Detection and functional characterization of a novel MEF2A variation responsible for familial dilated cardiomyopathy

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Qi Qiao ◽  
Cui-Mei Zhao ◽  
Chen-Xi Yang ◽  
Jia-Ning Gu ◽  
Yu-Han Guo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dilated Cardiomyopathy ◽  
Sanger Sequencing ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Functional Characterization ◽  
Luciferase Assay ◽  
Chinese Family ◽  
Sequencing Analysis ◽  
Loss Of Function

AbstractObjectivesDilated cardiomyopathy (DCM) represents the most frequent form of cardiomyopathy, leading to heart failure, cardiac arrhythmias and death. Accumulating evidence convincingly demonstrates the crucial role of genetic defects in the pathogenesis of DCM, and over 100 culprit genes have been implicated with DCM. However, DCM is of substantial genetic heterogeneity, and the genetic determinants underpinning DCM remain largely elusive.MethodsWhole-exome sequencing and bioinformatical analyses were implemented in a consanguineous Chinese family with DCM. A total of 380 clinically annotated control individuals and 166 more DCM index cases then underwent Sanger sequencing analysis for the identified genetic variation. The functional characteristics of the variant were delineated by utilizing a dual-luciferase assay system.ResultsA heterozygous variation in the MEF2A gene (encoding myocyte enhancer factor 2A, a transcription factor pivotal for embryonic cardiogenesis and postnatal cardiac adaptation), NM_001365204.1: c.718G>T; p. (Gly240*), was identified, and verified by Sanger sequencing to segregate with autosome-dominant DCM in the family with complete penetrance. The nonsense variation was neither detected in 760 control chromosomes nor found in 166 more DCM probands. Functional analyses revealed that the variant lost transactivation on the validated target genes MYH6 and FHL2, both causally linked to DCM. Furthermore, the variation nullified the synergistic activation between MEF2A and GATA4, another key transcription factor involved in DCM.ConclusionsThe findings firstly indicate that MEF2A loss-of-function variation predisposes to DCM in humans, providing novel insight into the molecular mechanisms of DCM and suggesting potential implications for genetic testing and prognostic evaluation of DCM patients.

scholarly journals Regulatory Network Analysis in Estradiol-Treated Human Endothelial Cells

2021 ◽  
Vol 22 (15) ◽  
pp. 8193
Author(s):  
Daniel Pérez-Cremades ◽  
Ana B. Paes ◽  
Xavier Vidal-Gómez ◽  
Ana Mompeón ◽  
Carlos Hermenegildo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Regulatory Network ◽  
Mirna Target ◽  
Target Genes ◽  
Functional Characterization ◽  
Human Endothelial Cells ◽  
Mrna Targets ◽  
Canonical Pathways

Background/Aims: Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a comprehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. Methods: miRNA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17β-estradiol (E2) (1 nmol/L, 24 h). miRNA–mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miRNA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software. Results: miRNA–target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship according to miRNA target prediction databases. The analysis identified 588 miRNA–target interactions between 102 miRNAs and 588 targets. Specifically, 63 upregulated miRNAs interacted with 295 downregulated targets, while 39 downregulated miRNAs were paired with 293 upregulated mRNA targets. Functional characterization of miRNA/mRNA association analysis highlighted hypoxia signaling, integrin, ephrin receptor signaling and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Transcription factors and downstream genes analysis revealed eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhesion molecule binding pathways. Conclusion: This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells.

scholarly journals Rolling Circle Amplification (RCA)-Mediated Genome-Wide ihpRNAi Mutant Library Construction in Brassica napus

2020 ◽  
Vol 21 (19) ◽  
pp. 7243
Author(s):  
Shengbo Zhao ◽  
Junling Luo ◽  
Xinhua Zeng ◽  
Keqi Li ◽  
Rong Yuan ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Functional Genomics ◽  
Insertional Mutagenesis ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Agronomic Traits ◽  
Rolling Circle Amplification ◽  
Functional Characterization ◽  
Rolling Circle ◽  
Genome Wide

