Pcsk6 mutant mice exhibit progressive loss of ovarian function, altered gene expression, and formation of ovarian pathology

Bioactivation of precursor proteins by members of the proprotein convertase (PC) family is essential for normal reproduction. ThePcsk6gene is a member of the PC family that is expressed in numerous ovarian cell types including granulosa cells and oocytes. We hypothesized that loss of PCSK6 would produce adverse effects in the mouse ovary. Mice incapable of expressing PCSK6 (Pcsk6tm1Rob) were obtained, and reproductive parameters (serum hormones, whelping interval, estrus cyclicity, and fertility) were compared toPcsk6+/+mice. WhilePcsk6tm1Robfemale mice are fertile, they manifest reduced reproductive capacity at an accelerated rate relative toPcsk6+/+mice. Reproductive senescence is typically reached by 9 months of age and is correlated with loss of estrus cyclicity, elevated serum FSH levels, and gross alterations in ovarian morphology. A wide range of ovarian morphologies were identified encompassing mild, such as an apparent reduction in follicle number, to moderate – ovarian atrophy with a complete absence of follicles – to severe, manifesting as normal ovarian structures replaced by benign ovarian tumors, including tubulostromal adenomas. Targeted gene expression profiling highlighted changes in RNA expression of molecules involved in processes such as steroidogenesis, gonadotropin signaling, transcriptional regulation, autocrine/paracrine signaling, cholesterol handling, and proprotein bioactivation. These results show that PCSK6 activity plays a role in maintaining normal cellular and tissue homeostasis in the ovary.

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RNA-Seq Data-Mining Allows the Discovery of Two Long Non-Coding RNA Biomarkers of Viral Infection in Humans

International Journal of Molecular Sciences ◽

10.3390/ijms21082748 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2748 ◽

Cited By ~ 1

Author(s):

Ruth Barral-Arca ◽

Alberto Gómez-Carballa ◽

Miriam Cebey-López ◽

María José Currás-Tuala ◽

Sara Pischedda ◽

...

Keyword(s):

Gene Expression ◽

Viral Infections ◽

Umbilical Vein ◽

Cell Types ◽

Dermal Fibroblasts ◽

Learning Approaches ◽

Rna Seq ◽

Wide Range ◽

Healthy Control ◽

Umbilical Vein Endothelial Cells

There is a growing interest in unraveling gene expression mechanisms leading to viral host invasion and infection progression. Current findings reveal that long non-coding RNAs (lncRNAs) are implicated in the regulation of the immune system by influencing gene expression through a wide range of mechanisms. By mining whole-transcriptome shotgun sequencing (RNA-seq) data using machine learning approaches, we detected two lncRNAs (ENSG00000254680 and ENSG00000273149) that are downregulated in a wide range of viral infections and different cell types, including blood monocluclear cells, umbilical vein endothelial cells, and dermal fibroblasts. The efficiency of these two lncRNAs was positively validated in different viral phenotypic scenarios. These two lncRNAs showed a strong downregulation in virus-infected patients when compared to healthy control transcriptomes, indicating that these biomarkers are promising targets for infection diagnosis. To the best of our knowledge, this is the very first study using host lncRNAs biomarkers for the diagnosis of human viral infections.

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HCR-FlowFISH: A flexible CRISPR screening method to identify cis-regulatory elements and their target genes

10.1101/2020.05.11.078675 ◽

2020 ◽

Author(s):

SK Reilly ◽

SJ Gosai ◽

A Gutierrez ◽

JC Ulirsch ◽

M Kanai ◽

...

Keyword(s):

Gene Expression ◽

Target Genes ◽

Screening Method ◽

Cell Types ◽

Regulatory Elements ◽

Hybridization Chain Reaction ◽

Genome Wide ◽

Wide Range ◽

Causal Variants ◽

Endogenous Loci

AbstractCRISPR screens for cis-regulatory elements (CREs) have shown unprecedented power to endogenously characterize the non-coding genome. To characterize CREs we developed HCR-FlowFISH (Hybridization Chain Reaction Fluorescent In-Situ Hybridization coupled with Flow Cytometry), which directly quantifies native transcripts within their endogenous loci following CRISPR perturbations of regulatory elements, eliminating the need for restrictive phenotypic assays such as growth or transcript-tagging. HCR-FlowFISH accurately quantifies gene expression across a wide range of transcript levels and cell types. We also developed CASA (CRISPR Activity Screen Analysis), a hierarchical Bayesian model to identify and quantify CRE activity. Using >270,000 perturbations, we identified CREs for GATA1, HDAC6, ERP29, LMO2, MEF2C, CD164, NMU, FEN1 and the FADS gene cluster. Our methods detect subtle gene expression changes and identify CREs regulating multiple genes, sometimes at different magnitudes and directions. We demonstrate the power of HCR-FlowFISH to parse genome-wide association signals by nominating causal variants and target genes.

