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Transcriptomic profiling of GATA4 Knock-Out in cardiac, lung, and tail tip fibroblasts

ABSTRACT: Fibroblasts from different anatomical origins exhibit distinct phenotypic properties. In this study, we aimed to determine the cell-type-specific transcriptional responses to GATA4 overexpression. We performed bulk RNA-seq on cardiac fibroblasts (CF), lung fibroblasts (LF), and tail tip fibroblasts (TTF) transduced with either Ad-GFP (control) or Ad-Cre (GATA4 KO). The goal is to delineate the general versus source-specific GATA4 regulatory networks.

ORGANISM(S): Mus musculus

PROVIDER: GSE325933 | GEO | 2026/07/01

REPOSITORIES: GEO

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Project description:How transcriptional regulators are linked to cell biological effectors of morphogenesis is not fully understood. To elucidate this linkage, we use the C. elegans male tail-tip as a model. The tail-tip is made of four cells that, in males, undergo changes in shape and position during the L4 stage. The transcription factor DMD-3 is the master regulator of this Tail-Tip Morphogenesis (TTM). To identify other genes involved in TTM, the lab has previously performed a genome-wide RNAi screen and conducted an RNA-seq differential expression (DE) analysis using tail tips isolated from L4 wild-type and dmd-3 mutant males. This yielded candidates for genes downstream of and regulated by DMD-3 directly or indirectly. To identify the direct targets of DMD-3, we conducted male-specific DMD-3 ChIP-seq on whole worms during TTM. We identified 1755 DMD-3 binding peaks representing 6061 genes and a de novo DMD-3-associated binding motif, sharing similarity to the chromatin binding factor, EOR-1. Integrating this data with the previous RNAi screen and the DE analysis, we found 270 candidates for direct DMD-3 targets in tail-tip cells. To validate these candidates, we inserted GFP into the endogenous loci using CRISPR and evaluated the expression of the fusion proteins. We then deleted the sequences associated with DMD-3 ChIP peaks or the DMD-3-associated binding motif and assessed if this resulted in a protein expression change or defective TTM. We found four genes where deletion of a peak had an effect: The transcription factor FOS-1 is expressed in nuclei of male tail tips during TTM and in the vulva. Deletion of a DMD-3 peak or DMD-3-associated motif sites abolishes expression in male tail-tip nuclei. NMY-2 is localized as a “cap” at the tip of the tail during TTM. Removal of the peak reduces the level of tail tip NMY-2 expression. The pan-1 locus has two peaks. PAN-1::GFP is first seen in puncta at the cell surface, then at basal lateral membranes. Removal of the distal peak causes TTM defects in 10% of screened males, while removal of both peaks leads to global, previously reported, pan-1 phenotypes. HMR-1 is expressed at adherens junctions at the onset of TTM and later forms puncta at the cell surface. Deleting the peak has little effect on expression but results in epidermal bulges in both sexes, including the mail tail region. Because of the global phenotypes observed in peak mutants, we hypothesize the peak regions contain binding sites for other factors. Additionally, using extrachromosomal arrays, we tested and confirmed the DMD-3-associated motif’s contribution to wild-type TTM.

Project description:The transcription factor (TF) GATA4 is a key mediator of cardiogenesis. GATA4 regulates cardiogenesis through the expression of its target genes, only some of which have been identified. We have used a gain of function model based on pluripotent embryonic ectoderm explants from Xenopus embryos expressing GATA4, to identify a set of downstream targets of GATA4 which are also regulated by Nodal, a known cardiogenic signal. GATA4 was shown to be required for the expression of target genes Tbx2 and Prdm1 in vivo, likely acting in a direct fashion by interacting with their regulatory regions. In addition, Tbx2 and Prdm1 are shown to have roles of their own in vivo, as downregulation of Tbx2, a positive target, and overexpression of Prdm1, a negative target, interferes with cardiac development in Xenopus embryos. The conservation of the GATA4-TBX2-PRDM1 regulatory relationship was shown in human iPSC-derived cardiomyocytes. Loss of function of GATA4 lead to downregulation of TBX2, upregulation of PRDM1 expression and failure of cardiogenesis. GATA4 deficient cells failed to form normal cardiomyocytes, with most cells likely adopting alternative fates such as smooth muscle cells and fibroblasts, and only a small minority expressing an aberrant cardiomyocyte phenotype. Genome wide transcriptomic analysis documented severe reduction of cardiomyocyte and endothelial cell transcriptomes and upregulation of transcriptional profiles of smooth muscle cells and fibroblasts. Disruption of TBX2 function did not alter cardiomyocyte differentiation efficiency but led to the formation of abnormal, hypertrophic cardiomyocytes, characterised by defective sarcomeres and upregulation of cardiomyocyte Gene Ontology terms. In addition, we show that PRDM1 is not essential for formation of cardiomyocytes, implying suppression of alternative fates and fine-tuning the expression of the cardiomyocyte program. The results presented establish a conserved regulatory relationship between GATA4 and its target genes TBX2 and PRDM1, expanding the cardiac GRN and providing further insight into how cardiogenesis proceeds

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Transcriptomic profiling of GATA4 Knock-Out in cardiac, lung, and tail tip fibroblasts

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