Cell Type-specific Gene Expression Research Articles

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.

DNA Methyltransferase 3b Accelerates the Process of Atherosclerosis.

Background DNA methylation plays a key role in establishing cell type–specific gene expression profiles and patterns in atherosclerosis. The underlying mechanism remains unclear. Previous studies have shown that DNA methyltransferase 3b (DNMT3b) may play an important role in atherosclerosis. This study aimed to establish the regulatory role of DNMT3b in the development of atherosclerosis. Methods We constructed a viral vector carrying Dnmt3b shRNA to transduce ApoE−/− mice. Meanwhile, healthy human peripheral blood Treg cells were treated with inhibitor of DNMT3b (AZA and EGCG) or transduced with DNMT3b shRNA. Results It showed that Dnmt3b silencing attenuated atherosclerosis, including decreased lesion size and macrophage content and increased collagen and smooth muscle cells content in ApoE−/− mice. To further investigate the possible mechanisms, combined with previous studies by our group, we showed that Foxp3-TSDR methylation level was significantly reduced Foxp3 expression and peripheral blood Treg levels were significantly increased by Dnmt3b shRNA vector transduction in animals committed to western diet for 12 and 18 weeks. Consistently, inhibition of DNMT3b (AZA and EGCG) decreased the expression levels of DNMT3b, which can increase the expression levels of FOXP3, and increase the levels of TGF-β and IL-10 and decrease the levels of IL-β and IFN-γ. After transduction with DNMT3b shRNA, the effect was more obvious. Conclusions DNMT3b accelerated atherosclerosis, and may be associated with FOXP3 hypermethylation status in regulatory T cells.

Combined Single Cell Transcriptome and Surface Epitope Profiling Identifies Potential Biomarkers of Psoriatic Arthritis and Facilitates Diagnosis via Machine Learning.

Early diagnosis of psoriatic arthritis (PSA) is important for successful therapeutic intervention but currently remains challenging due, in part, to the scarcity of non-invasive biomarkers. In this study, we performed single cell profiling of transcriptome and cell surface protein expression to compare the peripheral blood immunocyte populations of individuals with PSA, individuals with cutaneous psoriasis (PSO) alone, and healthy individuals. We identified genes and proteins differentially expressed between PSA, PSO, and healthy subjects across 30 immune cell types and observed that some cell types, as well as specific phenotypic subsets of cells, differed in abundance between these cohorts. Cell type-specific gene and protein expression differences between PSA, PSO, and healthy groups, along with 200 previously published genetic risk factors for PSA, were further used to perform machine learning classification, with the best models achieving AUROC ≥ 0.87 when either classifying subjects among the three groups or specifically distinguishing PSA from PSO. Our findings thus expand the repertoire of gene, protein, and cellular biomarkers relevant to PSA and demonstrate the utility of machine learning-based diagnostics for this disease.

H3K27ac nucleosomes facilitate HMGN localization at regulatory sites to modulate chromatin binding of transcription factors

Nucleosomes containing acetylated H3K27 are a major epigenetic mark of active chromatin and identify cell-type specific chromatin regulatory regions which serve as binding sites for transcription factors. Here we show that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 bind preferentially to H3K27ac nucleosomes at cell-type specific chromatin regulatory regions. HMGNs bind directly to the acetylated nucleosome; the H3K27ac residue and linker DNA facilitate the preferential binding of HMGNs to the modified nucleosomes. Loss of HMGNs increases the levels of H3K27me3 and the histone H1 occupancy at enhancers and promoters and alters the interaction of transcription factors with chromatin. These experiments indicate that the H3K27ac epigenetic mark enhances the interaction of architectural protein with chromatin regulatory sites and identify determinants that facilitate the localization of HMGN proteins at regulatory sites to modulate cell-type specific gene expression.

Immune cell gene expression signatures in diffuse glioma are associated with IDH mutation status, patient outcome and malignant cell state, and highlight the importance of specific cell subsets in glioma biology

