Cell Type-specific Gene Expression Research Articles

Single‐cell nucleic acid sequencing reinforces to reveal signal‐ dependent pathways contributing to cellular heterogeneity

Single‐cell nucleic acid sequencing reinforces to reveal signal‐ dependent pathways contributing to cellular heterogeneity

Luminal and Glandular Epithelial Cells from the Porcine Endometrium maintain Cell Type-Specific Marker Gene Expression in Air–Liquid Interface Culture

Two different types of epithelial cells constitute the inner surface of the endometrium. While luminal epithelial cells line the uterine cavity and build the embryo-maternal contact zone, glandular epithelial cells form tubular glands reaching deeply into the endometrial stroma. To facilitate investigations considering the functional and molecular differences between the two populations of epithelial cells and their contribution to reproductive processes, we aimed at establishing differentiated in vitro models of both the luminal and the glandular epithelium of the porcine endometrium using an air–liquid interface (ALI) approach. We first tested if porcine luminal endometrium epithelial cells (PEEC-L) reproducibly form differentiated epithelial monolayers under ALI conditions by monitoring the morphology and the trans-epithelial electrical resistance (TEER). Subsequently, luminal (PEEC-L) and glandular epithelial cells (PEEC-G) were consecutively isolated from the endometrium of the uterine horn. Both cell types were characterized by marker gene expression analysis immediately after isolation. Cells were separately grown at the ALI and assessed by means of histomorphometry, TEER, and marker gene expression after 3 weeks of culture. PEEC-L and PEEC-G formed polarized monolayers of differentiated epithelial cells with a moderate TEER and in vivo-like morphology at the ALI. They exhibited distinct patterns of functional and cell type-specific marker gene expression after isolation and largely maintained these patterns during the culture period. The here presented cell culture procedure for PEEC-L and -G offers new opportunities to study the impact of embryonic signals, endocrine effectors, and reproductive toxins on both porcine endometrial epithelial cell types under standardized in vitro conditions.Graphical Created with BioRender.com.

Characterizing cis-regulatory elements using single-cell epigenomics.

Cell type-specific gene expression patterns and dynamics during development or in disease are controlled by cis-regulatory elements (CREs), such as promoters and enhancers. Distinct classes of CREs can be characterized by their epigenomic features, including DNA methylation, chromatin accessibility, combinations of histone modifications and conformation of local chromatin. Tremendous progress has been made in cataloguing CREs in the human genome using bulk transcriptomic and epigenomic methods. However, single-cell epigenomic and multi-omic technologies have the potential to provide deeper insight into cell type-specific gene regulatory programmes as well as into how they change during development, in response to environmental cues and through disease pathogenesis. Here, we highlight recent advances in single-cell epigenomic methods and analytical tools and discuss their readiness for human tissue profiling.

Generating specificity in genome regulation through transcription factor sensitivity to chromatin.

Cell type-specific gene expression relies on transcription factors (TFs) binding DNA sequence motifs embedded in chromatin. Understanding how motifs are accessed in chromatin is crucial to comprehend differential transcriptional responses and the phenotypic impact of sequence variation. Chromatin obstacles to TF binding range from DNA methylation to restriction of DNA access by nucleosomes depending on their position, composition and modification. In vivo and in vitro approaches now enable the study of TF binding in chromatin at unprecedented resolution. Emerging insights suggest that TFs vary in their ability to navigate chromatin states. However, it remains challenging to link binding and transcriptional outcomes to molecular characteristics of TFs or the local chromatin substrate. Here, we discuss our current understanding of how TFs access DNA in chromatin and novel techniques and directions towards a better understanding of this critical step in genome regulation.