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Identification of Split-GAL4 Drivers and Enhancers That Allow Regional Cell Type Manipulations of the Drosophila melanogaster Intestine

Genetics ◽

10.1534/genetics.120.303625 ◽

2020 ◽

Vol 216 (4) ◽

pp. 891-903

Author(s):

Ishara S. Ariyapala ◽

Jessica M. Holsopple ◽

Ellen M. Popodi ◽

Dalton G. Hartwick ◽

Lily Kahsai ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Expression Patterns ◽

Cell Types ◽

Enteroendocrine Cells ◽

Epithelial Tissue ◽

Cell Type ◽

Cell Functions ◽

Distinct Subset ◽

Targeted Gene Expression

The Drosophila adult midgut is a model epithelial tissue composed of a few major cell types with distinct regional identities. One of the limitations to its analysis is the lack of tools to manipulate gene expression based on these regional identities. To overcome this obstacle, we applied the intersectional split-GAL4 system to the adult midgut and report 653 driver combinations that label cells by region and cell type. We first identified 424 split-GAL4 drivers with midgut expression from ∼7300 drivers screened, and then evaluated the expression patterns of each of these 424 when paired with three reference drivers that report activity specifically in progenitor cells, enteroendocrine cells, or enterocytes. We also evaluated a subset of the drivers expressed in progenitor cells for expression in enteroblasts using another reference driver. We show that driver combinations can define novel cell populations by identifying a driver that marks a distinct subset of enteroendocrine cells expressing genes usually associated with progenitor cells. The regional cell type patterns associated with the entire set of driver combinations are documented in a freely available website, providing information for the design of thousands of additional driver combinations to experimentally manipulate small subsets of intestinal cells. In addition, we show that intestinal enhancers identified with the split-GAL4 system can confer equivalent expression patterns on other transgenic reporters. Altogether, the resource reported here will enable more precisely targeted gene expression for studying intestinal processes, epithelial cell functions, and diseases affecting self-renewing tissues.

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Identification of split-GAL4 drivers and enhancers that allow regional cell type manipulations of the Drosophila melanogaster intestine

10.1101/2020.08.24.264887 ◽

2020 ◽

Author(s):

Ishara S. Ariyapala ◽

Jessica M. Holsopple ◽

Ellen M. Popodi ◽

Dalton G. Hartwick ◽

Lily Kahsai ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Expression Patterns ◽

Cell Types ◽

Enteroendocrine Cells ◽

Epithelial Tissue ◽

Cell Type ◽

Cell Functions ◽

Distinct Subset ◽

Targeted Gene Expression

ABSTRACTThe Drosophila adult midgut is a model epithelial tissue composed of a few major cell types with distinct regional identities. One of the limitations to its analysis is the lack of tools to manipulate gene expression based on these regional identities. To overcome this obstacle, we applied the intersectional split-GAL4 system to the adult midgut and report 653 driver combinations that label cells by region and cell type. We first identified 424 split-GAL4 drivers with midgut expression from over 7,300 drivers screened, and then evaluated the expression patterns of each of these 424 when paired with three reference drivers that report activity specifically in progenitor cells, enteroendocrine cells, or enterocytes. We also evaluated a subset of the drivers expressed in progenitor cells for expression in enteroblasts using another reference driver. We show that driver combinations can define novel cell populations by identifying a driver that marks a distinct subset of enteroendocrine cells expressing genes usually associated with progenitor cells. The regional cell type patterns associated with the entire set of driver combinations are documented in a freely available website, providing information for the design of thousands of additional driver combinations to experimentally manipulate small subsets of intestinal cells. In addition, we show that intestinal enhancers identified with the split-GAL4 system can confer equivalent expression patterns on other transgenic reporters. Altogether, the resource reported here will enable more precisely targeted gene expression for studying intestinal processes, epithelial cell functions, and diseases affecting self-renewing tissues.

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Putative positive role of inflammatory genes in fat deposition supported by altered gene expression in purified human adipocytes and preadipocytes from lean and obese adipose tissues

10.21203/rs.3.rs-15748/v1 ◽

2020 ◽

Author(s):

Sang-Hyeop Lee ◽

Nak-Hyeon Choi ◽

In-Uk Koh ◽

Bong-Jo Kim ◽

Song Lee ◽

...

