Pane, Luna Simona (2010) "Mechanisms of transcriptional regulation by Tbx1". [Tesi di dottorato] (Unpublished)

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Deletion 22q11.2 syndrome (22q11DS) is the most common microdeletion syndrome in man, with an incidence of approximately 1:4000 live births (1); the major malformations include congenital heart defects such as truncus arteriosus (TA) and interrupted aortic arch type B (IAA-B), hypo/aplasia of the parathyroid and thymus glands, and craniofacial dysmorphism. Velo-cardio-facial (VCFS) and DiGeorge syndromes (DGS) are other diagnoses commonly made in affected individuals (1). The gene encoding the T-box transcription factor Tbx1, which is required for pharyngeal and cardiovascular development, has been identified as the gene haploinsufficient in mouse and human. We and others have identified a number of genes potentially targeted by Tbx1, but the mechanisms by which it can regulate the transcription of these genes and how it controls developmental pathways, are moslty unclear. One of the best studied molecular functions of Tbx1 is in heart development, where it is required to sustain proliferation of mesodermally-derived precursors of the second heart field (SHF), a cardiac progenitor cell population that contributes to the development of most of the heart, including the outflow tract and right ventricle. To better understand how it works during embryonic development, we evaluated Tbx1-dosage dependent gene expression changes in vivo using a novel dosage gradient approach. Among genes sensitive to Tbx1 level, we found the one encoding the cardiogenic transcription factor Mef2c which is involved in cardiomyocyte differentiation. Interestingly, this gene was anti-correlated to Tbx1 dosage; in situ hybridization on mutant mouse embryos also corroborated quantitative expression data. These results suggest that Tbx1 may negatively regulate cardiac muscle cell differentiation through a mechanism involving Mef2c transcriptional repression; this would be consistent with recent data showing that loss of function of Tbx1 is associated with increased expression of differentiation markers of the myocardium. It has also been shown that Mef2c is a direct transcriptional target of Gata4 in the SHF, during mouse embryonic development (2); accordingly our in vitro data, suggest that Tbx1 could negatively regulate Mef2c expression, somehow interfering with Gata4-dependent Mef2c activation. Virtually all the mechanistic data obtained so far derive from murine models of 22q11DS; there is the need of a system to validate these data on human material. Since human Embryonic Stem cells can differentiate in vitro, into multiple somatic tissues, including cardiac progenitors, we generated DiGeorge syndrome-specific Pluripotent Stem cells by reprogramming adult patient fibroblasts. Developing of this system as human model of the disease, will help us to investigate its underlying molecular mechanism on a cellular level. Tbx1 loss of function in mice, and, to a lesser extent, TBX1 haploinsufficency in DiGeorge syndrome patients, is associated with hypoplasia or aplasia of several organs and tissues; so it is possible that Tbx1 function in regulating the balance between proliferation and differentiation in the SHF, may also apply to other tissues where Tbx1 is expressed. Availability of DiGeorge syndrome-specific Pluripotent Stem cells, will help us to speculate whether that disregulation of the balance between proliferation and differentiation of different types of progenitor cells or stem cells, may be a basic pathogenic mechanism for the loss of function phenotype.

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