The choice of negative control antisense oligonucleotides dramatically impacts downstream analysis depending on the cellular background

Abstract Background The lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism’s physiological function. Despite their common developmental origin, they present distinct functional fates in adulthood that rely on robust lineage-specific regulatory programs. The recent technological boost in sequencing approaches unveiled long noncoding RNAs (lncRNAs) as prominent regulatory players of various gene expression levels in a cell-type-specific manner. Results To investigate the potential roles of lncRNAs in vascular biology, we performed antisense oligonucleotide (ASO) knockdowns of lncRNA candidates specifically expressed either in human lymphatic or blood vascular endothelial cells (LECs or BECs) followed by Cap Analysis of Gene Expression (CAGE-Seq). Here, we describe the quality control steps adopted in our analysis pipeline before determining the knockdown effects of three ASOs per lncRNA target on the LEC or BEC transcriptomes. In this regard, we especially observed that the choice of negative control ASOs can dramatically impact the conclusions drawn from the analysis depending on the cellular background. Conclusion In conclusion, the comparison of negative control ASO effects on the targeted cell type transcriptomes highlights the essential need to select a proper control set of multiple negative control ASO based on the investigated cell types.

Download Full-text

Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

Communications Biology ◽

10.1038/s42003-020-01418-x ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 1

Author(s):

Ana J. Chucair-Elliott ◽

Sarah R. Ocañas ◽

David R. Stanford ◽

Victor A. Ansere ◽

Kyla B. Buettner ◽

...

Keyword(s):

Gene Expression ◽

Affinity Purification ◽

Regulation Of Gene Expression ◽

Cell Types ◽

Tissue Level ◽

Cell Phenotype ◽

Specific Gene ◽

Cell Type ◽

A Cell ◽

Cell Type Specific

AbstractEpigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific central nervous system cell types. Analysis of gene expression and DNA modifications in astrocytes or microglia from the same animal demonstrates differential usage of DNA methylation and hydroxymethylation in CpG and non-CpG contexts that corresponds to cell type-specific gene expression. Application of this approach in LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model and the validation approaches presented can be applied to any brain cell type for which a cell type-specific cre is available.

Download Full-text

Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

10.1101/862722 ◽

2019 ◽

Author(s):

Ana J. Chucair-Elliott ◽

Sarah R. Ocañas ◽

David R. Stanford ◽

Victor A. Ansere ◽

Kyla B. Buettner ◽

...

Keyword(s):

Gene Expression ◽

Regulation Of Gene Expression ◽

Cell Types ◽

Tissue Level ◽

Cell Phenotype ◽

Specific Gene ◽

Cell Type ◽

Current Cell ◽

A Cell ◽

Cell Type Specific

AbstractEpigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific CNS cell types. Paired analysis of the transcriptome and DNA modifications in astrocytes and microglia demonstrates differential usage of DNA methylation and hydroxymethylation in CG and non-CG contexts that corresponds to cell type-specific gene expression. Application of this approach in LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model and the validation approaches presented can be applied to any CNS cell type for which a cell type-specific cre is available.

Download Full-text

Alignment of single-cell RNA-seq samples without over-correction using kernel density matching

10.1101/2020.01.05.895136 ◽

2020 ◽

Cited By ~ 2

Author(s):

Mengjie Chen ◽

Qi Zhan ◽

Zepeng Mu ◽

Lili Wang ◽

Zhaohui Zheng ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Rna Sequencing ◽

Kernel Density ◽

Cell Types ◽

Clinical Samples ◽

Cell Type ◽

Inflammatory Monocytes ◽

A Cell ◽

Cell Type Specific

AbstractSingle-cell RNA sequencing (scRNA-seq) technology is poised to replace bulk cell RNA sequencing for most biological and medical applications as it allows users to measure gene expression levels in a cell-type-specific manner. However, data produced by scRNA-seq often exhibit batch effects that can be specific to a cell-type, to a sample, or to an experiment, which prevent integration or comparisons across multiple experiments. Here, we present Dmatch, a method that leverages an external expression atlas of human primary cells and kernel density matching to align multiple scRNA-seq experiments for downstream biological analysis. Dmatch facilitates alignment of scRNA-seq datasets with cell-types that may overlap only partially, and thus allows integration of multiple distinct scRNA-seq experiments to extract biological insights. In simulation, Dmatch compares favorably to other alignment methods, both in terms of reducing sample-specific clustering, and in terms of avoiding over-correction. When applied to scRNA-seq data collected from clinical samples in a healthy individual and five autoimmune disease patients, Dmatch enabled cell-type-specific differential gene expression comparisons across biopsy sites and disease conditions, and uncovered a shared population of pro-inflammatory monocytes across biopsy sites in RA patients. We further show that Dmatch increases the number of eQTLs mapped from population scRNA-seq data. Dmatch is fast, scalable, and improves the utility of scRNA-seq for several important applications. Dmatch is freely available online (https://qzhan321.github.io/dmatch/).