Keyword(s):

Gene Expression ◽

Inflammatory Responses ◽

Cell Types ◽

Deposition Process ◽

Fat Deposition ◽

Low Grade ◽

Positive Role ◽

Inflammatory Genes ◽

Altered Gene

Abstract BackgroundObesity is a chronic low-grade inflammatory disease that is generally characterized by enhanced inflammation in obese adipose tissue (AT). Here, we investigated alterations in gene expression between lean and obese conditions using mRNA-Seq data derived from human purified adipocytes (ACs) and preadipocytes (preACs). ResultsWe defined four classes of differentially expressed genes (DEGs) by comparing gene expression between 1) lean and obese ACs, 2) lean and obese preACs, 3) lean ACs and lean preACs, and 4) obese ACs and obese preACs. Based on an analysis of comparison 1, numerous canonical obesity-related genes, particularly inflammatory genes including IL6, TNF- and IL-1, i.e., the genes that are expected to be upregulated in obesity conditions, were found to be expressed at significantly lower levels in obese ACs than in lean ACs. In contrast, some inflammatory genes were found to be expressed at higher levels in obese preACs than lean preACs in the analysis of comparison 2. These two results indicate that (1) up-/downregulation of genes in ACs and preACs is inversely controlled during the fat deposition process and (2) preACs rather than ACs have increased inflammatory response genes in comparisons of lean and obese conditions for each of these cell types. Analysis of comparisons 3 and 4 showed that inflammatory gene classes were expressed at higher levels in differentiated ACs than undifferentiated preACs under both lean and obese conditions; however, the degree of upregulation was greater for lean than for obese conditions.ConclusionsTaken together, our analyses may suggest that lean fat differentiation involves even greater enhancement of inflammatory responses than does obese fat differentiation.

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Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915974116 ◽

2019 ◽

Vol 116 (52) ◽

pp. 27001-27010 ◽

Cited By ~ 7

Author(s):

Kyle B. Fischer ◽

Hannah K. Collins ◽

Edward M. Callaway

Keyword(s):

Gene Expression ◽

Transgenic Mouse ◽

Transgene Expression ◽

Cell Types ◽

Cell Type ◽

Recognition Sites ◽

Highly Sensitive ◽

Plasmid Production ◽

Targeted Gene Expression ◽

Mouse Lines

In combination with transgenic mouse lines expressing Cre or Flp recombinases in defined cell types, recombinase-dependent adeno-associated viruses (AAVs) have become the tool of choice for localized cell-type-targeted gene expression. Unfortunately, applications of this technique when expressing highly sensitive transgenes are impeded by off-target, or “leak” expression, from recombinase-dependent AAVs. We investigated this phenomenon and find that leak expression is mediated by both infrequent transcription from the inverted transgene in recombinant-dependent AAV designs and recombination events during bacterial AAV plasmid production. Recombination in bacteria is mediated by homology across the antiparallel recombinase-specific recognition sites present in recombinase-dependent designs. To address both of these issues we designed an AAV vector that uses mutant “cross-over insensitive” recognition sites combined with an “ATG-out” design. We show that these CIAO (cross-over insensitive ATG-out) vectors virtually eliminate leak expression. CIAO vectors provide reliable and targeted transgene expression and are extremely useful for recombinase-dependent expression of highly sensitive transgenes.

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Calcium-mediated actin reset (CaAR) mediates acute cell adaptations

eLife ◽

10.7554/elife.19850 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 60

Author(s):

Pauline Wales ◽

Christian E Schuberth ◽

Roland Aufschnaiter ◽

Johannes Fels ◽

Ireth García-Aguilar ◽

...

Keyword(s):

Gene Expression ◽

Dynamic Equilibrium ◽

Calcium Influx ◽

Cell Spreading ◽

Cell Types ◽

Cell Cortex ◽

Cortical Actin ◽

Cellular Morphogenesis ◽

Wide Range ◽

A Cell

Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress.

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Aspects of Ovarian Function in a Line of Sheep with a Novel X-Linked Maternally-Imprinted Gene that is Associated with an Increased Ovulation Rate

10.26686/wgtn.16945789 ◽

2021 ◽

Author(s):

◽

Elisabeth Sheinach Feary

Keyword(s):