With the successful completion of genomic sequencing for Brassica napus, identification of novel genes, determination of functions performed by genes, and exploring the molecular mechanisms underlying important agronomic traits were challenged. Mutagenesis-based functional genomics techniques including chemical, physical, and insertional mutagenesis have been used successfully in the functional characterization of genes. However, these techniques had their disadvantages and inherent limitations for allopolyploid Brassica napus, which contained a large number of homologous and redundant genes. Long intron-spliced hairpin RNA (ihpRNA) constructs which contained inverted repeats of the target gene separated by an intron, had been shown to be very effective in triggering RNAi in plants. In the present study, the genome-wide long ihpRNA library of B. napus was constructed with the rolling circle amplification (RCA)-mediated technology. Using the phytoene desaturase (PDS) gene as a target control, it was shown that the RCA-mediated long ihpRNA construct was significantly effective in triggering gene silence in B. napus. Subsequently, the resultant long ihpRNA library was transformed into B. napus to produce corresponding RNAi mutants. Among the obtained transgenic ihpRNA population of B. napus, five ihpRNA lines with observable mutant phenotypes were acquired including alterations in the floral model and the stamen development. The target genes could be quickly identified using specific primers. These results showed that the RCA-mediated ihpRNA construction method was effective for the genome-wide long ihpRNA library of B. napus, therefore providing a platform for study of functional genomics in allopolyploid B. napus.

LncRNA-H19 silencing suppresses synoviocytes proliferation and attenuates collagen-induced arthritis progression by modulating miR-124a

Rheumatology ◽  
2020 ◽  
Vol 60 (1) ◽  
pp. 430-440
Author(s):  
Xiaohong Fu ◽  
Guojing Song ◽  
Rongrong Ni ◽  
Han Liu ◽  
Zhizhen Xu ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Therapeutic Potential ◽  
Flow Cytometric Analysis ◽  
Cartilage Destruction ◽  
Luciferase Assay ◽  
Flow Cytometric ◽  
Non Coding Rna ◽  
Novel Strategy ◽  
Long Non Coding Rna

Abstract Objectives Long non-coding RNA H19 (lncRNA-H19) is highly expressed in fibroblast-like synoviocytes (FLS) from patients with RA. The present study aimed to clarify the pathological significance and regulatory mechanisms of lncRNA-H19 in FLS. Methods Mice with CIA were locally injected with LV-shH19. The progression of CIA was explored by measuring arthritic index (AI), paw thickness (PT) and histologic analysis. The growth and cell cycle of human synoviocyte MH7A were assessed by CCK-8 and flow cytometric analysis. The putative binding sites between lncRNA-H19 and miR-124a were predicted online, and the binding was identified by luciferase assay. RT-qPCR, Western blot and luciferase assay were performed to explore the molecular mechanisms between liver X receptor (LXR), lncRNA-H19, miR-124a and its target genes. Results The expression of lncRNA-H19 was closely associated with the proliferation of synoviocytes and knockdown of lncRNA-H19 significantly ameliorated the progression of CIA, reflected by decreased AI, PT and cartilage destruction. Notably, lncRNA-H19 competitively bound to miR-124a, which directly targets CDK2 and MCP-1. It was confirmed that lncRNA-H19 regulates the proliferation of synoviocytes by acting as a sponge of miR-124a to modulate CDK2 and MCP-1 expression. Furthermore, the agonists of LXR inhibited lncRNA-H19-mediated miR-124a-CDK2/MCP-1 signalling pathway in synoviocytes. The ‘lncRNA-H19-miR-124a-CDK2/MCP-1’ axis plays an important role in LXR anti-arthritis. Conclusion Regulation of the miR-124a-CDK2/MCP-1 pathway by lncRNA-H19 plays a crucial role in the proliferation of FLS. Targeting this axis has therapeutic potential in the treatment of RA and may represent a novel strategy for RA treatment.

scholarly journals A Novel Homozygous VPS13B Splice-Site Mutation Causing the Skipping of Exon 38 in a Chinese Family With Cohen Syndrome

2021 ◽  
Vol 9 ◽  
Author(s):  
Liangshan Li ◽  
Xiangmao Bu ◽  
Yuhua Ji ◽  
Ping Tan ◽  
Shiguo Liu
Keyword(s):  
Splice Site ◽  
Reverse Transcription ◽  
Sanger Sequencing ◽  
Splice Site Mutation ◽  
Chinese Family ◽  
Sequencing Analysis ◽  
Site Mutation ◽  
Transcription Analysis ◽  
Cohen Syndrome ◽  
The Impact

