Lysolipid receptor cross-talk regulates lymphatic endothelial junctions in lymph nodes

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) activate G protein–coupled receptors (GPCRs) to regulate biological processes. Using a genome-wide CRISPR/dCas9–based GPCR signaling screen, LPAR1 was identified as an inducer of S1PR1/β-arrestin coupling while suppressing Gαi signaling. S1pr1 and Lpar1-positive lymphatic endothelial cells (LECs) of lymph nodes exhibit constitutive S1PR1/β-arrestin signaling, which was suppressed by LPAR1 antagonism. Pharmacological inhibition or genetic loss of function of Lpar1 reduced the frequency of punctate junctions at sinus-lining LECs. Ligand activation of transfected LPAR1 in endothelial cells remodeled junctions from continuous to punctate structures and increased transendothelial permeability. In addition, LPAR1 antagonism in mice increased lymph node retention of adoptively transferred lymphocytes. These data suggest that cross-talk between LPAR1 and S1PR1 promotes the porous junctional architecture of sinus-lining LECs, which enables efficient lymphocyte trafficking. Heterotypic inter-GPCR coupling may regulate complex cellular phenotypes in physiological milieu containing many GPCR ligands.

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Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

10.1101/435776 ◽

2018 ◽

Author(s):

Yu Hisano ◽

Mari Kono ◽

Eric Engelbrecht ◽

Koki Kawakami ◽

Keisuke Yanagida ◽

...

Keyword(s):

Endothelial Cells ◽

Lymph Nodes ◽

Cross Talk ◽

Transcriptional Activation ◽

Endothelial Permeability ◽

Lipid Mediator ◽

Protein Signaling ◽

A Genome ◽

Sphingosine 1 Phosphate ◽

A Site

AbstractLysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) activate G protein-coupled receptors (GPCRs) to regulate key pathobiological processes. Here we report a novel lipid mediator GPCR cross-talk mechanism that modulates lymphatic endothelial junctional architecture in lymph nodes. LPAR1 was identified as an inducer of S1PR1/ ß-arrestin coupling from a genome-wide CRISPR/ Cas9 transcriptional activation screen. LPAR1 activation induced S1PR1 ß-arrestin recruitment while suppressing Gαi protein signaling. Lymphatic endothelial cells from cortical and medullary sinuses of lymph nodes which express LPAR1 and S1PR1, exhibit porous junctional architecture and constitutive S1PR1 coupling to ß-arrestin which was suppressed by the LPAR1 antagonist AM095. In endothelial cells, LPAR1-activation increased trans-endothelial permeability and junctional remodeling from zipper-like structures to puncta of adhesion plaques that terminate at actin-rich stress fibers with abundant intercellular gaps. Cross-talk between LPA and S1P receptors regulates complex junctional architecture of lymphatic sinus endothelial cells, a site of high lymphocyte traffic and lymph flow.

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A Nonsense Variant in Hephaestin Like 1 (HEPHL1) Is Responsible for Congenital Hypotrichosis in Belted Galloway Cattle

Genes ◽

10.3390/genes12050643 ◽

2021 ◽

Vol 12 (5) ◽

pp. 643

Author(s):

Thibaud Kuca ◽

Brandy M. Marron ◽

Joana G. P. Jacinto ◽

Julia M. Paris ◽

Christian Gerspach ◽

...

Keyword(s):

Genome Wide Association Study ◽

Homozygosity Mapping ◽

Mendelian Inheritance ◽

Large Animal Model ◽

Large Animal ◽

Loss Of Function ◽

Protein Coding ◽

Positional Candidate ◽

Genome Wide ◽

A Genome

Genodermatosis such as hair disorders mostly follow a monogenic mode of inheritance. Congenital hypotrichosis (HY) belong to this group of disorders and is characterized by abnormally reduced hair since birth. The purpose of this study was to characterize the clinical phenotype of a breed-specific non-syndromic form of HY in Belted Galloway cattle and to identify the causative genetic variant for this recessive disorder. An affected calf born in Switzerland presented with multiple small to large areas of alopecia on the limbs and on the dorsal part of the head, neck, and back. A genome-wide association study using Swiss and US Belted Galloway cattle encompassing 12 cases and 61 controls revealed an association signal on chromosome 29. Homozygosity mapping in a subset of cases refined the HY locus to a 1.5 Mb critical interval and subsequent Sanger sequencing of protein-coding exons of positional candidate genes revealed a stop gain variant in the HEPHL1 gene that encodes a multi-copper ferroxidase protein so-called hephaestin like 1 (c.1684A>T; p.Lys562*). A perfect concordance between the homozygous presence of this most likely pathogenic loss-of-function variant and the HY phenotype was found. Genotyping of more than 700 purebred Swiss and US Belted Galloway cattle showed the global spread of the mutation. This study provides a molecular test that will permit the avoidance of risk matings by systematic genotyping of relevant breeding animals. This rare recessive HEPHL1-related form of hypotrichosis provides a novel large animal model for similar human conditions. The results have been incorporated in the Online Mendelian Inheritance in Animals (OMIA) database (OMIA 002230-9913).

