CRISPR gene editing, cardiac disease modelling and transcriptomics interpretation of a novel GATA4 patient genetic variant
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA956514 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Other | ||||
| SRR24198258_1.fastq.gz | Fastqsanger.gz | |||
| SRR24198258_2.fastq.gz | Fastqsanger.gz | |||
| SRR24198259_1.fastq.gz | Fastqsanger.gz | |||
| SRR24198259_2.fastq.gz | Fastqsanger.gz |
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Project description:Over 400 million people worldwide are living with a rare disease, with genetic variants determining 80% of cases. Next Generation Sequencing identifies potential disease causative genetic variants, however many of these are classified as variants of uncertain significance (VUS). Each VUS requires functional validation as pathogenic or benign in disease pathology in specialist laboratories creating major delays in patient diagnosis. In this study we test a rapid genetic variant assessment pipeline using an EHMT1 (Euchromatin histone methyltransferase 1; EHMT1 p.Gln1144Ter) genetic variant that is pathogenic for Kleefstra Syndrome. Precise CRISPR homology directed (HDR) gene editing introduced the single nucleotide genetic variant in iPS cells and EHMT1_SNV cell clones were rapidly identified with amplicon sequencing. Induction of neural differentiation and RNA sequencing determined differences in differentiation at the gene and transcription factor level. The applied CRISPR HDR methodology was rapid and reliable for the introduction of SNVs in iPSCs for subsequent neuronal cell differentiation. Key features of Kleefstra Syndrome were identified, with involvement of key transcription factors REST and SP1 in disease mechanisms. This study indicates that precise iPSC gene editing and changes in disease modelling pathways can contribute to disease diagnosis and understanding of mechanisms.
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Project description:GATA4 is frequently epigenetically silenced in lung cancers. However, the impact of GATA4 inactivation on tumorigenesis and related therapeutic strategy remain to be determined. Through the genome-wide screening of tumor suppressing transcription factors, we demonstrate that GATA4 functions as an essential tumor suppressor in lung cancer in vitro and in vitro. Interestingly, ectopic GATA4 expression resulted in cellular senescence. Mechanistically, GATA4 up-regulates multiple miRNAs (miRNA-32, miRNA-301b, miR-320a, and miR-590) targeting TGFB2 mRNA and causes ensuing downregulation of WNT7B level to induce senescence. TGFBRI inhibitor synergizes with MEK1/2 inhibitor to promote lung cancer regression in Kras G12D/GATA4-/- mouse models. Decreased GATA4 level in clinical specimens negatively correlates with WNT7B or TGFB2 level and is significantly associated with poor prognosis.
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Project description:Analysis of changes in gene expression following hepatocyte specific deletion of GATA4 in adult mice. Results showed that the subset of differentially expressed genes had liver specific ontologies. Total RNA isolated from hepatocytes of AAV8-Tbg-Cre injected GATA4 fl/fl mice was compared to total RNA isolated from AAV-Tbg-GFP injected GATA4 fl/fl mice.
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Project description:GATA4 is a transcription factor known for its crucial role in the development of many tissues, including liver; however, its role in adult liver metabolism is unknown. Here, using high-throughput sequencing technologies, including assay for transposase-accessible chromatin with sequencing (ATAC-Seq), we identified GATA4 as a transcriptional regulator of metabolism in liver. GATA4 expression is elevated in response to refeeding, and its occupancy is increased at enhancers of genes linked to fatty acid and lipoprotein metabolism. Knocking out GATA4 in adult liver (Gata4LKO) decreased transcriptional activity at GATA4 binding sites especially during feeding. Gata4LKO mice have reduced plasma HDL cholesterol and increased liver triglyceride levels. The expression of a panel of genes involved in cholesterol export and triglyceride hydrolysis was downregulated and the expression of those involved in lipid uptake were upregulated in Gata4LKO liver, We further demonstrate that GATA4 collaborates with LXR liver. GATA4 shares a number of binding sites and direct transcriptional targets with LXRs, and loss of GATA4 impairs the hepatic transcriptional response to LXR agonist. Collectively, these results show that hepatic GATA4 contributes to the transcriptional control of hepatic and systemic lipid homeostasis.
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