Background: Cohen syndrome (CS) is a clinically heterogeneous disorder characterized by extensive phenotypic variation with autosomal recessive inheritance. VPS13B was identified to be the disease-causing gene for CS. The objectives of the present study were to screen likely pathogenic mutations of the patient with developmental delay and mental retardation, and to determinate the effect of this splice-site mutation by reverse transcription analysis.Methods: Whole exome sequencing (WES) in combination with Sanger sequencing were performed to identify the causative mutations of this CS family. Subsequently, the impact of the intronic variant on splicing was analyzed by reverse transcription and the construction of expression vector.Results: A novel homozygous splice-site mutation (c.6940+1G>T) in the VPS13B gene was identified in this proband. Sanger sequencing analysis of the cDNA demonstrated that the c.6940+1G>T variant could cause the skipping of entire exon 38, resulting in the loss of 208 nucleotides and further give rise to the generation of a premature in-frame stop codon at code 2,247.Conclusions: The homozygous VPS13B splicing variant c.6940+1G>T was co-segregated with the CS phenotypes in this family and was identified to be the cause of CS after comprehensive consideration of the clinical manifestations, genetic analysis and cDNA sequencing result.

scholarly journals Paupar LncRNA Promotes KAP1 Dependent Chromatin Changes And Regulates Subventricular Zone Neurogenesis

2017 ◽  
Author(s):  
Ioanna Pavlaki ◽  
Farah Alammari ◽  
Bin Sun ◽  
Neil Clark ◽  
Tamara Sirey ◽  
...  
Keyword(s):  
Subventricular Zone ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Neuroblastoma Cell ◽  
Regulatory Protein ◽  
Regulatory Elements ◽  
Loss Of Function ◽  
Transcriptional Regulatory Elements ◽  
Genome Wide

ABSTRACTMany long non-coding RNAs (lncRNAs) are expressed during central nervous system (CNS) development, yet their in vivo roles and molecular mechanisms of action remain poorly understood. Paupar, a CNS expressed lncRNA, controls neuroblastoma cell growth by binding and modulating the activity of genome-wide transcriptional regulatory elements. We show here that Paupar transcript directly binds KAP1, an essential epigenetic regulatory protein, and thereby regulates the expression of shared target genes important for proliferation and neuronal differentiation. Paupar promotes KAP1 chromatin occupancy and H3K9me3 deposition at a subset of distal targets, through formation of a DNA binding ribonucleoprotein complex containing Paupar, KAP1 and the PAX6 transcription factor. Paupar-KAP1 genome-wide co-occupancy reveals a 4-fold enrichment of overlap between Paupar and KAP1 bound sequences. Furthermore, both Paupar and Kap1 loss of function in vivo accelerates lineage progression in the mouse postnatal subventricular zone (SVZ) stem cell niche and disrupts olfactory bulb neurogenesis. These observations provide important conceptual insights into the trans-acting modes of lncRNA-mediated epigenetic regulation, the mechanisms of KAP1 genomic recruitment and identify Paupar and Kap1 as regulators of SVZ neurogenesis.

scholarly journals MIR822 modulates monosporic female gametogenesis through an ARGONAUTE9-dependent pathway in Arabidopsis thaliana

2021 ◽  
Author(s):  
ANDREA TOVAR AGUILAR ◽  
Daniel GRIMANELLI ◽  
Gerardo Acosta Garcia ◽  
Jean Philippe Vielle Calzada ◽  
Jesus Agustin Badillo-Corona ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Flowering Plants ◽  
Loss Of Function ◽  
Genes Encoding ◽  
Functional Megaspore ◽  
Dependent Pathway ◽  
Female Gametogenesis