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Generation and Transcriptome Profiling of Slr1-d7 and Slr1-d8 Mutant Lines with a New Semi-Dominant Dwarf Allele of SLR1 Using the CRISPR/Cas9 System in Rice

International Journal of Molecular Sciences ◽

10.3390/ijms21155492 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5492 ◽

Cited By ~ 1

Author(s):

Yu Jin Jung ◽

Jong Hee Kim ◽

Hyo Ju Lee ◽

Dong Hyun Kim ◽

Jihyeon Yu ◽

...

Keyword(s):

Gene Expression Analysis ◽

Transcriptome Profiling ◽

Rna Seq ◽

Loss Of Function ◽

Amino Acid Motif ◽

Dwarf Phenotype ◽

Genome Wide ◽

A Genome ◽

Mutant Lines ◽

Genome Wide Gene Expression

The rice SLR1 gene encodes the DELLA protein (protein with DELLA amino acid motif), and a loss-of-function mutation is dwarfed by inhibiting plant growth. We generate slr1-d mutants with a semi-dominant dwarf phenotype to target mutations of the DELLA/TVHYNP domain using CRISPR/Cas9 genome editing in rice. Sixteen genetic edited lines out of 31 transgenic plants were generated. Deep sequencing results showed that the mutants had six different mutation types at the target site of the TVHYNP domain of the SLR1 gene. The homo-edited plants selected individuals without DNA (T-DNA) transcribed by segregation in the T1 generation. The slr1-d7 and slr1-d8 plants caused a gibberellin (GA)-insensitive dwarf phenotype with shrunken leaves and shortened internodes. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two GA-related genes, GA20OX2 (Gibberellin oxidase) and GA3OX2, were increased in the edited mutant plants, suggesting that GA20OX2 acts as a convert of GA12 signaling. These mutant plants are required by altering GA responses, at least partially by a defect in the phytohormone signaling system process and prevented cell elongation. The new mutants, namely, the slr1-d7 and slr1-d8 lines, are valuable semi-dominant dwarf alleles with potential application value for molecule breeding using the CRISPR/Cas9 system in rice.

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Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy

Science Translational Medicine ◽

10.1126/scitranslmed.aay0271 ◽

2020 ◽

Vol 12 (536) ◽

pp. eaay0271 ◽

Cited By ~ 6

Author(s):

Angela Lek ◽

Yuanfan Zhang ◽

Keryn G. Woodman ◽

Shushu Huang ◽

Alec M. DeSimone ◽

...

Keyword(s):

Cell Death ◽

Muscular Dystrophy ◽

Phenotypic Selection ◽

Facioscapulohumeral Muscular Dystrophy ◽

Loss Of Function ◽

Hypoxia Response ◽

Genome Wide ◽

A Genome ◽

Structural And Functional Properties ◽

Cellular Hypoxia

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

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A Genome-wide Analysis of Human Pluripotent Stem Cell-Derived Endothelial Cells in 2D or 3D Culture

Stem Cell Reports ◽

10.1016/j.stemcr.2017.02.014 ◽

2017 ◽

Vol 8 (4) ◽

pp. 907-918 ◽

Cited By ~ 22

Author(s):

Jue Zhang ◽

Michael P. Schwartz ◽

Zhonggang Hou ◽

Yongsheng Bai ◽

Hamisha Ardalani ◽

...

Keyword(s):

Endothelial Cells ◽

Stem Cell ◽

Pluripotent Stem Cell ◽

3D Culture ◽

Human Pluripotent Stem Cell ◽

Genome Wide Analysis ◽

Genome Wide ◽

A Genome

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CUX2 mutations in temporal lobe epilepsy; increased kainate susceptibility and excitatory input to hippocampus in deficient mice

10.1101/2021.09.17.460803 ◽

2021 ◽

Author(s):

Toshimitsu Suzuki ◽

Tetsuya Tatsukawa ◽

Genki Sudo ◽

Caroline Delandre ◽

Yun Jin Pai ◽

...

