Cell Type-specific Transcriptomes Research Articles

Integration of RNA-seq and ATAC-seq analyzes the effect of low dose neutron-γ radiation on gene expression of lymphocytes from oilfield logging workers

Objective: Although radiation workers are exposed to much lower doses of neutron-γ rays than those suffered in nuclear explosions and accidents, it does not mean that their health is not affected by radiation. Lower doses of radiation do not always cause morphological aberrations in chromosomes, so more sophisticated tests must be sought to specific alterations in the exposed cells. Our goal was to characterize the specific gene expression in lymphocytes from logging workers who were continuously exposed to low doses of neutron-γ radiation. We hypothesized that the combination of cell type-specific transcriptomes and open chromatin profiles would identify lymphocyte-specific gene alterations induced by long-term radiation with low-dose neutron-γ-rays and discover new regulatory pathways and transcriptional regulatory elements.Methods: Lymphocytes were extracted from workers who have been occupationally exposed to neutron-γ and workers unexposed to radiation in the same company. mRNA-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) were performed, followed integrative analysis to identify specific gene regulatory regions induced by neutron-γ radiation. A qPCR assay was then performed to verify the downregulation of RNA coding for ribosomal proteins and flow cytometry was used to detect ribosomal protein expression and cell cycle alterations.Results: We identified transcripts that were specifically induced by neutron-γ radiation and discovered differential open chromatin regions that correlated with these gene activation patterns. Notably, we observed a downward trend in the expression of both differentially expressed genes and open chromatin peaks. Our most significant finding was that the differential peak upregulated in ATAC-seq, while the differential gene was downregulated in the ribosome pathway. We confirmed that neutron-γ radiation leads to transcriptional inhibition by analyzing the most enriched promoters, examining RPS18 and RPS27A expression by qPCR, and analyzing protein-protein interactions of the differential genes. Ribosomal protein expression and cell cycle were also affected by neutron-γ as detected by flow cytometry.Conclusion: We have comprehensively analyzed the genetic landscape of human lymphocytes based on chromatin accessibility and transcript levels, enabling the identification of novel neutron-γ induced signature genes not previously known. By comparing fine-mapping of open chromatin and RNA reads, we have determined that neutron-γ specifically leads to downregulation of genes in the ribosome pathway, with pseudogenes potentially playing a crucial role.

Single-Cell Transcriptional Analysis of Lamina Propria Lymphocytes in the Jejunum Reveals Innate Lymphoid Cell-like Cells in Pigs.

Pigs are the most suitable model to study various therapeutic strategies and drugs for human beings, although knowledge about cell type-specific transcriptomes and heterogeneity is poorly available. Through single-cell RNA sequencing and flow cytometry analysis of the types in the jejunum of pigs, we found that innate lymphoid cells (ILCs) existed in the lamina propria lymphocytes (LPLs) of the jejunum. Then, through flow sorting of live/dead-lineage (Lin)-CD45+ cells and single-cell RNA sequencing, we found that ILCs in the porcine jejunum were mainly ILC3s, with a small number of NK cells, ILC1s, and ILC2s. ILCs coexpressed IL-7Rα, ID2, and other genes and differentially expressed RORC, GATA3, and other genes but did not express the CD3 gene. ILC3s can be divided into four subgroups, and genes such as CXCL8, CXCL2, IL-22, IL-17, and NCR2 are differentially expressed. To further detect and identify ILC3s, we verified the classification of ILCs in the porcine jejunum subgroup and the expression of related hallmark genes at the protein level by flow cytometry. For systematically characterizing ILCs in the porcine intestines, we combined our pig ILC dataset with publicly available human and mice ILC data and identified that the human and pig ILCs shared more common features than did those mouse ILCs in gene signatures and cell states. Our results showed in detail for the first time (to our knowledge) the gene expression of porcine jejunal ILCs, the subtype classification of ILCs, and the markers of various ILCs, which provide a basis for an in-depth exploration of porcine intestinal mucosal immunity.

Opposing roles for ADAMTS2 and ADAMTS14 in myofibroblast differentiation and function.

Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor β availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

OR09-04 Ovarian Stimulation Disrupts Expression Of The Retinoic Acid (RA) Signaling Pathway In The Human Endometrium

Abstract Disclosure: Q. Zhao: None. P. Timmins: None. M. Saad-Naguib: None. C. Samuels: None. R. Loia: None. T. Wu: None. A. Chemerinski: None. S.S. Morelli: None. A.V. Babwah: None. N. Douglas: None. Ovarian stimulation (OS) is employed to recruit multiple dominant follicles and mature oocytes for in vitro fertilization (IVF). OS followed by a fresh embryo transfer (ET) is associated with decreased pregnancy rates. By better understanding the processes which prepare the endometrium for implantation, we hope to improve outcomes following OS. To understand the impact of OS on the endometrium, we performed an in-depth comparative analysis of the human endometrium under natural vs OS conditions during the periovulatory (PO) and mid-secretory (MSE) phase of the menstrual cycle. Our data showed that OS, which significantly elevated serum E2 and P4 levels, was associated with dyssynchronous glandular-stromal development, increased glandular epithelial volume, and increased macrophage and B cell abundance. We then performed bulk and single-cell RNA sequencing (RNA-seq) to identify the molecular pathways and cell-type specific transcriptomes associated with these changes. We found that OS significantly changed the expression of numerous genes in the retinoic acid (RA) signaling pathway, a pathway known to be crucial for a successful pregnancy. Our data showed that OS affected the fibroblast and epithelial cell-specific expression of genes that regulate RA signaling, with increased expression of RA synthesis genes and RA target genes, suggesting that OS increases endometrial RA signaling. RA is the metabolically active form of vitamin A which regulates the transcription of over 500 genes and adversely affects pregnancy when deficient or in excess. Thus, these findings led us to hypothesize that OS disrupts normal endometrial RA signaling and thereby endometrial differentiation that is required for implantation. We investigated whether RA is required for endometrial differentiation in humans and the establishment of pregnancy in mice. Using siRNAs against the RA receptor RARA, we observed that reduced expression of RARA mRNA in human endometrial stromal cells reduced their ability to undergo decidualization, an essential requirement for successful embryo implantation. We found that administration of BMS93, a pan RAR receptor antagonist, to pregnant dams prior to implantation from E0.5 or E2.5 until E7.5 resulted in pregnancy failure. Whereas BMS493 administration to pregnant dams from E4.5 to E7.5, resulted in pregnancies with reduced decidual vascular development in the anti-mesometrial region. Together, these data suggest that RA is required for the proper preparation of the endometrium for embryo implantation and loss of RA signaling in the endometrium, not the embryo, is a cause of pregnancy failure. Importantly, OS induced disruptions of RA signaling might contribute to reduced implantation rates in fresh ET, uncovering a novel pharmacologic target for improving pregnancy outcomes in fresh ET following OS. Presentation: Friday, June 16, 2023

Single-cell RNA sequencing profiles reveal cell type-specific transcriptional regulation networks conditioning fungal invasion in maize roots.

Stalk rot caused by Fusarium verticillioides (Fv) is one of the most destructive diseases in maize production. The defence response of root system to Fv invasion is important for plant growth and development. Dissection of root cell type-specific response to Fv infection and its underlying transcription regulatory networks will aid in understanding the defence mechanism of maize roots to Fv invasion. Here, we reported the transcriptomes of 29 217 single cells derived from root tips of two maize inbred lines inoculated with Fv and mock condition, and identified seven major cell types with 21 transcriptionally distinct cell clusters. Through the weighted gene co-expression network analysis, we identified 12 Fv-responsive regulatory modules from 4049 differentially expressed genes (DEGs) that were activated or repressed by Fv infection in these seven cell types. Using a machining-learning approach, we constructed six cell type-specific immune regulatory networks by integrating Fv-induced DEGs from the cell type-specific transcriptomes, 16 known maize disease-resistant genes, five experimentally validated genes (ZmWOX5b, ZmPIN1a, ZmPAL6, ZmCCoAOMT2, and ZmCOMT), and 42 QTL or QTN predicted genes that are associated with Fv resistance. Taken together, this study provides not only a global view of maize cell fate determination during root development but also insights into the immune regulatory networks in major cell types of maize root tips at single-cell resolution, thus laying the foundation for dissecting molecular mechanisms underlying disease resistance in maize.