In the ovule of flowering plants, the establishment of the haploid generation occurs when a somatic cell differentiates into a Megaspore Mother Cell (MMC) and initiates meiosis. As most flowering plants, Arabidopsis thaliana undergoes a monosporic type of gametogenesis; three meiotically derived cells degenerate without further division, and a single one, the functional megaspore (FM), divides mitotically to form the female gametophyte. In Arabidopsis, the ARGONAUTE4 clade proteins are involved in the control of megasporogenesis. In particular, mutations in ARGONAUTE9 (AGO9) lead to the ectopic differentiation of gametic precursors that can give rise female gametophytes. However, the genetic basis and molecular mechanisms that control monosporic gametogenesis remain largely unknown. Here, we show that Arabidopsis plants carrying loss-of-function mutations in the AGO9-interacting miR822a give rise to extranumerary surviving megaspores that acquire a FM identity and divide without giving rise to differentiated female gametophytes. The overexpression of three miR822a target genes encoding Cysteine/Histidine-Rich C1 domain proteins (DC1) phenocopy mir822a plants. The miR822a targets are overexpressed in ago9 mutant ovules, confirming that miR822a acts through an AGO9-dependent pathway to negatively regulate DC1 domain proteins. Our results identify a new role of miRNAs in the most prevalent form of female gametogenesis in flowering plants

scholarly journals MicroRNA Sequencing Analysis in Obstructive Sleep Apnea and Depression: Anti-Oxidant and MAOA-Inhibiting Effects of miR-15b-5p and miR-92b-3p through Targeting PTGS1-NF-κB-SP1 Signaling

Antioxidants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1854
Author(s):  
Yung-Che Chen ◽  
Po-Yuan Hsu ◽  
Mao-Chang Su ◽  
Ting-Wen Chen ◽  
Chang-Chun Hsiao ◽  
...  
Keyword(s):  
Obstructive Sleep Apnea ◽  
Sleep Apnea ◽  
Target Genes ◽  
Luciferase Assay ◽  
Apnea Hypopnea Index ◽  
Cox1 Gene ◽  
Sequencing Analysis ◽  
Gene Expressions ◽  
Obstructive Sleep

The aim of this study was to identify novel microRNAs related to obstructive sleep apnea (OSA) characterized by intermittent hypoxia with re-oxygenation (IHR) injury. Illumina MiSeq was used to identify OSA-associated microRNAs, which were validated in an independent cohort. The interaction between candidate microRNA and target genes was detected in the human THP-1, HUVEC, and SH-SY5Y cell lines. Next-generation sequencing analysis identified 22 differentially expressed miRs (12 up-regulated and 10 down-regulated) in OSA patients. Enriched predicted target pathways included senescence, adherens junction, and AGE-RAGE/TNF-α/HIF-1α signaling. In the validation cohort, miR-92b-3p and miR-15b-5p gene expressions were decreased in OSA patients, and negatively correlated with an apnea hypopnea index. PTGS1 (COX1) gene expression was increased in OSA patients, especially in those with depression. Transfection with miR-15b-5p/miR-92b-3p mimic in vitro reversed IHR-induced early apoptosis, reactive oxygen species production, MAOA hyperactivity, and up-regulations of their predicted target genes, including PTGS1, ADRB1, GABRB2, GARG1, LEP, TNFSF13B, VEGFA, and CXCL5. The luciferase assay revealed the suppressed PTGS1 expression by miR-92b-3p. Down-regulated miR-15b-5p/miR-92b-3p in OSA patients could contribute to IHR-induced oxidative stress and MAOA hyperactivity through the eicosanoid inflammatory pathway via directly targeting PTGS1-NF-κB-SP1 signaling. Over-expression of the miR-15b-5p/miR-92b-3p may be a new therapeutic strategy for OSA-related depression.

scholarly journals GENETICS IN ENDOCRINOLOGY: Glucocorticoid resistance syndrome

2020 ◽  
Vol 182 (2) ◽  
pp. R15-R27 ◽  
Author(s):  
Géraldine Vitellius ◽  
Marc Lombes
Keyword(s):  
Target Gene ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Cushing Syndrome ◽  
Functional Characterization ◽  
Clinical Signs ◽  
Cell Types ◽  
Glucocorticoid Resistance ◽  
Loss Of Function ◽  
Adrenal Hyperplasia