Keyword(s):

Synaptic Transmission ◽

Temporal Lobe Epilepsy ◽

Entorhinal Cortex ◽

Temporal Lobe ◽

Genome Wide Association Study ◽

De Novo ◽

Cell Number ◽

Loss Of Function ◽

Genome Wide ◽

A Genome

CUX2 gene encodes a transcription factor that controls neuronal proliferation, dendrite branching and synapse formation, locating at the epilepsy-associated chromosomal region 12q24 that we previously identified by a genome-wide association study (GWAS) in Japanese population. A CUX2 recurrent de novo variant p.E590K has been described in patients with rare epileptic encephalopathies and the gene is a candidate for the locus, however the mutation may not be enough to generate the genome-wide significance in the GWAS and whether CUX2 variants appear in other types of epilepsies and physiopathological mechanisms are remained to be investigated. Here in this study, we conducted targeted sequencings of CUX2, a paralog CUX1 and its short isoform CASP harboring a unique C-terminus on 271 Japanese patients with a variety of epilepsies, and found that multiple CUX2 missense variants, other than the p.E590K, and some CASP variants including a deletion, predominantly appeared in patients with temporal lobe epilepsy (TLE). Human cell culture and fly dendritic arborization analyses revealed loss-of- function properties for the CUX2 variants. Cux2- and Casp-specific knockout mice both showed high susceptibility to kainate, increased excitatory cell number in the entorhinal cortex, and significant enhancement in glutamatergic synaptic transmission to the hippocampus. CASP and CUX2 proteins physiologically bound to each other and co-expressed in excitatory neurons in brain regions including the entorhinal cortex. These results suggest that CUX2 and CASP variants contribute to the TLE pathology through a facilitation of excitatory synaptic transmission from entorhinal cortex to hippocampus.

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Genome-wide synthetic lethal CRISPR screen identifies FIS1 as a genetic interactor of ALS-linked C9ORF72

10.1101/778118 ◽

2019 ◽

Author(s):

Noori Chai ◽

Michael S. Haney ◽

Julien Couthouis ◽

David W. Morgens ◽

Alyssa Benjamin ◽

...

Keyword(s):

Genetic Interaction ◽

Mitochondrial Fission ◽

Loss Of Function ◽

Synthetic Lethal ◽

Genome Wide ◽

A Genome ◽

Crispr Screen ◽

Insight Into ◽

Mitochondrial Membrane Protein ◽

Lateral Sclerosis

AbstractMutations in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis (ALS). Both toxic gain of function and loss of function pathogenic mechanisms have been proposed. Accruing evidence from mouse knockout studies point to a role for C9ORF72 as a regulator of immune function. To provide further insight into its cellular function, we performed a genome-wide synthetic lethal CRISPR screen in human myeloid cells lacking C9ORF72. We discovered a strong synthetic lethal genetic interaction between C9ORF72 and FIS1, which encodes a mitochondrial membrane protein involved in mitochondrial fission and mitophagy. Mass spectrometry experiments revealed that in C9ORF72 knockout cells, FIS1 strongly bound to a class of immune regulators that activate the receptor for advanced glycation end (RAGE) products and trigger inflammatory cascades. These findings present a novel genetic interactor for C9ORF72 and suggest a compensatory role for FIS1 in suppressing inflammatory signaling in the absence of C9ORF72.

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Genomic Characterization of Endothelial Enhancers Reveals a Multifunctional Role for NR2F2 in Regulation of Arteriovenous Gene Expression

Circulation Research ◽

10.1161/circresaha.119.316075 ◽

2020 ◽

Vol 126 (7) ◽

pp. 875-888 ◽

Cited By ~ 6

Author(s):

Samir Sissaoui ◽

Jun Yu ◽

Aimin Yan ◽

Rui Li ◽

Onur Yukselen ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Transcription Factor ◽