The tumor micro-environment (TME) plays an important role in various cancers, including gliomas. We estimated immune cell type-specific gene expression profiles in 3 large clinically annotated glioma datasets using CIBERSORTx and LM22/LM10 blood-based immune signatures and found that the proportions and estimated gene expression patterns of specific immune cells significantly varied according to IDH mutation status. When IDH-WT and IDH-MUT tumors were considered separately, cluster-of-cluster analyses of immune cell gene expression identified groups with distinct survival outcomes. We confirmed and extended these findings by applying a signature matrix derived from single-cell RNA-sequencing data derived from 19 glioma tumor samples to the bulk profiling data, validating findings from the LM22/LM10 results. To link immune cell signatures with outcomes in checkpoint therapy, we then showed a significant association of monocytic lineage cell gene expression clusters with patient survival and with mesenchymal gene expression scores. Integrating immune cell-based gene expression with previously described malignant cell states in glioma demonstrated that macrophage M0 abundance significantly correlated with mesenchymal state in IDH-WT gliomas, with evidence of a previously implicated role of the Oncostatin-M receptor and macrophages in the mesenchymal state. Among IDH-WT tumors that were enriched for the mesenchymal cell state, the estimated M0 macrophage expression signature coordinately also trended to a mesenchymal signature. We also examined IDH-MUT tumors stratified by 1p/19q status, showing that a mesenchymal gene expression signature the M0 macrophage fraction was enriched in IDH-MUT, non-codeleted tumors. Overall, these results highlight the biological and clinical significance of the immune cell environment related to IDH mutation status, patient prognosis and the mesenchymal state in diffuse gliomas.

Deconvolving Clinically Relevant Cellular Immune Cross-talk from Bulk Gene Expression Using CODEFACS and LIRICS Stratifies Patients with Melanoma to Anti-PD-1 Therapy.

This work presents two new computational methods that can deconvolve a large collection of bulk tumor gene expression profiles into their respective cell type-specific gene expression profiles and identify cell type-specific ligand-receptor interactions predictive of response to immune-checkpoint blockade therapy. This article is highlighted in the In This Issue feature, p. 873.

The Alzheimer's Cell Atlas (TACA): A single-cell molecular map for translational therapeutics accelerator in Alzheimer's disease.

IntroductionRecent advances in generating massive single‐cell/nucleus transcriptomic data have shown great potential for facilitating the identification of cell type–specific Alzheimer's disease (AD) pathobiology and drug‐target discovery for therapeutic development.MethodsWe developed The Alzheimer's Cell Atlas (TACA) by compiling an AD brain cell atlas consisting of over 1.1 million cells/nuclei across 26 data sets, covering major brain regions (hippocampus, cerebellum, prefrontal cortex, and so on) and cell types (astrocyte, microglia, neuron, oligodendrocytes, and so on). We conducted nearly 1400 differential expression comparisons to identify cell type–specific molecular alterations (e.g., case vs healthy control, sex‐specific, apolipoprotein E (APOE) ε4/ε4, and TREM2 mutations). Each comparison was followed by protein‐protein interaction module detection, functional enrichment analysis, and omics‐informed target and drug (over 700,000 perturbation profiles) screening. Over 400 cell‐cell interaction analyses using 6000 ligand‐receptor interactions were conducted to identify the cell‐cell communication networks in AD.ResultsAll results are integrated into TACA (https://taca.lerner.ccf.org/), a new web portal with cell type–specific, abundant transcriptomic information, and 12 interactive visualization tools for AD.DiscussionWe envision that TACA will be a highly valuable resource for both basic and translational research in AD, as it provides abundant information for AD pathobiology and actionable systems biology tools for drug discovery.Highlights We compiled an Alzheimer's disease (AD) brain cell atlas consisting of more than 1.1 million cells/nuclei transcriptomes from 26 data sets, covering major brain regions (cortex, hippocampus, cerebellum) and cell types (e.g., neuron, oligodendrocyte, astrocyte, and microglia).We conducted over 1400 differential expression (DE) comparisons to identify cell type–specific gene expression alterations. Major comparison types are (1) AD versus healthy control; (2) sex‐specific DE, (3) genotype‐driven DE (i.e., apolipoprotein E [APOE] ε4/ε4 vs APOE ε3/ε3; TREM2R47H vs common variants) analysis; and (4) others. Each comparison was further followed by (1) human protein‐protein interactome network module analysis, (2) pathway enrichment analysis, and (3) gene‐set enrichment analysis.For drug screening, we conducted gene set enrichment analysis for all the comparisons with over 700,000 drug perturbation profiles connecting more than 10,000 human genes and 13,000 drugs/compounds.A total of over 400 analyses of cell‐cell interactions against 6000 experimentally validated ligand‐receptor interactions were conducted to reveal the disease‐relevant cell‐cell communications in AD.