Glucocorticoids (GC) such as cortisol regulate multiple physiological functions, notably those involved in development, metabolism, inflammatory processes and stress, and exert their effects upon binding to the glucocorticoid receptor (GR, encoded by NR3C1 gene in humans). GC signaling follows several consecutive steps leading to target gene transactivation, including ligand binding, nuclear translocation of ligand-activated GR complexes, DNA binding, and recruitment of functional transcriptional machinery. Generalized glucocorticoid resistance syndrome, due to GR loss-of-function mutations, may be related to the impairment of one of the GC signaling steps. To date, 31 NR3C1 loss-of-function mutations have been reported in patients presenting with various clinical signs such as hypertension, adrenal hyperplasia, hirsutism or metabolic disorders associated with biological hypercortisolism but without Cushing syndrome signs and no negative regulatory feedback loop on the hypothalamic-pituitary-adrenal axis. Functional characterization of GR loss-of-function mutations often demonstrates GR haploinsufficiency and a decrease of GR target gene induction in relevant cell types. The main signs at presentation are very variable from resistant hypertension, bilateral adrenal hyperplasia likely related to increased ACTH levels but not exclusively, hirsutism to isolated renin-angiotensin-aldosterone system abnormalities in a context of 11βHSD2 deficiency. Some mutated GR patients are obese or overweight together with a healthier metabolic profile that remains to be further explored in future studies. Deciphering the molecular mechanisms altered by GR mutations should enhance our knowledge on GR signaling and ultimately facilitate management of GC-resistant patients. This review also focuses on the criteria facilitating identification of novel NR3C1 mutations in selected patients.

scholarly journals Control of mitotic chromosome condensation by the fission yeast transcription factor Zas1

2018 ◽  
Vol 217 (7) ◽  
pp. 2383-2401 ◽  
Author(s):  
Christoph Schiklenk ◽  
Boryana Petrova ◽  
Marc Kschonsak ◽  
Markus Hassler ◽  
Carlo Klein ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fission Yeast ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Chromosome Condensation ◽  
Condensation Process ◽  
Transactivation Domain ◽  
Promoter Regions ◽  
Live Cell Microscopy ◽  
Mitotic Chromosome Condensation

Although the formation of rod-shaped chromosomes is vital for the correct segregation of eukaryotic genomes during cell divisions, the molecular mechanisms that control the chromosome condensation process have remained largely unknown. Here, we identify the C2H2 zinc-finger transcription factor Zas1 as a key regulator of mitotic condensation dynamics in a quantitative live-cell microscopy screen of the fission yeast Schizosaccharomyces pombe. By binding to specific DNA target sequences in their promoter regions, Zas1 controls expression of the Cnd1 subunit of the condensin protein complex and several other target genes, whose combined misregulation in zas1 mutants results in defects in chromosome condensation and segregation. Genetic and biochemical analysis reveals an evolutionarily conserved transactivation domain motif in Zas1 that is pivotal to its function in gene regulation. Our results suggest that this motif, together with the Zas1 C-terminal helical domain to which it binds, creates a cis/trans switch module for transcriptional regulation of genes that control chromosome condensation.

scholarly journals Cleidocranial dysplasia. A molecular and clinical review.

2018 ◽  
Vol 8 (1) ◽  
pp. 35-38
Author(s):  
Andrea Avendaño ◽  
Francisco Cammarata-Scalisi ◽  
Mochamad Fahlevi Rizal ◽  
Sarworini Bagio Budiardjo ◽  
Margaretha Suharsini ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Autosomal Dominant ◽  
Cleidocranial Dysplasia ◽  
Supernumerary Teeth ◽  
Phenotypic Characteristics ◽  
Autosomal Dominant Disorder ◽  
Dental Abnormalities ◽  
Related Transcription Factor

Cleidocranial dysplasia (CCD) is a rare autosomal dominant disorder characterized by skeletal and dental abnormalities primarily, short stature, aplasia or hypoplasia of clavicles, open fontanelles and supernumerary teeth. Heterozygous mutations of the runt related transcription factor 2 (RUNX2) gene have been found in approximately 60-70% of cases leaving a large number of cases with no defined genetic cause which led us to delve into molecular mechanisms underlying CCD and thus to detect potential target genes to be explored in these patients. In this review we also highlight very broadly the phenotypic characteristics of previously reported patients with CCD.

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