Biology subjects

Ferrentino, R.

Publications and source records attributed to Ferrentino, R..

6 recordsLinked to original sources

bioRxiv2026

Regulatory mutants of the Tbx1 gene alter transcription programs of lineage determination and patterning in early mesoderm.

The Tbx1 gene is haploinsufficient in mice and in humans, where it causes a DiGeorge syndrome phenotype characterized by developmental deficits of the pharyngeal apparatus. TBX1 plays a critical role in the differentiation and regionalization of the cardiopharyngeal mesoderm lineage and its derivatives. Nevertheless, its regulation is incompletely understood. Here we used a combination of computational and wet-lab approaches to identify regulatory sequences of the Tbx1 gene, and we use single-cell molecular analysis as a read-out and to establish the consequences of their deletion. Results revealed a cluster of regulatory sequences with at least three distinct elements. Elimination of the entire cluster caused a near shut down of the gene, while individual deletions had milder, quantitative effects. Transcriptomic analyses of the deletion mutants revealed the down regulation of genes related to cardiopharyngeal lineage specification and, more surprisingly, up regulation and anteriorization of genes related to embryonic patterning, thereby providing a rationale for the severe dysmorphogenesis of the posterior pharyngeal apparatus observed in Tbx1 mutant mice.

developmental biology↗

bioRxiv2024

Tbx1 stabilizes differentiation of the cardiopharyngeal mesoderm and drives morphogenesis in the pharyngeal apparatus

BackgroundTBX1 is required for the development of the pharyngeal apparatus. In the mouse, fish, and ascidian, Tbx1 is a marker of cardiopharyngeal mesoderm (CPM), a cell population that provides progenitors to the heart and branchiomeric muscles. However, in mammals: a) the molecular cascade that drives the diversification of this multipotent cell population, and b) the role of Tbx1 therein, are not well defined. Material and methodsWe used in vitro differentiation of WT and Tbx1-/- mouse embryonic stem cells into precardiac mesoderm, and performed single cell RNA-seq and ATAC-seq at two differentiation stages. We then used WT and Tbx1-/- mouse embryos for in vivo validation of the key findings. Results and conclusionsWe found that the response to loss of TBX1 is cell sub-population-specific, both in terms of gene expression and chromatin remodeling. We show that Tbx1 regulates chromatin accessibility and gene expression of an ancient transcriptional module that orchestrates the development of the trunk, pharynx and heart across evolution. This module is co-regulated and includes genes encoding the conserved transcription factor families of Tea Shirt (Tshz), Sine Oculis (Six), Eye absent (Eya), and Ebf/Collier. Analysis of putative regulatory regions of these genes, which were selected using a machine-learning computational procedure, predicted a feed-forward regulatory relationship between TBX1 and SIX factors that drives or stabilizes the module. Most surprisingly, we found a drift in the differentiation trajectory of the Tbx1 mutant CPM that led to a relative expansion of cells with epithelial-like transcriptional features in the cell culture model and in mouse embryos. We conclude that TBX1 is a critical factor for maintaining the transcriptional profile of the CPM.

developmental biology↗

bioRxiv2024

Cardiac Outflow tract septation defects in a DiGeorge syndrome model respond to Minoxidil treatment.

BackgroundThe T-BOX transcription factor TBX1 is essential for the development of the pharyngeal apparatus and it is haploinsufficient in DiGeorge syndrome (DGS), a developmental anomaly associated with congenital heart disease and other abnormalities. The murine model recapitulates the heart phenotype and showed collagen accumulation. MethodsWe first used a cellular model to study gene expression during cardiogenic differentiation of WT and Tbx1-/- mouse embryonic stem cells. Then we used a mouse model of DGS to test whether interfering with collagen accumulation using an inhibitor of lysyl hydroxylase would modify the cardiac phenotype of the mutant. Results and conclusionsIn the cell differentiation model, loss of Tbx1 was associated with up regulation of a subset of ECM-related genes, including several collagen genes. In the in vivo model, early prenatal treatment with Minoxidil, a lysyl hydroxylase inhibitor, ameliorated the cardiac outflow tract septation phenotype in Tbx1 mutant fetuses, but it had no effect on septation in WT fetuses. We conclude that TBX1 suppresses a subset of ECM-related genes. The partial rescue of the septation phenotype through Minoxidil treatment suggests that inhibiting collagen cross-linking reduces the impact of the phenotypic consequences of Tbx1 mutation.

