scMMO-atlas: a single cell multimodal omics atlas and portal for exploring fine cell heterogeneity and cell dynamics.

Single-cell multimodal sequencing parallelly captures multiple modalities of the same cell, providing unparalleled insights into cell heterogeneity and cell dynamics. For example, joint profiling of chromatin accessibility and transcriptome from the same single cell (scATAC + RNA) identified new cell subsets within the well-defined clusters. However, lack of single-cell multimodal omics (scMMO) database has led to data fragmentation, seriously hindering access, utilization and mining of scMMO data. Here, we constructed a scMMO atlas by collecting and integrating various scMMO data, then constructed scMMO database and portal called scMMO-atlas (https://www.biosino.org/scMMO-atlas/). scMMO-atlas includes scATAC + RNA (ISSAAS-seq, SNARE-seq, paired-seq, sci-CAR, scCARE-seq, 10X Multiome and so on), scRNA + protein, scATAC + protein and scTri-modal omics data, with 3 168 824 cells from 27 cell tissues/organs. scMMO-atlas offered an interactive portal for visualization and featured analysis for each modality and the integrated data. Integrated analysis of scATAC + RNA data of mouse cerebral cortex in scMMO-atlas identified more cell subsets compared with unimodal omics data. Among these new cell subsets, there is an early astrocyte subset highly expressed Grm3, called Astro-Grm3. Furthermore, we identified Ex-L6-Tle4-Nrf1, a progenitor of Ex-L6-Tle4, indicating the statistical power provided by the big data in scMMO-atlas. In summary, scMMO-atlas offers cell atlas, database and portal to facilitate data utilization and biological insight.

Decreased GATA3 levels cause changed mouse cutaneous innate lymphoid cell fate, facilitating hair follicle recycling.

In mice, skin-resident type 2 innate lymphoid cells (ILC2s) exhibit some ILC3-like characteristics. However, the underlying mechanism remains elusive. Here, we observed lower expression of the ILC2 master regulator GATA3 specifically in cutaneous ILC2s (cILC2s) compared with canonical ILC2s, in line with its functionally divergent role in transcriptional control in cILC2s. Decreased levels of GATA3 enabled the expansion of RORγt fate-mapped (RORγtfm+) cILC2s after postnatal days, displaying certain similarities to ILC3s. Single-cell trajectory analysis showed a sequential promotion of the RORγtfm+ cILC2 divergency by RORγt and GATA3. Notably, during hair follicle recycling, these RORγtfm+ cILC2s accumulated around the hair follicle dermal papilla (DP) region to facilitate the process. Mechanistically, we found that GATA3-mediated integrin α3ß1 upregulation on RORγtfm+ cILC2s was required for their positioning around the DP. Overall, our study demonstrates a distinct regulatory role of GATA3 in cILC2s, particularly in promoting the divergence of RORγtfm+ cILC2s to facilitate hair follicle recycling.

Progress in single-cell multimodal sequencing and multi-omics data integration.

With the rapid advance of single-cell sequencing technology, cell heterogeneity in various biological processes was dissected at different omics levels. However, single-cell mono-omics results in fragmentation of information and could not provide complete cell states. In the past several years, a variety of single-cell multimodal omics technologies have been developed to jointly profile multiple molecular modalities, including genome, transcriptome, epigenome, and proteome, from the same single cell. With the availability of single-cell multimodal omics data, we can simultaneously investigate the effects of genomic mutation or epigenetic modification on transcription and translation, and reveal the potential mechanisms underlying disease pathogenesis. Driven by the massive single-cell omics data, the integration method of single-cell multi-omics data has rapidly developed. Integration of the massive multi-omics single-cell data in public databases in the future will make it possible to construct a cell atlas of multi-omics, enabling us to comprehensively understand cell state and gene regulation at single-cell resolution. In this review, we summarized the experimental methods for single-cell multimodal omics data and computational methods for multi-omics data integration. We also discussed the future development of this field.

Single cell analyses of cancer cells identified two regulatorily and functionally distinct categories in differentially expressed genes among tumor subclones.