Download Full-text

Endothelial cell-type-specific molecular requirements for angiogenesis drive fenestrated vessel development in the brain

eLife ◽

10.7554/elife.64295 ◽

2021 ◽

Vol 10 ◽

Author(s):

Sweta Parab ◽

Rachael E Quick ◽

Ryota L Matsuoka

Keyword(s):

Vascular Endothelial Cells ◽

Cell Types ◽

Vascular Endothelial ◽

Cell Type ◽

Vessel Formation ◽

Brain Vasculature ◽

Cell Type Specific ◽

Vessel Development ◽

The Brain ◽

Brain Vessel

Vascular endothelial cells (vECs) in the brain exhibit structural and functional heterogeneity. Fenestrated, permeable brain vasculature mediates neuroendocrine function, body-fluid regulation, and neural immune responses; however, its vascular formation remains poorly understood. Here, we show that specific combinations of vascular endothelial growth factors (Vegfs) are required to selectively drive fenestrated vessel formation in the zebrafish myelencephalic choroid plexus (mCP). We found that the combined, but not individual, loss of Vegfab, Vegfc, and Vegfd causes severely impaired mCP vascularization with little effect on neighboring non-fenestrated brain vessel formation, demonstrating fenestrated-vEC-specific angiogenic requirements. This Vegfs-mediated vessel-selective patterning also involves Ccbe1. Expression analyses, cell-type-specific ablation, and paracrine activity-deficient vegfc mutant characterization suggest that vEC-autonomous Vegfc and meningeal fibroblast-derived Vegfab and Vegfd are critical for mCP vascularization. These results define molecular cues and cell types critical for directing fenestrated CP vascularization and indicate that vECs’ distinct molecular requirements for angiogenesis underlie brain vessel heterogeneity.

Download Full-text

Histone modifications form a cell-type-specific chromosomal bar code that persists through the cell cycle

Scientific Reports ◽

10.1038/s41598-021-82539-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

John A. Halsall ◽

Simon Andrews ◽

Felix Krueger ◽

Charlotte E. Rutledge ◽

Gabriella Ficz ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Histone Modifications ◽

Expression Patterns ◽

Cell Types ◽

Cell Type ◽

Bar Code ◽

Genes Encoding ◽

Cell Type Specific ◽

Rolling Windows

AbstractChromatin configuration influences gene expression in eukaryotes at multiple levels, from individual nucleosomes to chromatin domains several Mb long. Post-translational modifications (PTM) of core histones seem to be involved in chromatin structural transitions, but how remains unclear. To explore this, we used ChIP-seq and two cell types, HeLa and lymphoblastoid (LCL), to define how changes in chromatin packaging through the cell cycle influence the distributions of three transcription-associated histone modifications, H3K9ac, H3K4me3 and H3K27me3. We show that chromosome regions (bands) of 10–50 Mb, detectable by immunofluorescence microscopy of metaphase (M) chromosomes, are also present in G1 and G2. They comprise 1–5 Mb sub-bands that differ between HeLa and LCL but remain consistent through the cell cycle. The same sub-bands are defined by H3K9ac and H3K4me3, while H3K27me3 spreads more widely. We found little change between cell cycle phases, whether compared by 5 Kb rolling windows or when analysis was restricted to functional elements such as transcription start sites and topologically associating domains. Only a small number of genes showed cell-cycle related changes: at genes encoding proteins involved in mitosis, H3K9 became highly acetylated in G2M, possibly because of ongoing transcription. In conclusion, modified histone isoforms H3K9ac, H3K4me3 and H3K27me3 exhibit a characteristic genomic distribution at resolutions of 1 Mb and below that differs between HeLa and lymphoblastoid cells but remains remarkably consistent through the cell cycle. We suggest that this cell-type-specific chromosomal bar-code is part of a homeostatic mechanism by which cells retain their characteristic gene expression patterns, and hence their identity, through multiple mitoses.

