer cells (MCF-7 and MDA-MB-231) in monolayer (2D) and spheroid (3D) cultures. The biosynthesized AgNPs were characterized by UV-Vis spectrophotometer (the peaks of resonances at 432nm), scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDS). 1-20µM/mL AgNPs were applied to MCF-7 and MDA-MB-231 cell lines to determine IC50 values at 24, 48 and 72nd h and were found to be 10µM/mL for both cell lines. Immunohistochemical staining results of BrdU, TUNEL, caspase-3 and Endo G in both 2D and 3D cultures and gene expression levels of caspases (caspase-3, -8 and -9) and Endo G were evaluated. Moreover, the total oxidant/antioxidant status (TOS-TAS) due to AgNPs application in both cell culture mediums was evaluated. AgNPs treatment results in both cell lines in both 2D and 3D cultures showed a significant decrease in the BrdU labeling index, while large amounts of cells were labelled with TUNEL and Endo G. In 2D culture, Endo G expression increased in MCF-7 cells at 48 and 72nd hours, while it increased significantly in MDA-MB-231 cells at all hours. OSI results show that ROS production is increased in cell medium treated with AgNPs. In conclusion, AgNPs mediated by Phlomis armeniaca, synthesized by a green method, successfully induced damage to mitochondria, resulting in cell cycle arrest and consequent cell proliferation blockade and death in both MCF-7 and MDA-MB-231 cells.

3D chromatin structure in human macrophages and iPSC‐derived microglia identifies putative Alzheimer’s Disease risk genes

AbstractBackgroundAlzheimer’s disease (AD) genome‐wide association studies (GWAS) identified 75 disease‐associated loci. The vast majority of risk variants are located in non‐coding genomic regions, suggesting a regulatory function (i.e. enhancer activity). Enhancers can regulate distal genes via DNA loops, bringing elements millions of base pairs away into close physical proximity in three‐dimensional (3D) space. Mapping 3D chromatin contact frequencies using Hi‐C can reveal enhancer‐promoter connections and assign disease‐associated variants to putative causal genes. Because enhancer and 3D chromatin structures are cell‐type specific, it is critical to map these features in the relevant cell type and biological context. Genetic risk factors for AD are located within microglia and macrophages chromatin‐accessible regions. The aim of this work was to map 3D chromatin structures in human microglia and macrophages and intersect them with AD GWAS to identify novel genes regulated by non‐coding AD risk variants.MethodWe performed deeply sequenced Hi‐C, RNA‐seq, ATAC‐seq, and Cut&amp;Run targeting histone H3K27 acetylation to map regulatory networks in THP‐1 macrophages and human iPSC‐derived microglia‐like cells (IMGLs). Next, we performed differential analysis to identify cell‐type specific differences in 3D chromatin structure, enhancer activity, and gene expression. Finally, we intersected these regulatory networks with AD GWAS to identify variants that may disrupt these networks and reveal novel AD risk genes for further study.ResultWe identified 19,608 chromatin loops, 766 of which were specific to either cell type. These loops connected 14,413 enhancers (8,023 of which were cell‐type specific) to 7,548 genes (2,280 of which differed between cell types). Cell‐type specific regulatory features correlated with cell‐type specific gene expression. For example, a microglia‐specific loop connected a distal regulatory loci to the promoter of PER1, which was also upregulated in microglia and has a known role in circadian rhythm regulation, underscoring microglia’s known circadian‐regulated behavior.ConclusionWe identified cell‐type specific regulatory networks in human macrophages and microglia. While these networks exhibited similarities, we identified thousands of enhancer‐promoter connections that differed. Intersecting with AD GWAS enables us to identify cell‐type specific genes regulated by non‐coding AD risk variants and reveal novel regulatory connections that would be otherwise unknown.

Subtype-specific 3D genome alteration in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

Multiscale 3D genome organization underlies ILC2 ontogenesis and allergic airway inflammation.

Innate lymphoid cells (ILCs) are well-characterized immune cells that play key roles in host defense and tissue homeostasis. Yet, how the three-dimensional (3D) genome organization underlies the development and functions of ILCs is unknown. Herein, we carried out an integrative analysis of the 3D genome structure, chromatin accessibility and gene expression in mature ILCs. Our results revealed that the local 3D configuration of the genome is rewired specifically at loci associated with ILC biology to promote their development and functional differentiation. Importantly, we demonstrated that the ontogenesis of ILC2s and the progression of allergic airway inflammation are determined by a unique local 3D configuration of the region containing the ILC-lineage-defining factor Id2, which is characterized by multiple interactions between the Id2 promoter and distal regulatory elements bound by the transcription factors GATA-3 and RORα, unveiling the mechanism whereby the Id2 expression is specifically controlled in group 2 ILCs.

A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data

Cellular heterogeneity in growth and differentiation results in organ patterning. Single-cell transcriptomics allows characterization of gene expression heterogeneity in developing organs at unprecedented resolution. However, the original physical location of the cell is lost during this methodology. To recover the original location of cells in the developing organ is essential to link gene activity with cellular identity and function in plants. Here, we propose a method to reconstruct genome-wide gene expression patterns of individual cells in a 3D flower meristem by combining single-nuclei RNA-seq with microcopy-based 3D spatial reconstruction. By this, gene expression differences among meristematic domains giving rise to different tissue and organ types can be determined. As a proof of principle, the method is used to trace the initiation of vascular identity within the floral meristem. Our work demonstrates the power of spatially reconstructed single cell transcriptome atlases to understand plant morphogenesis. The floral meristem 3D gene expression atlas can be accessed at http://threed-flower-meristem.herokuapp.com.

Functional genomics atlas of synovial fibroblasts defining rheumatoid arthritis heritability

BackgroundGenome-wide association studies have reported more than 100 risk loci for rheumatoid arthritis (RA). These loci are shown to be enriched in immune cell-specific enhancers, but the analysis so far has excluded stromal cells, such as synovial fibroblasts (FLS), despite their crucial involvement in the pathogenesis of RA. Here we integrate DNA architecture, 3D chromatin interactions, DNA accessibility, and gene expression in FLS, B cells, and T cells with genetic fine mapping of RA loci.ResultsWe identify putative causal variants, enhancers, genes, and cell types for 30–60% of RA loci and demonstrate that FLS account for up to 24% of RA heritability. TNF stimulation of FLS alters the organization of topologically associating domains, chromatin state, and the expression of putative causal genes such as TNFAIP3 and IFNAR1. Several putative causal genes constitute RA-relevant functional networks in FLS with roles in cellular proliferation and activation. Finally, we demonstrate that risk variants can have joint-specific effects on target gene expression in RA FLS, which may contribute to the development of the characteristic pattern of joint involvement in RA.ConclusionOverall, our research provides the first direct evidence for a causal role of FLS in the genetic susceptibility for RA accounting for up to a quarter of RA heritability.