To explore the feature of cancer cells and tumor subclones, we analyzed 101,065 single-cell transcriptomes from 12 colorectal cancer (CRC) patients and 92 single cell genomes from one of these patients. We found cancer cells, endothelial cells and stromal cells in tumor tissue expressed much more genes and had stronger cell-cell interactions than their counterparts in normal tissue. We identified copy number variations (CNVs) in each cancer cell and found correlation between gene copy number and expression level in cancer cells at single cell resolution. Analysis of tumor subclones inferred by CNVs showed accumulation of mutations in each tumor subclone along lineage trajectories. We found differentially expressed genes (DEGs) between tumor subclones had two populations: DEGCNV and DEGreg. DEGCNV, showing high CNV-expression correlation and whose expression differences depend on the differences of CNV level, enriched in housekeeping genes and cell adhesion associated genes. DEGreg, showing low CNV-expression correlation and mainly in low CNV variation regions and regions without CNVs, enriched in cytokine signaling genes. Furthermore, cell-cell communication analyses showed that DEGCNV tends to involve in cell-cell contact while DEGreg tends to involve in secreted signaling, which further support that DEGCNV and DEGreg are two regulatorily and functionally distinct categories.

The chromatin accessibility dynamics during cell fate specifications in zebrafish early embryogenesis.

Chromatin accessibility plays a critical role in the regulation of cell fate decisions. Although gene expression changes have been extensively profiled at the single-cell level during early embryogenesis, the dynamics of chromatin accessibility at cis-regulatory elements remain poorly studied. Here, we used a plate-based single-cell ATAC-seq method to profile the chromatin accessibility dynamics of over 10 000 nuclei from zebrafish embryos. We investigated several important time points immediately after zygotic genome activation (ZGA), covering key developmental stages up to dome. The results revealed key chromatin signatures in the first cell fate specifications when cells start to differentiate into enveloping layer (EVL) and yolk syncytial layer (YSL) cells. Finally, we uncovered many potential cell-type specific enhancers and transcription factor motifs that are important for the cell fate specifications.

Sirpα on tumor-associated myeloid cells restrains antitumor immunity in colorectal cancer independent of its interaction with CD47.

Immunosuppressive myeloid cells hinder immunotherapeutic efficacy in tumors, but the precise mechanisms remain undefined. Here, by performing single-cell RNA sequencing in colorectal cancer tissues, we found tumor-associated macrophages and granulocytic myeloid-derived suppressor cells increased most compared to their counterparts in normal tissue and displayed the highest immune-inhibitory signatures among all immunocytes. These cells exhibited significantly increased expression of immunoreceptor tyrosine-based inhibitory motif-bearing receptors, including SIRPA. Notably, Sirpa-/- mice were more resistant to tumor progression than wild-type mice. Moreover, Sirpα deficiency reprogramed the tumor microenvironment through expansion of TAM_Ccl8hi and gMDSC_H2-Q10hi subsets showing strong antitumor activity. Sirpa-/- macrophages presented strong phagocytosis and antigen presentation to enhance T cell activation and proliferation. Furthermore, Sirpa-/- macrophages facilitated T cell recruitment via Syk/Btk-dependent Ccl8 secretion. Therefore, Sirpα deficiency enhances innate and adaptive immune activation independent of expression of CD47 and Sirpα blockade could be a promising strategy to improve cancer immunotherapy efficacy.

The bone-liver interaction modulates immune and hematopoietic function through Pinch-Cxcl12-Mbl2 pathway.