The protein kinases of Dictyostelia and their incorporation into a signalome

Protein kinases are major regulators of cellular processes, but the roles of most kinases remain unresolved. Dictyostelid social amoebas have been useful in identifying functions for 30% of its kinases in cell migration, cytokinesis, vesicle trafficking, gene regulation and other processes but their upstream regulators and downstream effectors are mostly unknown. Comparative genomics can assist to distinguish between genes involved in deeply conserved core processes and those involved in species-specific innovations, while co-expression of genes as evident from comparative transcriptomics can provide cues to the protein complement of regulatory networks. Genomes and developmental and cell-type specific transcriptomes are available for species that span the 0.5 billion years of evolution of Dictyostelia from their unicellular ancestors. In this work we analysed conservation and change in the abundance, functional domain architecture and developmental regulation of protein kinases across the 4 major taxon groups of Dictyostelia. All data are summarized in annotated phylogenetic trees of the kinase subtypes and accompanied by functional information of all kinases that were experimentally studied. We detected 393 different protein kinase domains across the five studied genomes, of which 212 were fully conserved. Conservation was highest (71%) in the previously defined AGC, CAMK, CK1, CMCG, STE and TKL groups and lowest (26%) in the “other” group of typical protein kinases. This was mostly due to species-specific single gene amplification of “other” kinases. Apart from the AFK and α-kinases, the atypical protein kinases, such as the PIKK and histidine kinases were also almost fully conserved. The phylogeny-wide developmental and cell-type specific expression profiles of the protein kinase genes were combined with profiles from the same transcriptomic experiments for the families of G-protein coupled receptors, small GTPases and their GEFs and GAPs, the transcription factors and for all genes that upon lesion generate a developmental defect. This dataset was subjected to hierarchical clustering to identify clusters of co-expressed genes that potentially act together in a signalling network. The work provides a valuable resource that allows researchers to identify protein kinases and other regulatory proteins that are likely to act as intermediates in a network of interest.

Abstract 709: Implementation Of Ribosomal Tagging Strategies To Analyze Smooth Muscle Specific Rna Expression In Mouse Models Of Atherosclerosis

Background: Atherosclerotic cardiovascular disease (CVD) is the leading cause of mortality worldwide. Smooth muscle cells (SMCs) make up the bulk of the healthy arterial wall and atherosclerotic lesions. During atherosclerosis progression, we and others have reported on the emergence of multiple harmful and protective types of SMC-derived cell types (SDCs) in lesions, and the use of fluorescence lineage-tracing animal models have made it possible to isolate and analyze cell-type specific transcriptomes by flow cytometry and scRNA-seq. However, these manipulations are known to carry risk for artifacts, alter transcription, and introduce cell type bias. Therefore, alternative approaches that circumvent the need for single cell isolation may be beneficial. Hypothesis: Introduction of an isolation-free method for SMC translatome retrieval may be beneficial in confirming the presence of SDCs correlating with disease. Methods: SMC-specific translatomes were isolated by immunoprecipitation (IP) of epitope-tagged ribosomes from the aortic arch following the “RiboTag approach” developed by McKnight et al. and sequenced to test against libraries created by flow isolation of SMCs and bulk RNA-sequencing. Results: Here we report the creation of SMC-specific atherosclerotic RiboTag mouse model and initial efforts made to assess the SMC/SDC translatome during atherosclerosis. Side-by-side comparison of translatomes isolated by IP from the aortic arch and transcriptomes from sorted SDCs will be used to identify advantages and disadvantages of the respective approaches. Conclusions: This work will determine if and how the use of cell isolation may impact the transcriptome of SMCs and SDCs from mouse atherosclerotic models and establish if translatome analysis of these cell types can be used to complement standard sort-based profiling strategies in the study of atherosclerosis.