The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's Disease, a chronic enteritis of ruminants prevalent across the world. It is estimated that approximately 50% of UK dairy herds are infected with MAP, but this is likely an underestimate of the true prevalence. Infection can result in reduced milk yield, infertility and premature culling of the animal, leading to significant losses to the farming economy and negatively affecting animal welfare. Understanding the initial interaction between MAP and the host is critical to develop improved diagnostic tools and novel vaccines. Here we describe the characterisation of three different multicellular in vitro models derived from bovine intestinal tissue, and their use for the study of cellular interactions with MAP. In addition to the previously described basal-out 3D bovine enteroids, we have established viable 2D monolayers and 3D apical-out organoids. The apical-out enteroids differ from previously described bovine enteroids as the apical surface is exposed on the exterior surface of the 3D structure, enabling study of host-pathogen interactions at the epithelial surface without the need for microinjection. We have characterised the cell types present in each model system using RT-qPCR to detect predicted cell type-specific gene expression, and confocal microscopy for cell type-specific protein expression. Each model contained the cells present in the original bovine intestinal tissue, confirming they were representative of the bovine gut. Exposure of the three model systems to the K10 reference strain of MAP K10, and a recent Scottish isolate referred to as C49, led to the observation of intracellular bacteria by confocal microscopy. Enumeration of the bacteria by quantification of genome copy number, indicated that K10 was less invasive than C49 at early time points in infection in all model systems. This study shows that bovine enteroid-based models are permissive to infection with MAP and that these models may be useful in investigating early stages of MAP pathogenesis in a physiologically relevant in vitro system, whilst reducing the use of animals in scientific research.Bos taurus: urn:lsid:zoobank.org:act:4C90C4FA-6296-4972-BE6A-5EF578677D64

SCADIE: simultaneous estimation of cell type proportions and cell type-specific gene expressions using SCAD-based iterative estimating procedure

A challenge in bulk gene differential expression analysis is to differentiate changes due to cell type-specific gene expression and cell type proportions. SCADIE is an iterative algorithm that simultaneously estimates cell type-specific gene expression profiles and cell type proportions, and performs cell type-specific differential expression analysis at the group level. Through its unique penalty and objective function, SCADIE more accurately identifies cell type-specific differentially expressed genes than existing methods, including those that may be missed from single cell RNA-Seq data. SCADIE has robust performance with respect to the choice of deconvolution methods and the sources and quality of input data.

Abstract 5007: Discovery of stromal targets in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with limited treatment options. Development of stroma targeting therapeutics for PDAC is gaining attention because PDAC has a unique tumor microenvironment with dense tumor stroma and the tumor stroma is a critical mediator of PDAC progression. Herein, we discovered pancreatic cancer stroma-specific cell surface markers by integrative analysis of single cell RNA sequencing and cell surface marker database. Methods: Single cell RNA sequencing data of PDAC tissue (n = 3, GSE154778) and normal pancreas tissue (n = 3, GSE131886) was obtained from Gene Expression Omnibus database. Using Seurat package, the data from each sample integrated and the cells were clustered. We selected stroma cell cluster for differential analysis to obtain the genes enriched in PDAC stroma. Then, the intersection of the PDAC stroma specific genes with the gene set of SurfaceomeDB was obtained, which allowed us to obtain the stroma cell surface markers that are highly expressed in PDAC. The marker genes were validated in 1) 36 pairs of PDAC and normal tissue samples from a RNA sequencing data (GSE15471), and 2) 143 PDAC samples in The Cancer Genome Atlas (TCGA), 328 normal pancreatic tissues in Genotype-Tissue Expression (GTEx) database (https://www.gtexportal.org). Results: The cells from PDAC and normal pancreas were clustered into ductal/tumor, stromal and immune cell clusters according to the expression of cell-type specific marker genes. We identified variably expressed genes between PDAC and normal cells within the stromal cell cluster. Among the PDAC stroma-specific genes, we could obtain 360 cell surface markers using SurfaceomeDB. The top ten PDAC stroma-specific cell surface marker genes were MXRA8, ANTXR1, THY1, SCD1, LY6E, BST2, GPNMB, CDH11, GJB2, and CD55. To validate the results, we compared the expression levels of the top 10 genes between pancreatic cancer tissue and adjacent normal pancreatic tissue. We found that the expression levels of the genes were significantly higher in the cancer tissue compared to the normal pancreatic tissue. We also confirmed that the expression levels of 10 genes were significantly higher in all stages of PDAC compared to normal tissues in a combined RNA sequencing dataset from healthy human pancreatic tissue from GTEx and pancreatic cancer patients from TCGA. Conclusion: We identified pancreatic cancer stroma-specific cell surface markers using single-cell RNA sequencing data and SurfaceomeDB. The markers can be utilized in development of various pancreatic cancer stroma targeted therapeutics including antibodies, antibody-drug conjugates, peptides, cell therapeutic and nanoparticles. Citation Format: Shengjun Li, Jeong-Bin Park, Hyung-Jun Im. Discovery of stromal targets in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5007.