Deep Sequencing ◽

Chromatin Immunoprecipitation ◽

Regulatory Elements ◽

Bhlh Transcription Factor ◽

Genome Wide ◽

A Genome

Rationale: Significant progress has revealed transcriptional inputs that underlie regulation of artery and vein endothelial cell fates. However, little is known concerning genome-wide regulation of this process. Therefore, such studies are warranted to address this gap. Objective: To identify and characterize artery- and vein-specific endothelial enhancers in the human genome, thereby gaining insights into mechanisms by which blood vessel identity is regulated. Methods and Results: Using chromatin immunoprecipitation and deep sequencing for markers of active chromatin in human arterial and venous endothelial cells, we identified several thousand artery- and vein-specific regulatory elements. Computational analysis revealed that NR2F2 (nuclear receptor subfamily 2, group F, member 2) sites were overrepresented in vein-specific enhancers, suggesting a direct role in promoting vein identity. Subsequent integration of chromatin immunoprecipitation and deep sequencing data sets with RNA sequencing revealed that NR2F2 regulated 3 distinct aspects related to arteriovenous identity. First, consistent with previous genetic observations, NR2F2 directly activated enhancer elements flanking cell cycle genes to drive their expression. Second, NR2F2 was essential to directly activate vein-specific enhancers and their associated genes. Our genomic approach further revealed that NR2F2 acts with ERG (ETS-related gene) at many of these sites to drive vein-specific gene expression. Finally, NR2F2 directly repressed only a small number of artery enhancers in venous cells to prevent their activation, including a distal element upstream of the artery-specific transcription factor, HEY2 (hes related family bHLH transcription factor with YRPW motif 2). In arterial endothelial cells, this enhancer was normally bound by ERG, which was also required for arterial HEY2 expression. By contrast, in venous endothelial cells, NR2F2 was bound to this site, together with ERG, and prevented its activation. Conclusions: By leveraging a genome-wide approach, we revealed mechanistic insights into how NR2F2 functions in multiple roles to maintain venous identity. Importantly, characterization of its role at a crucial artery enhancer upstream of HEY2 established a novel mechanism by which artery-specific expression can be achieved.

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A genome-wide DNA methylation signature for SETD1B-related syndrome

Clinical Epigenetics ◽

10.1186/s13148-019-0749-3 ◽

2019 ◽

Vol 11 (1) ◽

Cited By ~ 12

Author(s):

I. M. Krzyzewska ◽

S. M. Maas ◽

P. Henneman ◽

K. v. d. Lip ◽

A. Venema ◽

...

Keyword(s):

Clinical Utility ◽

De Novo ◽

Histone H3 ◽

Missense Mutations ◽

Loss Of Function ◽

Variant Of Uncertain Significance ◽

Epigenetic Marker ◽

Genome Wide ◽

A Genome ◽

Uncertain Significance

Abstract SETD1B is a component of a histone methyltransferase complex that specifically methylates Lys-4 of histone H3 (H3K4) and is responsible for the epigenetic control of chromatin structure and gene expression. De novo microdeletions encompassing this gene as well as de novo missense mutations were previously linked to syndromic intellectual disability (ID). Here, we identify a specific hypermethylation signature associated with loss of function mutations in the SETD1B gene which may be used as an epigenetic marker supporting the diagnosis of syndromic SETD1B-related diseases. We demonstrate the clinical utility of this unique epi-signature by reclassifying previously identified SETD1B VUS (variant of uncertain significance) in two patients.

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Efficient genome-wide first-generation phenotypic screening system in mice using thepiggyBactransposon

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906354116 ◽

2019 ◽

Vol 116 (37) ◽

pp. 18507-18516 ◽

Cited By ~ 3

Author(s):

Hao Chang ◽

Yukun Pan ◽

Sean Landrette ◽

Sheng Ding ◽

Dong Yang ◽

...

Keyword(s):

First Generation ◽

Cranial Nerves ◽

Model Organisms ◽

Biological Traits ◽

Screening System ◽

Loss Of Function ◽

Developmental Defects ◽

Genome Wide ◽

A Genome ◽

Milk Ingestion

Genome-wide phenotypic screens provide an unbiased way to identify genes involved in particular biological traits, and have been widely used in lower model organisms. However, cost and time have limited the utility of such screens to address biological and disease questions in mammals. Here we report a highly efficientpiggyBac(PB) transposon-based first-generation (F1) dominant screening system in mice that enables an individual investigator to conduct a genome-wide phenotypic screen within a year with fewer than 300 cages. ThePBscreening system uses visually trackable transposons to induce both gain- and loss-of-function mutations and generates genome-wide distributed new insertions in more than 55% of F1 progeny. Using this system, we successfully conducted a pilot F1 screen and identified 5 growth retardation mutations. One of these mutants, a Six1/4PB/+mutant, revealed a role in milk intake behavior. The mutant animals exhibit abnormalities in nipple recognition and milk ingestion, as well as developmental defects in cranial nerves V, IX, and X. ThisPBF1 screening system offers individual laboratories unprecedented opportunities to conduct affordable genome-wide phenotypic screens for deciphering the genetic basis of mammalian biology and disease pathogenesis.

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