Single-nuclei transcriptome analysis of the shoot apex vascular system differentiation in Populus.

Differentiation of stem cells in the plant apex gives rise to aerial tissues and organs. Presently, we lack a lineage map of the shoot apex cells in woody perennials - a crucial gap considering their role in determining primary and secondary growth. Here, we used single-nuclei RNA-sequencing to determine cell type-specific transcriptomes of the Populus vegetative shoot apex. We identified highly heterogeneous cell populations clustered into seven broad groups represented by 18 transcriptionally distinct cell clusters. Next, we established the developmental trajectories of the epidermis, leaf mesophyll and vascular tissue. Motivated by the high similarities between Populus and Arabidopsis cell population in the vegetative apex, we applied a pipeline for interspecific single-cell gene expression data integration. We contrasted the developmental trajectories of primary phloem and xylem formation in both species, establishing the first comparison of vascular development between a model annual herbaceous and a woody perennial plant species. Our results offer a valuable resource for investigating the principles underlying cell division and differentiation conserved between herbaceous and perennial species while also allowing us to examine species-specific differences at single-cell resolution.

Network analysis of hepatocellular carcinoma liquid biopsies augmented by single-cell sequencing data.

Liquid biopsy, the analysis of body fluids, represents a promising approach for disease diagnosis and prognosis with minimal intervention. Sequencing cell-free RNA derived from liquid biopsies has been very promising for the diagnosis of several diseases. Cancer research, in particular, has emerged as a prominent candidate since early diagnosis has been shown to be a critical determinant of disease prognosis. Although high-throughput analysis of liquid biopsies has uncovered many differentially expressed genes in the context of cancer, the functional connection between these genes is not investigated in depth. An important approach to remedy this issue is the construction of gene networks which describes the correlation patterns between different genes, thereby allowing to infer their functional organization. In this study, we aimed at characterizing extracellular transcriptome gene networks of hepatocellular carcinoma patients compared to healthy controls. Our analysis revealed a number of genes previously associated with hepatocellular carcinoma and uncovered their association network in the blood. Our study thus demonstrates the feasibility of performing gene co-expression network analysis from cell-free RNA data and its utility in studying hepatocellular carcinoma. Furthermore, we augmented cell-free RNA network analysis with single-cell RNA sequencing data which enables the contextualization of the identified network modules with cell-type specific transcriptomes from the liver.

A transcriptome atlas of the mouse iris at single-cell resolution defines cell types and the genomic response to pupil dilation.

The iris controls the level of retinal illumination by controlling pupil diameter. It is a site of diverse ophthalmologic diseases and it is a potential source of cells for ocular auto-transplantation. The present study provides foundational data on the mouse iris based on single nucleus RNA sequencing. More specifically, this work has (1) defined all of the major cell types in the mouse iris and ciliary body, (2) led to the discovery of two types of iris stromal cells and two types of iris sphincter cells, (3) revealed the differences in cell type-specific transcriptomes in the resting vs. dilated states, and (4) identified and validated antibody and in situ hybridization probes that can be used to visualize the major iris cell types. By immunostaining for specific iris cell types, we have observed and quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre-expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre-expressing progenitors contribute only to stromal cells. This work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.

Interactome and evolutionary conservation of Dictyostelid small GTPases and their direct regulators

ABSTRACT GTP binding proteins known as small GTPases make up one of the largest groups of regulatory proteins and control almost all functions of living cells. Their activity is under, respectively, positive and negative regulation by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which together with their upstream regulators and the downstream targets of the small GTPases form formidable signalling networks. While genomics has revealed the large size of the GTPase, GEF and GAP repertoires, only a small fraction of their interactions and functions have yet been experimentally explored. Dictyostelid social amoebas have been particularly useful in unravelling the roles of many proteins in the Rac-Rho and Ras-Rap families of GTPases in directional cell migration and regulation of the actin cytoskeleton. Genomes and cell-type specific and developmental transcriptomes are available for Dictyostelium species that span the 0.5 billion years of evolution of the group from their unicellular ancestors. In this work, we identified all GTPases, GEFs and GAPs from genomes representative of the four major taxon groups and investigated their phylogenetic relationships and evolutionary conservation and changes in their functional domain architecture and in their developmental and cell-type specific expression. We performed a hierarchical cluster analysis of the expression profiles of the ~2000 analysed genes to identify putative interacting sets of GTPases, GEFs and GAPs, which highlight sets known to interact experimentally and many novel combinations. This work represents a valuable resource for research into all fields of cellular regulation.