Download Full-text

Rel Induces Interferon Regulatory Factor 4 (IRF-4) Expression in Lymphocytes

Journal of Experimental Medicine ◽

10.1084/jem.191.8.1281 ◽

2000 ◽

Vol 191 (8) ◽

pp. 1281-1292 ◽

Cited By ~ 146

Author(s):

Raelene J. Grumont ◽

Steve Gerondakis

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Nuclear Factor Κb ◽

Wild Type ◽

Cell Type ◽

Regulatory Factor ◽

Specific Manner ◽

A Cell ◽

Cell Type Specific ◽

Interferon Regulatory Factor 4

In lymphocytes, the Rel transcription factor is essential in establishing a pattern of gene expression that promotes cell proliferation, survival, and differentiation. Here we show that mitogen-induced expression of interferon (IFN) regulatory factor 4 (IRF-4), a lymphoid-specific member of the IFN family of transcription factors, is Rel dependent. Consistent with IRF-4 functioning as a repressor of IFN-induced gene expression, the absence of IRF-4 expression in c-rel−/− B cells coincided with a greater sensitivity of these cells to the antiproliferative activity of IFNs. In turn, enforced expression of an IRF-4 transgene restored IFN modulated c-rel−/− B cell proliferation to that of wild-type cells. This cross-regulation between two different signaling pathways represents a novel mechanism that Rel/nuclear factor κB can repress the transcription of IFN-regulated genes in a cell type–specific manner.

Download Full-text

A scalable platform for the development of cell-type-specific viral drivers

eLife ◽

10.7554/elife.48089 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 12

Author(s):

Sinisa Hrvatin ◽

Christopher P Tzeng ◽

M Aurel Nagy ◽

Hume Stroud ◽

Charalampia Koutsioumpa ◽

...

Keyword(s):

Gene Expression ◽

Heterologous Gene Expression ◽

High Specificity ◽

Cell Types ◽

Regulatory Elements ◽

Cell Type ◽

Cell Type Specificity ◽

Cell Type Specific ◽

The Many ◽

Dna Regulatory Elements

Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression. Recent studies characterizing individual enhancers have revealed their potential to direct heterologous gene expression in a highly cell-type-specific manner. However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism. We have developed PESCA, a scalable and generalizable method that leverages ATAC- and single-cell RNA-sequencing protocols, to characterize cell-type-specific enhancers that should enable genetic access and perturbation of gene function across mammalian cell types. Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity. This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.

Download Full-text

Epigenetic mechanisms mediating cell state transitions in chondrocytes

10.1101/2020.10.26.319822 ◽

2020 ◽

Author(s):

Manuela Wuelling ◽

Christoph Neu ◽

Andrea M. Thiesen ◽

Simo Kitanovski ◽

Yingying Cao ◽

...

Keyword(s):

Metabolic Pathways ◽

Cell Lineage ◽

Cell Types ◽

State Transitions ◽

Epigenetic Mechanisms ◽

Cell Type ◽

Transition Analysis ◽

Lineage Differentiation ◽

A Cell ◽

Cell Type Specific

AbstractEpigenetic modifications play critical roles in regulating cell lineage differentiation, but the epigenetic mechanisms guiding specific differentiation steps within a cell lineage have rarely been investigated. To decipher such mechanisms, we used the defined transition from proliferating (PC) into hypertrophic chondrocytes (HC) during endochondral ossification as a model. We established a map of activating and repressive histone modifications for each cell type. ChromHMM state transition analysis and Pareto-based integration of differential levels of mRNA and epigenetic marks revealed that differentiation associated gene repression is initiated by the addition of H3K27me3 to promoters still carrying substantial levels of activating marks. Moreover, the integrative analysis identified genes specifically expressed in cells undergoing the transition into hypertrophy.Investigation of enhancer profiles detected surprising differences in enhancer number, location, and transcription factor binding sites between the two closely related cell types. Furthermore, cell type-specific upregulation of gene expression was associated with a shift from low to high H3K27ac decoration. Pathway analysis identified PC-specific enhancers associated with chondrogenic genes, while HC-specific enhancers mainly control metabolic pathways linking epigenetic signature to biological functions.