Abstract 1458: Single cell resolution chromatin accessibility and transcriptomic atlas of genetic ancestry mapped healthy breast tissues

Abstract Single cell mRNA atlases of multiple organs have been reported using scRNA-seq. We had previously reported single cell mRNA atlas of the breast. In this study, we performed scATAC-seq and scRNA-seq of breast tissues from 22 women of Ashkenazi (A) ancestry (19,935 nuclei), 20 women of European (non-A) ancestry (19,090 nuclei), six BRCA1 (6,261 nuclei) and five BRCA2 (3,400 nuclei) mutation carriers to correlate cell type-specific gene expression with chromatin accessibility. An integrated analysis revealed 17 cell clusters including basal, luminal progenitor, and mature luminal epithelial cells, two endothelial subtypes and two fibroblast/mesenchymal stem-like cell types. Basal cells could be further subdivided into two subclusters that differentially expressed genes such as TSHZ2, POSTN and ETV6. Similarly, there were four luminal progenitor subclusters that differentially expressed genes such as MALAT1, SOX13, and MDM4. Although ESR1 expression was observed predominantly in mature luminal cells, binding sites for ESR1 were accessible in multiple epithelial and non-epithelial cells with the exception of basal cells. FOXA1 expression and binding site enrichment were observed mostly in mature luminal cells. GATA3 expression was observed in all epithelial cell types with highest expression in mature luminal cells but binding sites for GATA3 were accessible in multiple cell types. Expression and binding site enrichment of alveolar differentiation-associated EHF were predominant in luminal progenitor cells. TP63 expression and binding site enrichment were restricted to basal cells, which is consistent with the proposed role of TP63 in maintaining stem cells among the basal cell populations. EMT-associated ZEB1 showed an intriguing pattern. Similar to our published immunohistochemistry-based analysis of breast tissues, ZEB1 expression was detected in fibroblasts/mesenchymal cells, endothelial cells and adipocytes but not in epithelial cells. However, binding sites for this transcription factor were enriched in epithelial cell types and in T-cells. ZEB1 mRNA stability is tightly controlled by the epithelial cell-enriched miR-200 family of microRNAs, and it is possible that ZEB1 transcribed in epithelial cells is immediately targeted for degradation through miR-200. To gain insight into this possibility, we examined the chromatin accessibility status of ZEB1 regulatory regions in fibroblasts/mesenchymal cells and in epithelial cells. ZEB1 regulatory regions displayed open chromatin status in both epithelial cells and fibroblasts. With the exception of a few genes such as SIGLEC1 in macrophages and IL7R in T cells, cell type-specific expression of many genes is not controlled at the level of chromatin accessibility of their regulatory regions. Collectively, these results reveal complexities in gene expression in different cell types of the breast. Citation Format: Poornima Bhat-Nakshatri, Duojiao Chen, Hongyu Gao, Yunlong Liu, Anna Maria Storniolo, Harikrishna Nakshatri. Single cell resolution chromatin accessibility and transcriptomic atlas of genetic ancestry mapped healthy breast tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1458.

Single-cell RNA sequencing sheds light on cell-type specific gene expression in immune cells

Single-cell RNA sequencing sheds light on cell-type specific gene expression in immune cells

Single cell transcriptomics reveals dysregulated cellular and molecular networks in a fragile X syndrome model.

Despite advances in understanding the pathophysiology of Fragile X syndrome (FXS), its molecular basis is still poorly understood. Whole brain tissue expression profiles have proved surprisingly uninformative, therefore we applied single cell RNA sequencing to profile an FMRP deficient mouse model with higher resolution. We found that the absence of FMRP results in highly cell type specific gene expression changes that are strongest among specific neuronal types, where FMRP-bound mRNAs were prominently downregulated. Metabolic pathways including translation and respiration are significantly upregulated across most cell types with the notable exception of excitatory neurons. These effects point to a potential difference in the activity of mTOR pathways, and together with other dysregulated pathways, suggest an excitatory-inhibitory imbalance in the Fmr1-knock out cortex that is exacerbated by astrocytes. Our data demonstrate that FMRP loss affects abundance of key cellular communication genes that potentially affect neuronal synapses and provide a resource for interrogating the biological basis of this disorder.

Single-Cell Transcriptomic Analysis of the Mouse Pancreas: Characteristic Features of Pancreatic Ductal Cells in Chronic Pancreatitis.