RNA-Sequencing of Isolated Cell Populations Expressing Human APOL1 Risk Variants Reveals Molecular Correlates of Sickle Cell Nephropathy in Zebrafish Podocytes

Background: Kidney failure occurs in 5-13% of individuals with sickle cell disease (SCD), and is associated with early mortality. We have shown previously that genetic variation in apolipoprotein L1 (APOL1) is associated with increased risk for developing SCD nephropathy (Ashley-Koch et al, 2011). Two APOL1 alleles (G1 and G2) have been identified as risk factors for SCD nephropathy, as well as chronic kidney disease (CKD) in African Americans without SCD (Limou et al, 2014). Both risk alleles are highly prevalent in individuals of African descent. Despite the strong association of G1 and G2 with kidney dysfunction, the mechanisms by which these variants contribute to increased CKD risk remain poorly understood. However, the observation of podocyte effacement in CKD patients suggests that podocytes and/or endothelial cells are likely implicated in pathogenesis. We established zebrafish models of these human risk alleles previously for the purpose of better understanding the underlying pathology contributing to SCD nephropathy (Anderson et al, 2015). Here, we have utilized these models to explore further the cell-type specific transcriptomic perturbations contributing to nephropathy.Methods: To investigate the effects of the APOL1 G2 risk variant on cell-type specific transcriptomes, we injected human APOL1 mRNA into embryos from 3 different zebrafish lines expressing cell-type specific reporters to permit the isolation of podocytes (pod:NTR-mCherry), endothelial cells (fli1:egfp), or peripheral neurons (hb9:gfp; control). Three populations from each line were utilized: (1) uninjected controls; (2) embryos injected with canonical human APOL1 (G0); and (3) embryos injected with human APOL1 (G2) mRNA. Larvae were dissociated at 4 days post-fertilization (4 dpf), and fluorescent cell types were isolated via fluorescence activated cell-sorting (FACS). Messenger RNA was extracted from sorted cell populations (n=3 FACS sorted cell populations per condition) and subsequently amplified to obtain a sufficient amount of RNA for sequencing. The amplified RNA was prepared into cDNA libraries, and sequenced on an Illumina HiSeq 2500. We performed pairwise comparisons between uninjected and G0 injected larvae in each cell type to ascertain the effects of canonical human APOL1 on the zebrafish kidney transcriptome. Additionally, to measure the effects of APOL1 G2 expression on the transcriptome, we compared APOL1 G0 and APOL1 G2 samples. Transcripts were considered to be differentially expressed if they achieved an FDR-adjusted q-value of less than 0.05. To understand better the biological processes altered by injection condition, we conducted gene ontology enrichment analyses (GOEA) on sets of differentially expressed genes.Results: Our analysis of uninjected and APOL1 G0 libraries identified 38, 154, and 204 differentially expressed (DE) genes in endothelial cells, podocytes, and motor neurons, respectively. No significant enrichment of gene ontology terms was detected, suggesting that canonical G0 APOL1 did not have a substantial impact on the zebrafish transcriptome. In contrast, the comparison of APOL1 G0 and APOL1 G2 samples found 95, 7400 (Figure below), and 652 DE genes in endothelial cells, podocytes, and motor neurons, respectively. GOEA of the podocyte DE gene list showed enrichment for several autophagy-associated terms, including “Lysosome” and “Phagosome”. Close inspection of these autophagy-associated genes revealed that the majority are up-regulated in G2 relative to G0 (68/75 genes for “Lysosome” and 52/78 genes for “Phagosome”). Thus, the G2 allele appears to induce transcription of genes involved in autophagy in podocytes.Summary: Taken together, these data suggest that podocytes are the predominant cell type affected by the expression of APOL1 G2. The comparison of podocytes injected with APOL1 G0 or APOL1 G2 yielded an order of magnitude more DE transcripts than any other comparison. The GOEA results of the podocyte comparison are consistent with a recent study of transgenic mice expressing the G1 or G2 APOL1 high risk variants, which showed alterations in endocytic vesicle content and autophagic flux (Beckerman et al 2017). Ongoing work includes molecular analysis to characterize the effects of the APOL1 G1 allele. This work will continue to shed light on the molecular pathology of SCD nephropathy and provide insight into new therapeutic strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 3443: Atlas of clinically-distinct cell states and cellular ecosystems across human solid tumors