Download Full-text

Cell-type Specific Expression Quantitative Trait Loci Associated with Alzheimer Disease in Blood and Brain Tissue

10.1101/2020.11.23.20237008 ◽

2020 ◽

Author(s):

Devanshi Patel ◽

Xiaoling Zhang ◽

John J. Farrell ◽

Jaeyoon Chung ◽

Thor D. Stein ◽

...

Keyword(s):

Gene Expression ◽

Quantitative Trait ◽

Expression Patterns ◽

Regulation Of Gene Expression ◽

Cell Types ◽

Eqtl Analysis ◽

Cell Type ◽

Specific Expression ◽

Cell Type Specific Expression ◽

Cell Type Specific

ABSTRACTBecause regulation of gene expression is heritable and context-dependent, we investigated AD-related gene expression patterns in cell-types in blood and brain. Cis-expression quantitative trait locus (eQTL) mapping was performed genome-wide in blood from 5,257 Framingham Heart Study (FHS) participants and in brain donated by 475 Religious Orders Study/Memory & Aging Project (ROSMAP) participants. The association of gene expression with genotypes for all cis SNPs within 1Mb of genes was evaluated using linear regression models for unrelated subjects and linear mixed models for related subjects. Cell type-specific eQTL (ct-eQTL) models included an interaction term for expression of “proxy” genes that discriminate particular cell type. Ct-eQTL analysis identified 11,649 and 2,533 additional significant gene-SNP eQTL pairs in brain and blood, respectively, that were not detected in generic eQTL analysis. Of note, 386 unique target eGenes of significant eQTLs shared between blood and brain were enriched in apoptosis and Wnt signaling pathways. Five of these shared genes are established AD loci. The potential importance and relevance to AD of significant results in myeloid cell-types is supported by the observation that a large portion of GWS ct-eQTLs map within 1Mb of established AD loci and 58% (23/40) of the most significant eGenes in these eQTLs have previously been implicated in AD. This study identified cell-type specific expression patterns for established and potentially novel AD genes, found additional evidence for the role of myeloid cells in AD risk, and discovered potential novel blood and brain AD biomarkers that highlight the importance of cell-type specific analysis.

Download Full-text

Distinct CoREST complexes act in a cell-type-specific manner

Nucleic Acids Research ◽

10.1093/nar/gkz1050 ◽

2019 ◽

Author(s):

Igor Mačinković ◽

Ina Theofel ◽

Tim Hundertmark ◽

Kristina Kovač ◽

Stephan Awe ◽

...

Keyword(s):

Gene Expression ◽

Germ Line ◽

Protein Complexes ◽

Specific Gene ◽

S2 Cells ◽

Cell Type ◽

Specific Gene Expression ◽

Genome Wide ◽

A Cell ◽

Cell Type Specific

Abstract CoREST has been identified as a subunit of several protein complexes that generate transcriptionally repressive chromatin structures during development. However, a comprehensive analysis of the CoREST interactome has not been carried out. We use proteomic approaches to define the interactomes of two dCoREST isoforms, dCoREST-L and dCoREST-M, in Drosophila. We identify three distinct histone deacetylase complexes built around a common dCoREST/dRPD3 core: A dLSD1/dCoREST complex, the LINT complex and a dG9a/dCoREST complex. The latter two complexes can incorporate both dCoREST isoforms. By contrast, the dLSD1/dCoREST complex exclusively assembles with the dCoREST-L isoform. Genome-wide studies show that the three dCoREST complexes associate with chromatin predominantly at promoters. Transcriptome analyses in S2 cells and testes reveal that different cell lineages utilize distinct dCoREST complexes to maintain cell-type-specific gene expression programmes: In macrophage-like S2 cells, LINT represses germ line-related genes whereas other dCoREST complexes are largely dispensable. By contrast, in testes, the dLSD1/dCoREST complex prevents transcription of germ line-inappropriate genes and is essential for spermatogenesis and fertility, whereas depletion of other dCoREST complexes has no effect. Our study uncovers three distinct dCoREST complexes that function in a lineage-restricted fashion to repress specific sets of genes thereby maintaining cell-type-specific gene expression programmes.

Download Full-text