Chronic pancreatitis (CP) is a fibroinflammatory disorder of the pancreas. Our understanding of CP pathogenesis is partly limited by the incomplete characterization of pancreatic cell types. Here, we performed single-cell RNA sequencing on 3825 cells from the pancreas of one control mouse and mice with caerulein-induced CP. An analysis of the single-cell transcriptomes revealed 16 unique clusters and cell type-specific gene expression patterns in the mouse pancreas. Sub-clustering of the pancreatic mesenchymal cells from the control mouse revealed four clusters of cells with specific gene expression profiles (combinatorial expressions of Smoc2, Cxcl14, Tnfaip6, and Fn1). We observed that immune cells in the pancreas of the CP mice were abundant and diverse in cellular type. Compared to the control, 547 upregulated genes (including Mmp7, Ttr, Rgs5, Adh1, and Cldn2) and 257 downregulated genes were identified in ductal cells from the CP group. The elevated expression levels of MMP7 and TTR were further verified in the pancreatic ducts of CP patients. This study provides a preliminary description of the single-cell transcriptome profiles of mouse pancreata and accurately demonstrates the characteristics of pancreatic ductal cells in CP. The findings provide insight into novel disease-specific biomarkers and potential therapeutic targets of CP.

Subclinical endometritis differentially affects the transcriptomic profiles of endometrial glandular, luminal, and stromal cells of postpartum dairy cows

In postpartum dairy cows, subclinical endometritis (SCE) is characterized by persistent endometrial inflammation, which has profound detrimental effects on subsequent reproductive performance. To date, transcriptomic studies related to this condition were either based on biopsy-derived whole-endometrium tissue or endometrial swab or cytobrush samples, thus masking effects of disease on cell type-specific gene expression. This study tested the hypothesis that different endometrial health statuses are associated with distinct transcription profiles of endometrial stromal, glandular, and luminal epithelial cells. At 44 d postpartum (DPP), endometrial biopsies were taken from dairy cows (n = 24) classified as healthy, recovered from SCE, or affected by persistent SCE, according to endometrial cytology taken at 21 and 44 DPP. Stromal, glandular, and luminal epithelial cells were isolated from the whole-tissue biopsy by laser capture microdissection, and the cell-specific transcription profiles were determined by RNA sequencing. Differential gene expression was analyzed with DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html). Results demonstrated that global transcriptomic profiles and corresponding lists of differentially expressed genes between cows with different health statuses were distinct among cell types. Results also showed that although healthy and recovered cows presented similar endometrial clinically healthy phenotypes at 44 DPP, the prior presence of immune cells still affected the transcriptome of endometrial cells at this stage, delaying complete functional recovery. Recovery or persistence of inflammation was associated with gene expression patterns involved not only in immune function but also in tissue remodeling, cell adhesion, and uterine receptivity in a cell type-specific manner. Identifying these signatures may contribute to the development of novel diagnostic tools and therapeutic strategies. In addition, these results may help to define preventive measures or ways to stimulate recovery from endometrial inflammation, thus helping to restore the fertility of postpartum dairy cows.

Gastruloids: Pluripotent stem cell models of mammalian gastrulation and embryo engineering

Gastrulation is a fundamental and critical process of animal development whereby the mass of cells that results from the proliferation of the zygote transforms itself into a recognizable outline of an organism. The last few years have seen the emergence of a number of experimental models of early mammalian embryogenesis based on Embryonic Stem (ES) cells. One of this is the Gastruloid model. Gastruloids are aggregates of defined numbers of ES cells that, under defined culture conditions, undergo controlled proliferation, symmetry breaking, and the specification of all three germ layers characteristic of vertebrate embryos, and their derivatives. However, they lack brain structures and, surprisingly, reveal a disconnect between cell type specific gene expression and tissue morphogenesis, for example during somitogenesis. Gastruloids have been derived from mouse and human ES cells and several variations of the original model have emerged that reveal a hereto unknown modularity of mammalian embryos. We discuss the organization and development of gastruloids in the context of the embryonic stages that they represent, pointing out similarities and differences between the two. We also point out their potential as a reproducible, scalable and searchable experimental system and highlight some questions posed by the current menagerie of gastruloids.