Abstract Tumors are complex ecosystems consisting of malignant, immune, and stromal elements whose dynamic interactions drive patient survival and response to therapy. A comprehensive understanding of the diversity of cellular states within the tumor microenvironment (TME), and their patterns of co-occurrence, could provide new diagnostic tools for improved disease management and novel targets for therapeutic intervention. To address this challenge, we developed EcoTyper, a novel machine learning framework for large-scale identification of TME cell states and their co-association patterns from bulk, single-cell, and spatially resolved tumor expression data. EcoTyper starts by “purifying” cell type-specific gene expression profiles of epithelial cells, immune, and stromal cell types from bulk tissue transcriptomes using CIBERSORTx (Newman et al., Nat Biotechnol 2019). It then identifies transcriptional states for each cell type, validates them in scRNA-seq data, and uncovers co-occurrence patterns between cell states in order to define tumor cellular ecosystems. Applied to 6,475 tumor and adjacent normal samples from solid tumor types profiled by The Cancer Genome Atlas (TCGA), EcoTyper identified robust transcriptional states across 12 major cell types, including epithelial, fibroblast, endothelial, and 9 immune subsets. These states included both known and novel cellular phenotypes, nearly all of which could be validated in a compendium of scRNA-seq tumor atlases spanning ~140,000 cells. Most cell states were specific to neoplastic tissue, ubiquitous across tumor types, and significantly associated with overall survival, both in TCGA and in 9,062 held-out tumor specimens (Gentles/Newman et al., Nat Medicine 2015). We found that specific cell states co-occur in distinct cellular communities with characteristic patterns of ligand-receptor interactions, genomic features, clinical outcomes, and spatial organization. One such ecosystem defined a normal-like state that was strongly enriched in non-malignant samples. Others delineated novel pro- and anti-tumor inflammatory environments involving specific fibroblast, endothelial, and immune cell transcriptional programs. In summary, large-scale deconvolution of cell type-specific transcriptomes across thousands of solid tumors revealed a comprehensive atlas of TME cell states and cellular ecosystems. Our results provide a high-resolution portrait of cellular heterogeneity in the TME across multiple solid tumor types, with implications for novel diagnostics and immunotherapeutic targets.

Cell-specific expression of lung disease risk-related genes in the human small airway epithelium

BackgroundThe human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases.MethodsDrop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing.ResultsEleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes.ConclusionsThis data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.

Recapitulation of prostate tissue cell type-specific transcriptomes by an in vivo primary prostate tissue xenograft model.

Studies of the normal functions and diseases of the prostate request in vivo models that maintain the tissue architecture and the multiple-cell type compartments of human origin in order to recapitulate reliably the interactions of different cell types. Cell type-specific transcriptomes are critical to reveal the roles of each cell type in the functions and diseases of the prostate. A primary prostate tissue xenograft model was developed using fresh human prostate tissue specimens transplanted onto male mice that were castrated surgically and implanted with a device to maintain circulating testosterone levels comparable to adult human males. Endothelial cells and epithelial cells were isolated from 7 fresh human prostate tissue specimens and from primary tissue xenografts established from 9 fresh human prostate tissue specimens, using antibody-conjugated magnetic beads specific to human CD31 and human EpCAM, respectively. Transcriptomes of endothelial, epithelial and stromal cell fractions were obtained using RNA-Seq. Global and function-specific gene expression profiles were compared in inter-cell type and inter-tissue type manners. Gene expression profiles in the individual cell types isolated from xenografts were similar to those of cells isolated from fresh tissue, demonstrating the value of the primary tissue xenograft model for studies of the inter-relationships between prostatic cell types and the role of such inter-relationships in organ development, disease progression, and response to drug treatments.