Integrative analysis of the 3D genome structure reveals that CTCF maintains the properties of mouse female germline stem cells

The three-dimensional configuration of the genome ensures cell type-specific gene expression profiles by placing genes and regulatory elements in close spatial proximity. Here, we used in situ high-throughput chromosome conformation (in situ Hi-C), RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to characterize the high-order chromatin structure signature of female germline stem cells (FGSCs) and identify its regulating key factor based on the data-driven of multiple omics data. By comparison with pluripotent stem cells (PSCs), adult stem cells (ASCs), and somatic cells at three major levels of chromatin architecture, A/B compartments, topologically associating domains, and chromatin loops, the chromatin architecture of FGSCs was most similar to that of other ASCs and largely different from that of PSCs and somatic cells. After integrative analysis of the three-dimensional chromatin structure, active compartment-associating loops (aCALs) were identified as a signature of high-order chromatin organization in FGSCs, which revealed that CCCTC-binding factor was a major factor to maintain the properties of FGSCs through regulation of aCALs. We found FGSCs belong to ASCs at chromatin structure level and characterized aCALs as the high-order chromatin structure signature of FGSCs. Furthermore, CTCF was identified to play a key role in regulating aCALS to maintain the biological functions of FGSCs. These data provide a valuable resource for future studies of the features of chromatin organization in mammalian stem cells and further understanding of the fundamental characteristics of FGSCs.

Estimating cell type composition using isoform expression one gene at a time.

Human tissue samples are often mixtures of heterogeneous cell types, which can confound the analyses of gene expression data derived from such tissues. The cell type composition of a tissue sample may itself be of interest and is needed for proper analysis of differential gene expression. A variety of computational methods have been developed to estimate cell type proportions using gene-level expression data. However, RNA isoforms can also be differentially expressed across cell types, and isoform-level expression could be equally or more informative for determining cell type origin than gene-level expression. We propose a new computational method, IsoDeconvMM, which estimates cell type fractions using isoform-level gene expression data. A novel and useful feature of IsoDeconvMM is that it can estimate cell type proportions using only a single gene, though in practice we recommend aggregating estimates of a few dozen genes to obtain more accurate results. We demonstrate the performance of IsoDeconvMM using a unique data set with cell type-specific RNA-seq data across more than 135 individuals. This data set allows us to evaluate different methods given the biological variation of cell type-specific gene expression data across individuals. We further complement this analysis with additionalsimulations.

Tumor immune microenvironment lncRNAs.

Long non-coding ribonucleic acids (RNAs) (lncRNAs) are key players in tumorigenesis and immune responses. The nature of their cell type-specific gene expression and other functional evidence support the idea that lncRNAs have distinct cellular functions in the tumor immune microenvironment (TIME). To date, the majority of lncRNA studies have heavily relied on bulk RNA-sequencing data in which various cell types contribute to an averaged signal, limiting the discovery of cell type-specific lncRNA functions. Single-cell RNA-sequencing (scRNA-seq) is a potential solution for tackling this limitation despite the lack of annotations for low abundance yet cell type-specific lncRNAs. Hence, updated annotations and further understanding of the cellular expression of lncRNAs will be necessary for characterizing cell type-specific functions of lncRNA genes in the TIME. In this review, we discuss lncRNAs that are specifically expressed in tumor and immune cells, summarize the regulatory functions of the lncRNAs at the cell type level and highlight how a scRNA-seq approach can help to study the cell type-specific functions of TIME lncRNAs.

Dynamic transcription regulation at the single-molecule level

Cell fate changes during development, differentiation, and reprogramming are largely controlled at the transcription level. The DNA-binding transcription factors (TFs) often act in a combinatorial fashion to alter chromatin states and drive cell type-specific gene expression. Recent advances in fluorescent microscopy technologies have enabled direct visualization of biomolecules involved in the process of transcription and its regulatory events at the single-molecule level in living cells. Remarkably, imaging and tracking individual TF molecules at high temporal and spatial resolution revealed that they are highly dynamic in searching and binding cognate targets, rather than static and binding constantly. In combination with investigation using techniques from biochemistry, structure biology, genetics, and genomics, a more well-rounded view of transcription regulation is emerging. In this review, we briefly cover the technical aspects of live-cell single-molecule imaging and focus on the biological relevance and interpretation of the single-molecule dynamic features of transcription regulatory events observed in the native chromatin environment of living eukaryotic cells. We also discuss how these dynamic features might shed light on mechanistic understanding of transcription regulation.

APOE variant in the receptor binding domain confers cognitive resilience to familial Alzheimer's mutations and cell-type specific gene expression changes in the hippocampus.