Generative Deep Learning of the Single Cell Dose Response of 2,3,7,8 Tetrachlorodibenzo‐p‐dioxin in Mouse Liver

Single cell RNA‐seq provides a new opportunity to study the heterogeneity of chemical perturbation within tissues. However, exploring the space of all cell type – chemical combinations is experimentally and financially unfeasible. This space is significantly expanded by the dose axis of chemical perturbation. Computational tools are needed to predict responses not only across tissues, but also across doses while capturing the nuances of cell type specific gene expression. Generative deep learning architectures such as autoencoders simplify the single cell expression space allowing cross‐tissue, ‐organ, ‐organism and ‐dose predictions using simple vector arithmetic. However, differing sensitivities and non‐linearities make cell type specific gene expression predictions following treatment at higher doses challenging. Here, improvements to vector arithmetic in the latent space achieve high dose cell type specific predictions better than other state of the art algorithms. We specifically look at a well‐studied toxicant, 2,3,7,8 Tetrachlorodibenzo‐p‐dioxin (TCDD), in the mouse liver as a case study. TCDD binds to and activates the aryl hydrocarbon receptor (AhR) to regulate gene expression associated with several physiological processes including xenobiotic metabolism, development, and circadian rhythm. Prediction of the effects of TCDD across liver cell types is non‐trivial as AhR is not only differentially expressed across cell types but is also differentially expressed within certain cell types along the porto‐central axis of the liver lobule. Our model predicts dose‐dependent gene expression across liver zones (i.e., portal to central hepatocytes), and across liver cell types (e.g., endothelial to stellate cells) using basal gene expression data. The model is also able to predict low dose TCDD gene expression using high dose data. Finally, using outputs from the model, an algorithm was developed to order cells based on their sensitivities to a particular toxicant by assigning a “pseudo‐dose” value to each cell. Given the model’s high performance on these tasks, the tool developed here will aid the exploration of the gene expression space of chemical perturbation.

Cell type-specific differential expression in Alzheimer’s disease and cancer using spatial transcriptomics

Abstract Spatial transcriptomics enables spatially resolved gene expression measurements at near single-cell resolution. The detection of genes that are differentially expressed across tissue context for cell types of interest is an essential challenge for dissecting pathological mechanisms. However, changes in cell type composition across space and measurement contamination from other cell types introduce complex statistical challenges. Here, we introduce a statistical method, Generalized Linear Admixture Models for Differential Expression (GLAMDE), that estimates cell type-specific patterns of differential gene expression while accounting for localization of other cell types. By modeling spatial transcriptomics gene expression as an additive mixture, across cell types, of generalized log-linear functions, we provide a unified framework for identifying gene expression changes for a wide-range of relevant contexts: changes due to pathology, anatomical regions, physical proximity to specific cell types, and cellular microenvironment. Our approach enables statistical inference across multiple samples and replicates. We demonstrate, through simulations and validation experiments on Slide-seq and MERFISH data, that our approach accurately identifies cell type-specific differential gene expression with valid uncertainty quantification. Lastly, we apply GLAMDE to characterize spatially-localized tissue changes in the context of disease. In an Alzheimer’s mouse model Slide-seq dataset, we identify plaque-dependent patterns of cellular immune activity. We also find a putative interaction between tumor cells and myeloid immune cells in a Slide-seq tumor dataset. GLAMDE is available within the spacexr R package. D.C. was supported by a Fannie and John Hertz Foundation Fellowship and an NSF Graduate Research Fellowship. This work was supported by an NIH Early Independence Award (DP5, 1DP5OD024583 to F.C.), the NHGRI (R01, R01HG010647 to F.C.), as well as the Burroughs Wellcome Fund, the Searle Scholars Award, and the Merkin Institute to F.C.. R.A.I. was supported by NIH grants R35GM131802 and R01HG005220.

Combining Immunohistochemistry and In Situ Hybridization to Characterize Immunosuppression in the Tumor Microenvironment