Mesenchymal stromal cells (MSCs) are used to treat infectious and immune diseases and disorders; however, its mechanism(s) remain incompletely defined. Here we find that bone marrow stromal cells (BMSCs) lacking Pinch1/2 proteins display dramatically reduced ability to suppress lipopolysaccharide (LPS)-induced acute lung injury and dextran sulfate sodium (DSS)-induced inflammatory bowel disease in mice. Prx1-Cre; Pinch1f/f; Pinch2-/- transgenic mice have severe defects in both immune and hematopoietic functions, resulting in premature death, which can be restored by intravenous injection of wild-type BMSCs. Single cell sequencing analyses reveal dramatic alterations in subpopulations of the BMSCs in Pinch mutant mice. Pinch loss in Prx1+ cells blocks differentiation and maturation of hematopoietic cells in the bone marrow and increases production of pro-inflammatory cytokines TNF-α and IL-1ß in monocytes. We find that Pinch is critical for expression of Cxcl12 in BMSCs; reduced production of Cxcl12 protein from Pinch-deficient BMSCs reduces expression of the Mbl2 complement in hepatocytes, thus impairing the innate immunity and thereby contributing to infection and death. Administration of recombinant Mbl2 protein restores the lethality induced by Pinch loss in mice. Collectively, we demonstrate that the novel Pinch-Cxcl12-Mbl2 signaling pathway promotes the interactions between bone and liver to modulate immunity and hematopoiesis and may provide a useful therapeutic target for immune and infectious diseases.

Proline uptake promotes activation of lymphoid tissue inducer cells to maintain gut homeostasis.

Metabolic regulation is integral to the proper functioning of innate lymphoid cells, yet the underlying mechanisms remain elusive. Here, we show that disruption of exogenous proline uptake, either through dietary restriction or by deficiency of the proline transporter Slc6a7, in lymphoid tissue inducer (LTi) cells, impairs LTi activation and aggravates dextran sodium sulfate-induced colitis in mice. With an integrative transcriptomic and metabolomic analysis, we profile the metabolic characteristics of various innate lymphoid cell subsets and reveal a notable enrichment of proline metabolism in LTi cells. Mechanistically, defective proline uptake diminishes the generation of reactive oxygen species, previously known to facilitate LTi activation. Additionally, LTi cells deficient in Slc6a7 display downregulation of Cebpb and Kdm6b, resulting in compromised transcriptional and epigenetic regulation of interleukin-22. Furthermore, our study uncovers the therapeutic potential of proline supplementation in alleviating colitis. Therefore, these findings shed light on the role of proline in facilitating LTi activation and ultimately contributing to gut homeostasis.

Exploring the dynamics and influencing factors of CD4 T cell activation using single-cell RNA-seq.

T cell activation is a key event in adaptive immunity. However, the dynamics and influencing factors of T cell activation remain unclear. Here, we analyzed CD4 T cells that were stimulated with anti-CD3/CD28 under several conditions to explore the factors affecting T cell activation. We found a stimulated T subset (HSPhi T) highly expressing heat shock proteins, which was derived from stimulated naive T. We identified and characterized inert T, a stimulated T cell subset in transitional state from resting T to activated T. Interestingly, resting CXCR4low T responded to stimulation more efficiently than resting CXCR4hi T. Furthermore, stimulation of CD4 T in the presence of CD8 T resulted in more effector T and more homogeneous expressions of CD25, supporting that presence of CD8 T reduces the extreme response of T cells, which can be explained by regulation of CD4 T activation through CD8 T-initiated cytokine signaling and FAS/FASLG signaling.

A fast and globally optimal solution for RNA-seq quantification.

Alignment-based RNA-seq quantification methods typically involve a time-consuming alignment process prior to estimating transcript abundances. In contrast, alignment-free RNA-seq quantification methods bypass this step, resulting in significant speed improvements. Existing alignment-free methods rely on the Expectation-Maximization (EM) algorithm for estimating transcript abundances. However, EM algorithms only guarantee locally optimal solutions, leaving room for further accuracy improvement by finding a globally optimal solution. In this study, we present TQSLE, the first alignment-free RNA-seq quantification method that provides a globally optimal solution for transcript abundances estimation. TQSLE adopts a two-step approach: first, it constructs a k-mer frequency matrix A for the reference transcriptome and a k-mer frequency vector b for the RNA-seq reads; then, it directly estimates transcript abundances by solving the linear equation ATAx = ATb. We evaluated the performance of TQSLE using simulated and real RNA-seq data sets and observed that, despite comparable speed to other alignment-free methods, TQSLE outperforms them in terms of accuracy. TQSLE is freely available at https://github.com/yhg926/TQSLE.