Cognitive resilience to familial Alzheimer's Disease (FAD) is a phenomenon whereby cognitive functioning is better than predicted based on highly penetrant mutations in APP and PS1. This resilience is likely mediated by unidentified genetic factors, and we hypothesize that these factors provide key targets for treatment and prevention of AD. We previously discovered that the most commonly used background strain in AD model research (C57BL/6J, B6) is resilient to AD-related cognitive decline. Using genetic mapping, we determined that cognitive resilience is associated with a variant in the Apoe receptor binding domain and utilized CRISPR to generate a novel Apoe knockin mouse (Apoe KIB6/E163D ). Contextual fear conditioning (CFC), tau pathology, and single-nuclear RNAseq hippocampi analyses were performed in male and female Apoe KIB6/E163D x 5XFAD progeny that differed by only a single variant in Apoe leading to the E163D base pair change in the protein. We found that, indeed, cognitive resilience to the 5XFAD transgene was conferred in ApoeB6/B6 mice compared to Apoe KIB6/E163D mice tested on CFC. Although minimal differences in hyperphosphorylated tau abundance were observed between 5XFAD-ApoeWT/WT mice compared to 5XFAD-Apoe KIB6/E163D , we uncovered robust differences in the transcriptomes of hippocampal excitatory and inhibitory neurons and microglia using single-nuclear sequencing. Gene enrichment and pathway analyses of differentially expressed genes suggest that the 5XFAD-ApoeB6/B6 genotype promotes cation transport and suppression of amyloid fibril formation in excitatory neurons, promotes genes involved in ribosome assembly and protein translation in inhibitory neurons, and enhances gene expression associated with stress response in microglia. Our results suggest that 5XFAD-ApoeB6/B6 homozygotes exhibit cognitive resilience to causal FAD mutations that involve dynamic regulation of gene networks across multiple cell types, emphasizing the need for well-aligned cell culture models of cognitive resilience to validate targets and screen resilience-based interventions. Future work leveraging in vivo electrophysiology and imaging will establish mechanisms whereby Apoe variants alter neural and synaptic function. Understanding the relationship between protective genetic factors and cognitive resilience are crucial for determining mechanisms of resilience and uncovering novel targets for intervention.

Cell type-specific gene expression changes in Alzheimer's patients with varying levels of cerebral amyloid angiopathy.

Tau neurofibrillary tangles and senile plaques comprised of insoluble amyloid beta are the major histopathological hallmarks of Alzheimer's disease (AD). More than 85% of autopsy-confirmed AD cases also exhibit some degree of cerebral amyloid angiopathy (CAA), which is characterized by amyloid beta peptide deposits predominantly in blood vessels in the meningeal and intracerebral blood vessels. Consequently, CAA predisposes individuals with AD to cerebral infarction and hemorrhages, accounting for ∼20% of cases in the elderly, which could lead to faster cognitive decline through neurovascular pathway dysfunction. We sought to identify cellular composition changes and characterize cell type-specific expression associated with CAA pathology in AD patients. Using 10x Genomics single nuclei RNA sequencing, we profiled nuclei from frozen post-mortem temporal cortex tissue of 80 individuals with neuropathologic diagnosis of AD and varying levels of CAA, ranging from zero to severe CAA pathology. Braak and Thal measures were also obtained. Downstream analyses were performed in R using Seurat v3.2.3. Quality control filtering based on percentage of genes mapped to the mitochondrial genome and number of genes and UMIs detected resulted in 135,092 nuclei for analysis. Differential gene expression analyses were performed using R package MAST. We identified 30 clusters which were annotated using established cell type marker genes. There were 3 oligodendrocyte, 14 neuronal (2 excitatory and 3 inhibitory), 5 microglial, 3 endothelial, 4 astrocyte and 1 oligodendrocyte precursor cell cluster. We observed a significant increase in microglial cell proportions with increasing CAA severity, for 3 of the microglial clusters. Conversely, the excitatory neuronal proportion were decreased in individuals with more severe CAA. Further investigation is ongoing to identify the genes underlying the dysregulation of these cell types in CAA. We observed cellular proportion changes for multiple cell types with increasing CAA severity. Additional analyses to determine the effects of CAA independent of AD severity will be performed by adjusting for Braak and Thal measures. Deeper characterization of cell type-specific expression profiles is expected to provide novel insights into cell-type specific transcriptome changes associated with vascular risk factors in AD.