Abstract While immune-based cancer treatments have been successfully deployed in the clinic for hematological malignancies, treatment of solid tumors has proven more challenging. Comprehensive understanding of the dynamic landscape of the tumor microenvironment (TME) will lead to new therapies and improved efficacy of current therapies. Immunohistochemistry (IHC) and in situ hybridization (ISH) are the two techniques at the forefront of TME characterization. Combining RNA and protein detection with ISH and IHC reveals cell-type specific gene expression and the cellular sources of secreted proteins. Traditional ISH-IHC workflows require harsh pre-treatment of tissues prior to detection with primary antibodies, destroying the target epitope for some antibody clones. Bio-Techne’s RNAscopeTM RNA protein Codetection Assay improves the likelihood of successful antibody staining by incubating with primary antibody first, before initiating the RNAScope pretreatment step. With minimal optimization time, we were able to visualize protein expression using IHC validated antibodies for important immunosuppression biomarkers such as HIF-1 alpha, FoxP3, PD-L1, and CD11b. Using a cancer tissue microarray (TMA), we show this workflow is compatible with many tissue types, including pancreas, uterus, liver, breast, lung, prostate, and colon tissue. In combination with RNAScope probes CD8A, CD4, PD-1, TGFB1, and VEGF we were able to identify immunosuppressive cells in the TME, including Regulatory T cells (Tregs) and tumor associated macrophages (TAMs). Adoption of this workflow will streamline profiling of the TME, facilitating a more comprehensive evaluation of solid tumors for biomarker discovery and immunotherapy development.

Single Nuclear RNA Sequencing Reveals Activation of Neuroinflammation Within the Pre‐Bötzinger Complex Following Repeated Seizures

Epilepsy is one of the most common neurological diseases in the world. Treatment with anti‐epileptic drugs (AEDs) prevents recurrent seizures in ~70% of patients with epilepsy, but the remaining 30% of patients with refractory epilepsies continue to experience seizures. The consequences of repeated seizures include, but are not limited to, a high risk of post‐ictal cardiorespiratory failure and Sudden Unexpected Death in Epilepsy (SUDEP). Fundamental gaps exist in understanding how repeated seizures disrupt the vital cardiorespiratory control system in refractory epilepsy. A factor commonly identified in many neurological conditions is neuroinflammation, which may support beneficial health functions but is dysregulated in neurological disease. Key cells within the CNS that mediate beneficial and/or pathological neuroinflammation are resident microglia and astrocytes, which have been shown to be dysfunctional in human epilepsy and alter neuronal function. However, it is not known what mechanistic role they play in contributing to epilepsy‐related impairment in cardiorespiratory control or increased SUDEP risk. Here, we tested the hypothesis that repeated seizures lead to activation of neuroinflammation mediated by microglia and astrocytes in CNS regions of cardiorespiratory control, leading to a progressive decline in neurologic function. Preliminary data in our novel rat model with genetic mutations in Kcnj16, a gene encoding an inwardly‐rectifying potassium channel Kir5.1 in Dahl salt‐sensitive rat (SSkcnj16‐/‐) show that repeated sound‐induced seizures lead to a progressively more severe post‐ictal suppression of breathing frequency and heart rate. Single nuclear RNA sequencing of pre‐Bötzinger Complex/Nucleus Ambiguous cells (roughly 80,000) was performed from rats exposed to 0, 3, 7, or 10 seizures to measure cell type‐specific gene expression changes induced by seizures. Bioinformatic analyses of transcriptomic changes in microglia, neuronal, and glial cells within these regions point to activation of specific inflammatory pathways following repeated seizures such as IL‐1<img width=“7” height=“13” src=“file:////Users/wasifosmani/Library/Group%20Containers/UBF8T346G9.Office/TemporaryItems/msohtmlclip/clip_image002.png”= signaling and Toll‐like Receptor signaling. Afterwards, cytokine‐chemokine array was performed in brain regions regulating cardiorespiratory control after rats were exposed to 0, 3, 7, or 10 seizures and confirmed the presence of neuroinflammation after seizure induction. Identifying neuroinflammatory signals/pathways induced by repeated seizures within distinct neural circuits will enhance understanding of pathophysiological consequences of uncontrolled seizures in patients with refractory epilepsy and hold the potential for identifying new therapeutic targets aimed at improving cardiorespiratory function to reduce the risk of SUDEP.