Gene Expression ◽

Pituitary Gland ◽

Ovarian Function ◽

Follicular Development ◽

Cell Types ◽

Ovulation Rate ◽

Chromosome 2 ◽

Ovarian Follicular Development ◽

Er Alpha ◽

Large Ovary

<p>Fecundity is a term that refers to the number of offspring produced per female. It combines fertility (i.e. ability to produce offspring) and prolificacy (i.e. number of offspring). Ovulation rate i.e. the number of mature eggs released from the ovaries during one reproductive cycle in sheep, as with other mammals, is controlled by an exchange of hormonal signals between the pituitary gland and the ovary. Genetic mutations affecting ovulation are commonly referred to as the fecundity genes (Fec). The most obvious outcome is the number of offspring produced. There is already evidence of a number of major genes affecting the ovulation rate in sheep, specifically the Booroola, Inverdale, Hanna and more recently the Woodlands gene. The sheep carrying the Woodlands gene arose because the mutation was first recognised on a farm in Woodlands, Southland, New Zealand. Woodlands have a novel, X-linked maternally-imprinted, fecundity trait referred to as FecX2w, where Fec = fecundity, X = X chromosome, 2= 2nd mutation identified on X and W= Woodlands. The studies in this thesis investigated ovarian follicular development in both 4-week old Woodland carrier (W+) and non-carrier (++) lambs and adult ewes and evaluated some aspects of the endocrine interactions between the ovary and pituitary gland. The purpose was to identify potential physiological effects of the FecX2w gene on ovarian function. A confounding issue during these studies was the discovery that a large ovary phenotype (LOP) which was present in many of the W+ but not ++ lambs at 4 weeks of age was in fact a coincidence and not linked to the FecX2w mutation. The key findings from the studies of lambs and/or ewes that were carriers (W+) or non-carriers (++) of the FecX2w gene were: 1. No genotype differences were present either in the numbers of primordial (i.e. Type 1/1a follicles) or developing preantral (i.e. Types 2-4 follicles); 2. Significant genotype differences were present in the numbers of small antral (Type 5) follicles (W+>++; p<0.05); 3. An earlier onset of antral follicular development in W+ vs. ++ ewes with irregularities in morphology between the basement membrane and stroma in the former; 4. No genotype differences in the onset of gene expression during follicular development or in the cell-types expressing GDF9, BMP15, alpha inhibin, beta A inhibin and beta B inhibin, FSHR, ER alpha, or ER beta; 5. No genotype differences in the levels of GDF9 or BMP15 gene expression in oocytes throughout follicular growth; 6. No genotype difference in the diameters that follicles reached in W+ vs. ++ ewes; 7. Some lambs at 4-weeks of age had unusually large ovaries with an exceptional level of antral follicular development that is reminiscent of a polycystic ovarian condition. The underlying cause of this condition is unknown. In conclusion, the physiological characteristics of ovarian follicular development in ewes with the FecX2w gene is different from that in ewes with the Booroola, Inverdale, Hanna or other recently identified mutations.</p>

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Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2020-3111 ◽

2020 ◽

Vol 6 (3) ◽

pp. 430-433

Author(s):

Andreas Brietzke ◽

Rudolf Guthoff ◽

Thomas Stahnke ◽

Niels Grabow

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Human Fibroblasts ◽

Cell Types ◽

Vascular Endothelial ◽

Altered Gene ◽

Endothelial Growth Factor ◽

Tgf Β1

AbstractDespite decades of research, fibrosis still remains a significant challenge for medicine in many different fields. Although there is a general model of fibrosis, the causes and characteristics of the various pathologies are as diverse as the variety of organs and tissues that can be affected by fibrosis. Moreover, fibrosis also impedes the long-term prospects of success in implantation surgery. One possibility to address this challenge is the development of biocompatible implants featuring drug delivery systems loaded with antifibrotic pharmaceuticals. Due to diverse regulatory mechanisms in organs, tissues and also cell types, these active substances must consequentially be designed for diverse specific applications. Compared to fibrosis in organs like lung or liver, these mechanisms were poorly addressed in ophthalmologic research, but it is known that transforming growth factor beta (TGF-β) plays a key role. This gene expression study revealed 30 genes being upregulated more than two fold in TGF-β1 treated human primary tenon fibroblasts (hTF). Furthermore, 15 genes were found to be downregulated more than two fold. Tumor necrosis factor (TNF), vascular endothelial growth factor A (VEGFA) and inhibin beta (INHBE) were particular strongly regulated in TGF-β1 treated hTFs.

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COVID-19 genetic risk variants are associated with expression of multiple genes in diverse immune cell types

Nature Communications ◽

10.1038/s41467-021-26888-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Benjamin J. Schmiedel ◽

Job Rocha ◽

Cristian Gonzalez-Colin ◽

Sourya Bhattacharyya ◽

Ariel Madrigal ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Genetic Risk ◽

Immune Cell ◽

Severe Disease ◽

Cell Types ◽

Chromatin Interaction ◽

Risk Variants ◽

Wide Range ◽

Genetic Risk Variants

AbstractCommon genetic polymorphisms associated with COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Given the importance of immune cells in the pathogenesis of COVID-19 illness, here we assessed the effects of COVID-19-risk variants on gene expression in a wide range of immune cell types. Transcriptome-wide association study and colocalization analysis revealed putative causal genes and the specific immune cell types where gene expression is most influenced by COVID-19-risk variants. Notable examples include OAS1 in non-classical monocytes, DTX1 in B cells, IL10RB in NK cells, CXCR6 in follicular helper T cells, CCR9 in regulatory T cells and ARL17A in TH2 cells. By analysis of transposase accessible chromatin and H3K27ac-based chromatin-interaction maps of immune cell types, we prioritized potentially functional COVID-19-risk variants. Our study highlights the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations.

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