developmental biology↗

bioRxiv2023

ENDOTHELIAL GENE REGULATORY ELEMENTS ASSOCIATED WITH CARDIOPHARYNGEAL LINEAGE DIFFERENTIATION

Endothelial cells (EC) differentiate from multiple sources, including the cardiopharyngeal mesoderm, which gives rise also to cardiac and branchiomeric muscles. Here, we used a cardiogenic mesoderm cell differentiation model that also activates an endothelial transcription program to identify endothelial regulatory elements activated in early cardiogenic mesoderm. Integrating our chromatin remodeling and gene expression data with available single-cell RNA-seq data from mouse embryos, we identified 101 putative regulatory elements of EC genes. We then applied a machine-learning strategy, trained on validated enhancers, to predict the probability of the sequences to function as enhancers. The computational assay determined that 50% of these sequences were likely enhancers, some of which have been previously reported. We also identified a smaller set of regulatory elements of well-known EC genes and validated them using genetic and epigenetic perturbation. Finally, we used the integration of multiple data sources and computational tools to search for transcriptional factor binding motifs. In conclusion, we identified novel EC regulatory sequences with a high likelihood to be enhancers, and we validated a subset of them using computational and cell culture models. Motif analyses revealed that the core EC transcription factors GATA/ETS/FOS is a likely driver of EC differentiation in cardiopharyngeal mesoderm.

molecular biology↗

bioRxiv2020

Chromatin and transcriptional response to loss of TBX1 in differentiating mouse P19Cl6 and embryonic stem cells.

The T-box transcription factor TBX1 has critical roles in the cardiopharyngeal lineage and the gene is haploinsufficient in DiGeorge syndrome, a typical developmental anomaly of the pharyngeal apparatus. Despite almost two decades of research, if and how TBX1 function triggers chromatin remodeling is not known. Here, we explored genome-wide gene expression and chromatin remodeling in two independent cellular models of Tbx1 loss of function, mouse embryonic carcinoma cells P19Cl6, and mouse embryonic stem cells (mESCs). The results of our study revealed that the loss or knockdown of TBX1 caused extensive transcriptional changes, some of which were cell type-specific, some were in common between the two models. However, unexpectedly we observed only limited chromatin changes in both systems. In P19Cl6 cells, differentially accessible regions (DARs) were not enriched in T-BOX binding motifs; in contrast, in mESCs, 34% (n=47) of all DARs included a T-BOX binding motif and almost all of them gained accessibility in Tbx1-/- cells. In conclusion, despite a clear transcriptional response of our cell models to loss of TBX1 in early cell differentiation, chromatin changes were relatively modest.

genomics↗

bioRxiv2017

Tbx1 represses Mef2c gene expression by inducing histone 3 deacetylation of the anterior heart field enhancer

The TBX1 gene is haploinsufficient in the 22q11.2 deletion syndrome (22q11.2DS), and genetic evidence from human patients and mouse models points to a major role of this gene in the pathogenesis of this syndrome. Tbx1 can activate and repress transcription and previous work has shown that one of its functions is to negatively modulate cardiomyocyte differentiation. Tbx1 occupies the anterior heart field (AHF) enhancer of the Mef2c gene, which encodes a key cardiac differentiation transcription factor. Here we show that increased dosage of Tbx1 is associated with down regulation of Mef2c expression and reduced acetylation of its AHF enhancer in cultured mouse myoblasts. Consistently, 22q11.2DS-derived and in vitro differentiated induced pluripotent stem cells (hiPSCs) expressed higher levels of Mef2c and showed increased AHF acetylation, compared to hiPSCs from a healthy donor. Most importantly, we show that in mouse embryos, loss of Tbx1 enhances the expression of the Mef2c-AHF-Cre transgene in a specific region of the splanchnic mesoderm, and in a dosage-dependent manner, providing an in vivo correlate of our cell culture data. These results indicate that Tbx1 regulates the Mef2c-AHF enhancer by inducing histone deacetylation.

developmental biology↗