Abstract 397: Smooth Muscle Cell-specific Lncrna Carmn Is Regulated By Srf/myocd Complex

Background: Cell identify is established by strict control of cell type-specific gene expression programs through regulatory enhancers binding of transcriptional factors. Vascular smooth muscle cells (VSMCs) are critical in maintaining the vascular homeostasis by expressing an array of contractile genes that are primarily governed by SRF/MYOCD complex via binding to the DNA motif called CArG element. However, most of these previous studies regarding regulatory mechanism underlying SM-specific gene expression were performed in vitro, outside of their in situ genomic contexts. Methods and Results: To define the regulatory atlas of VSMC, we analyzed epigenetic datasets of multiple human tissues and cell types. We found CARMN , that we previously reported as a SMC-specific lncRNA and a critical regulator of SMC contractile phenotype by binding to MYOCD, is enriched with SMCs-specific enhancers. Co-expression analysis using RNA-seq datasets of different human tissues identified CARMN expression is positively correlated with both MYOCD and SRF . We further demonstrated overexpressing MYOCD activates, while silencing MYOCD or SRF inhibits CARMN expression in human coronary VSMCs. Subsequent analysis of SRF ChIP-seq data revealed 4 SRF binding peaks with CARMN locus, including 2 located within promoter (namely CArG 1 and 2) and the other 2 (namely CArG 3 and 4) located within distal enhancers. Further luciferase reporter assays revealed mutation of CArG 1 or 3 dramatically abolish MYOCD-induced reporter activity. To confirm these findings in vivo, we generated single CArG element mutant mice for each of the 4 CArG elements and CArG 3/4 double mutant mice by CRISPR-Cas9 genome editing approach. Analysis of Carmn expression in SMC-enriched tissues from these mutant mice revealed a ~50% decrease of Carmn expression in tissues of CArG 1 mutant mice while mutation of other CArG elements appears no effect on Carmn expression. Conclusions: Our results suggest the SM-specific expression of Carmn is dependent, at least partially, on SRF/MYOCD complex and CArG 1 is critical for this regulation. Together with our previous findings that CARMN potentiates MYOCD function, we propose CARMN and SRF/MYOCD complex form a feedforward loop promoting SMC contractile phenotype.

The molecular understanding of super-enhancer dysregulation in cancer.

ABSTRACTAbnormalities in the regulation of gene expression are associated with various pathological conditions. Among the distal regulatory elements in the genome, the activation of target genes by enhancers plays a central role in the formation of cell type–specific gene expression patterns. Super-enhancers are a subclass of enhancers that frequently contain multiple enhancer-like elements and are characterized by dense binding of master transcription factors and Mediator complexes and high signals of active histone marks. Super-enhancers have been studied in detail as important regulatory regions that control cell identity and contribute to the pathogenesis of diverse diseases. In cancer, super-enhancers have multifaceted roles by activating various oncogenes and other cancer-related genes and shaping characteristic gene expression patterns in cancer cells. Alterations in super-enhancer activities in cancer involve multiple mechanisms, including the dysregulation of transcription factors and the super-enhancer–associated genomic abnormalities. The study of super-enhancers could contribute to the identification of effective biomarkers and the development of cancer therapeutics targeting transcriptional addiction. In this review, we summarize the roles of super-enhancers in cancer biology, with a particular focus on hematopoietic malignancies, in which multiple super-enhancer alteration mechanisms have been reported.

Germacrene A Synthases for Sesquiterpene Lactone Biosynthesis Are Expressed in Vascular Parenchyma Cells Neighboring Laticifers in Lettuce.

Sesquiterpene lactone (STL) and natural rubber (NR) are characteristic isoprenoids in lettuce (Lactuca sativa). Both STL and NR co-accumulate in laticifers, pipe-like structures located along the vasculature. NR-biosynthetic genes are exclusively expressed in laticifers, but cell-type specific expression of STL-biosynthetic genes has not been studied. Here, we examined the expression pattern of germacrene A synthase (LsGAS), which catalyzes the first step in STL biosynthesis in lettuce. Quantitative PCR and Illumina read mapping revealed that the transcripts of two GAS isoforms (LsGAS1/LsGAS2) are expressed two orders of magnitude (~100–200) higher in stems than laticifers. This result implies that the cellular site for LsGAS1/2 expression is not in laticifers. To gain more insights, promoters of LsGAS1/2 were cloned and fused to β-glucuronidase (GUS), followed by transformations of lettuce with these promoter-GUS constructs. In in situ GUS assays, the GUS expression driven by the LsGAS1/2 promoters was tightly associated with vascular bundles. High-resolution microsections showed that GUS signals are not present in laticifers but are detected in the vascular parenchyma cells neighboring the laticifers. These results suggest that expression of LsGAS1/2 occurs in the parenchyma cells neighboring laticifers, while the resulting STL metabolites accumulate in laticifers. It can be inferred that active metabolite-trafficking occurs from the parenchyma cells to laticifers in lettuce.