2061-P: Islet Single Cell Transcriptomes Reveal Cellular and Gene Regulatory Alterations in Type 2 Diabetes Pathogenesis

Pancreatic islet (dys)function and failure is central to both type 2 diabetes (T2D) genetic risk and pathogenesis. Islet cellular heterogeneity has impeded precise understanding of the specific gene(s) and pathway(s) in each cell type that are altered by and/or may contribute to pathophysiology in the prediabetic (PD) and type 2 diabetic (T2D) states. To identify cellular and molecular features of islet dysfunction in PD and T2D states, we completed single cell transcriptome profiling of 137k human islet cells from 11 ND, 6 PD, and 11 T2D donors (⋃5k/individual). Single cell transcriptomes aggregated into distinct clusters of endocrine (alpha, beta, delta, PP/gamma), exocrine (acinar, stellate, ductal), endothelial, and immune cell types. Targeted analysis of each endocrine cell type identified a proliferative alpha cell sub-population comprising ∼0.15-0.3% of endocrine cells and variable insulin production and endoplasmic reticulum stress beta cell states. We compared the proportion of islet endocrine cell type (sub)populations sampled between T2D, PD, and ND donors and found that T2D samples contained significantly fewer beta cells and more alpha cells than ND and, to a lesser degree, PD samples. Delta and PP/gamma cell proportions were similar between disease states. Importantly, no (sub)populations were ND, PD, or T2D-specific. We compared aggregate T2D, PD, and ND cell type-specific transcriptomes to identify differentially expressed genes in each cell type. Genes related to exocytic granule assembly/organization, including those induced by T2D risk variants (DGKB) or T2D state (GAP43), were among 99 genes induced in T2D beta cells. 103 repressed genes included those associated with glucose transport and implicated as T2D GWAS effector genes such as SLC2A2, SLC5A1, and STARD10, a gene whose deletion in mice impairs islet insulin secretion. Studies are ongoing to assess the functional role(s) of these and additional genes in islet (dys)function. Disclosure N. Lawlor: None. R. Kursawe: None. M.L. Stitzel: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-18-ACE-15 to M.L.S.)

A transcriptome-wide antitermination mechanism sustaining identity of embryonic stem cells

Eukaryotic gene expression relies on extensive crosstalk between transcription and RNA processing. Changes in this composite regulation network may provide an important means for shaping cell type-specific transcriptomes. Here we show that the RNA-associated protein Srrt/Ars2 sustains embryonic stem cell (ESC) identity by preventing premature termination of numerous transcripts at cryptic cleavage/polyadenylation sites in first introns. Srrt interacts with the nuclear cap-binding complex and facilitates recruitment of the spliceosome component U1 snRNP to cognate intronic positions. At least in some cases, U1 recruited in this manner inhibits downstream cleavage/polyadenylation events through a splicing-independent mechanism called telescripting. We further provide evidence that the naturally high expression of Srrt in ESCs offsets deleterious effects of retrotransposable sequences accumulating in its targets. Our work identifies Srrt as a molecular guardian of the pluripotent cell state.