Spatiotemporal dynamics of SETD5-containing NCoR\u2013HDAC3 complex determines enhancer activation for adipogenesis

Enhancer activation is essential for cell-type specific gene expression during cellular differentiation, however, how enhancers transition from a hypoacetylated “primed” state to a hyperacetylated-active state is incompletely understood. Here, we show SET domain-containing 5 (SETD5) forms a complex with NCoR-HDAC3 co-repressor that prevents histone acetylation of enhancers for two master adipogenic regulatory genes Cebpa and Pparg early during adipogenesis. The loss of SETD5 from the complex is followed by enhancer hyperacetylation. SETD5 protein levels were transiently increased and rapidly degraded prior to enhancer activation providing a mechanism for the loss of SETD5 during the transition. We show that induction of the CDC20 co-activator of the ubiquitin ligase leads to APC/C mediated degradation of SETD5 during the transition and this operates as a molecular switch that facilitates adipogenesis.

Genome-wide expression of the residual lung reacting to experimental Pneumonectomy

BackgroundAcute or chronic irreversible respiratory failure may occur in patients undergoing pneumonectomy. Aim of this study was to determine transcriptome expression changes after experimental pneumonectomy in swine model. Experimental left pneumonectomy was performed in five pigs under general anaesthesia. Both the resected and the remaining lung, after 60 post-operative completely uneventful days, underwent genome-wide bulk RNA-Sequencing (RNA-Seq).ResultsHistological analysis showed dilation of air spaces and rupture of interalveolar septa. In addition, mild inflammation, no fibrosis, radial stretch of the bronchus, strong enlargement of airspaces and thinning of the blood supply were observed. Bioinformatic analyses of bulk RNA-Seq data identified 553 Differentially Expressed Genes (DEGs) at adjusted P-value below 0.001, between pre- and post-pneumonectomy. The top 10 up-regulated DEGs were Edn1, Areg, Havcr2, Gadd45g, Depp1, Cldn4, Atf3, Myc, Gadd45b, Socs3; the top 10 down-regulated DEGs were Obscn, Cdkn2b, ENSSSCG00000015738, Prrt2, Amer1, Flrt3, Efnb2, Tox3, Znf793, Znf365. Leveraging digital cytometry tools, no difference in cellular abundance was found between the two experimental groups, while the analysis of cell type-specific gene expression patterns highlighted a striking predominance of macrophage-specific genes among the DEGs. DAVID-based gene ontology analysis showed a significant enrichment of “Extrinsic apoptotic signaling pathway” (FDR q = 7.60 × 10− 3) and “Response to insulin” (FDR q = 7.60 × 10− 3) genes, along with an enrichment of genes involved as “Negative regulators of DDX58/IFIH1 signaling” (FDR q = 7.50 × 10− 4) found by querying the REACTOME pathway database. Gene network analyses indicated a general dysregulation of gene inter-connections.ConclusionThis translational genomics study highlighted the existence both of individual genes, mostly dysregulated in certain cellular populations (e.g., macrophages), and gene-networks involved in pulmonary reaction after left pneumonectomy. Their involvement in lung homeostasis is largely supported by previous studies, carried out both in humans and in other animal models (under homeostatic or disease-related conditions), that adopted candidate-gene approaches. Overall, the present findings represent a preliminary assessment for future, more focused, studies on compensatory lung adaptation, pulmonary regeneration and functional reload.

Exploring the Diverse Functional and Regulatory Consequences of Alternative Splicing in Development and Disease.

Alternative splicing is a fundamental mechanism of eukaryotic RNA regulation that increases the transcriptomic and proteomic complexity within an organism. Moreover, alternative splicing provides a framework for generating unique yet complex tissue- and cell type-specific gene expression profiles, despite using a limited number of genes. Recent efforts to understand the negative consequences of aberrant splicing have increased our understanding of developmental and neurodegenerative diseases such as spinal muscular atrophy, frontotemporal dementia and Parkinsonism linked to chromosome 17, myotonic dystrophy, and amyotrophic lateral sclerosis. Moreover, these studies have led to the development of innovative therapeutic treatments for diseases caused by aberrant splicing, also known as spliceopathies. Despite this, a paucity of information exists on the physiological roles and specific functions of distinct transcript spliceforms for a given gene. Here, we will highlight work that has specifically explored the distinct functions of protein-coding spliceforms during development. Moreover, we will discuss the use of alternative splicing of noncoding exons to regulate the stability and localization of RNA transcripts.