Single-cell RNA sequencing (scRNA-seq) identifies cell subpopulations within tissue but does not capture their spatial distribution nor reveal local networks of intercellular communication acting in ...situ. A suite of recently developed techniques that localize RNA within tissue, including multiplexed in situ hybridization and in situ sequencing (here defined as high-plex RNA imaging) and spatial barcoding, can help address this issue. However, no method currently provides as complete a scope of the transcriptome as does scRNA-seq, underscoring the need for approaches to integrate single-cell and spatial data. Here, we review efforts to integrate scRNA-seq with spatial transcriptomics, including emerging integrative computational methods, and propose ways to effectively combine current methodologies.
Methods to study RNA-protein interactions Ramanathan, Muthukumar; Porter, Douglas F; Khavari, Paul A
Nature methods,
03/2019, Letnik:
16, Številka:
3
Journal Article
Recenzirano
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Noncoding RNA sequences, including long noncoding RNAs, small nucleolar RNAs, and untranslated mRNA regions, accomplish many of their diverse functions through direct interactions with RNA-binding ...proteins (RBPs). Recent efforts have identified hundreds of new RBPs that lack known RNA-binding domains, thus underscoring the complexity and diversity of RNA-protein complexes. Recent progress has expanded the number of methods for studying RNA-protein interactions in two general categories: approaches that characterize proteins bound to an RNA of interest (RNA-centric), and those that examine RNAs bound to a protein of interest (protein-centric). Each method has unique strengths and limitations, which makes it important to select optimal approaches for the biological question being addressed. Here we review methods for the study of RNA-protein interactions, with a focus on their suitability for specific applications.
The ability of the skin to repair itself after injury is vital to human survival and is disrupted in a spectrum of disorders. The process of cutaneous wound healing is complex, requiring a ...coordinated response by immune cells, hematopoietic cells, and resident cells of the skin. We review the classic paradigms of wound healing and evaluate how recent discoveries have enriched our understanding of this process. We evaluate current and experimental approaches to treating cutaneous wounds, with an emphasis on cell-based therapies and skin transplantation.
Genome conformation is central to gene control but challenging to interrogate. Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP improves the yield of ...conformation-informative reads by over 10-fold and lowers the input requirement over 100-fold relative to that of ChIA-PET. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C.
To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging ...from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.
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•Profiling of 10 human skin SCCs and matched normals via scRNA-seq, ST, and MIBI•Tumor-specific keratinocytes (TSKs) reside within a fibrovascular niche at leading edges•Distinct ligand-receptor and spatial niche associations for tumor and stromal cells.•Subpopulation essential tumorigenic gene networks defined by in vivo CRISPR screening
Integration of high-dimensional multi-omics approaches to characterize human cutaneous squamous cell carcinoma identifies a tumor-specific keratinocyte population as well as the immune infiltrates and heterogeneity at tumor leading edges.
Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control ...them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.
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•Epidermal gene sets in a progenitor differentiation time course are identified•Module mapping identified MAF:MAFB as epidermal differentiation regulators•ANCR and TINCR lncRNAs regulate epidermal MAF:MAFB expression•GRHL3, ZNF750, KLF4, and PRDM1 are MAF:MAFB effectors
Lopez-Pajares et al. define dynamically altered genes during epidermal differentiation and use module mapping to identify transcription factors involved in this process. CRISPR/Cas9-mediated ablation of MAF and MAFB in primary human tissue shows they are essential differentiation regulators. MAF:MAFB is regulated by lncRNAs and controls downstream key effector TFs.
Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous ...detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.
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•A method to measure CRISPR perturbations and chromatin state in single cells•Mapping of dynamic chromatin regulatory networks through intercellular variation•Elucidating principles of epistatic interaction between trans-factors•Perturb-ATAC screen of TF function in epidermal differentiation trajectories
Perturb-ATAC combines CRISPR screening with chromatin accessibility profiling of single cells to uncover regulators of chromatin architecture and regulator occupancy and to determine epistatic relationships between regulatory factors in cell fate decisions.
The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for ...the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols.
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