Phylogeny-wide conservation and change in developmental expression, cell-type specificity and functional domains of the transcriptional regulators of social amoebas

BackgroundDictyostelid social amoebas self-organize into fruiting bodies, consisting of spores and up to four supporting cell types in the phenotypically most complex taxon group 4. High quality genomes and stage- and cell-type specific transcriptomes are available for representative species of each of the four taxon groups. To understand how evolution of gene regulation in Dictyostelia contributed to evolution of phenotypic complexity, we analysed conservation and change in abundance, functional domain architecture and developmental regulation of their transcription factors (TFs).ResultsWe detected 440 sequence-specific TFs across 33 families, of which 68% were upregulated in multicellular development and about half conserved throughout Dictyostelia. Prespore cells expressed two times more TFs than prestalk cells, but stalk cells expressed more TFs than spores, suggesting that gene expression events that define spores occur earlier than those that define stalk cells. Changes in TF developmental expression, but not in TF abundance or functional domains occurred more frequently between group 4 and groups 1–3, than between the more distant branches formed by groups 1 + 2 and 3 + 4.ConclusionsPhenotypic innovation is correlated with changes in TF regulation, rather than functional domain- or TF acquisition. The function of only 34 TFs is known. Of 12 TFs essential for cell differentiation, 9 are expressed in the cell type for which they are required. The information acquired here on conserved cell type specifity of 120 additional TFs can effectively guide further functional analysis, while observed evolutionary change in TF developmental expression may highlight how genotypic change caused phenotypic innovation.

NET-CAGE characterizes the dynamics and topology of human transcribed cis-regulatory elements.

Promoters and enhancers are key cis-regulatory elements, but how they operate to generate cell type-specific transcriptomes is not fully understood. We developed a simple and robust method, native elongating transcript-cap analysis of gene expression (NET-CAGE), to sensitively detect 5' ends of nascent RNAs in diverse cells and tissues, including unstable transcripts such as enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site level, characterizing the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without the influence of RNA turnover, and show that enhancer-promoter pairs are generally activated simultaneously on stimulation. By integrating NET-CAGE data with chromatin interaction maps, we show that cis-regulatory elements are topologically connected according to their cell type specificity. We identified new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our NET-CAGE dataset derived from human and mouse cells expands the FANTOM5 atlas of transcribed enhancers, with broad applicability to biomedical research.

Spatial Principles of Chromatin Architecture Associated With Organ-Specific Gene Regulation

Packaging of the genome in the nucleus is a non-random process that is thought to directly contribute to cell type-specific transcriptomes, although this hypothesis remains untested. Epigenome architecture, as assayed by chromatin conformation capture techniques, such as Hi-C, has recently been described in the mammalian cardiac myocyte and found to be remodeled in the setting of heart failure. In the present study, we sought to determine whether the structural features of the epigenome are conserved between different cell types by investigating Hi-C and RNA-seq data from heart and liver. Investigation of genes with enriched expression in heart or liver revealed nuanced interaction paradigms between organs: first, the log2 ratios of heart:liver (or liver:heart) intrachromosomal interactions are higher in organ-specific gene sets (p = 0.009), suggesting that organ-specific genes have specialized chromatin structural features. Despite similar number of total interactions between cell types, intrachromosomal interaction profiles in heart but not liver demonstrate that genes forming promoter-to-transcription-end-site loops in the cardiac nucleus tend to be involved in cardiac-related pathways. The same analysis revealed an analogous organ-specific interaction profile for liver-specific loop genes. Investigation of A/B compartmentalization (marker of chromatin accessibility) revealed that in the heart, 66.7% of cardiac-specific genes are in compartment A, while 66.1% of liver-specific genes are found in compartment B, suggesting that there exists a cardiac chromatin topology that allows for expression of cardiac genes. Analyses of interchromosomal interactions revealed a relationship between interchromosomal interaction count and organ-specific gene localization (p = 2.2 × 10−16) and that, for both organs, regions of active or inactive chromatin tend to segregate in 3D space (i.e., active with active, inactive with inactive). 3D models of topologically associating domains (TADs) suggest that TADs tend to interact with regions of similar compartmentalization across chromosomes, revealing trans structural interactions contributing to genomic compartmentalization at distinct structural scales. These models reveal discordant nuclear compaction strategies, with heart packaging compartment A genes preferentially toward the center of the nucleus and liver exhibiting preferential arrangement toward the periphery. Taken together, our data suggest that intra- and interchromosomal chromatin architecture plays a role in orchestrating tissue-specific gene expression.