A Wt1-Controlled Chromatin Switching Mechanism Underpins Tissue-Specific Wnt4 Activation and Repression

SummaryWt1 regulates the epithelial-mesenchymal transition (EMT) in the epicardium and the reverse process (MET) in kidney mesenchyme. The mechanisms underlying these reciprocal functions are unknown. Here, we show in both embryos and cultured cells that Wt1 regulates Wnt4 expression dichotomously. In kidney cells, Wt1 recruits Cbp and p300 as coactivators; in epicardial cells it enlists Basp1 as a corepressor. Surprisingly, in both tissues, Wt1 loss reciprocally switches the chromatin architecture of the entire Ctcf-bounded Wnt4 locus, but not the flanking regions; we term this mode of action “chromatin flip-flop.” Ctcf and cohesin are dispensable for Wt1-mediated chromatin flip-flop but essential for maintaining the insulating boundaries. This work demonstrates that a developmental regulator coordinates chromatin boundaries with the transcriptional competence of the flanked region. These findings also have implications for hierarchical transcriptional regulation in development and disease

Novel targets of the Wilms’ tumour 1 gene (Wt1) in the epicardium during development

Cardiovascular and heart diseases are the leading causes of death worldwide. In mammals, when heart damage occurs this organ is unable to regenerate itself. Understanding how to induce a regenerative process has been the focus of a great deal of attention recently. The understanding of heart development and the initial formation of several heart lineages could be used in finding a regenerative approach to heart damage that can mimic developmental processes. The Wilms’ tumour 1 gene (Wt1) is essential in the epicardium, the outer layer of cells around the heart, which during development has a multipotent potential and is the source of progenitors for several heart cell lineages such as: cells of the coronary vasculature, fibroblasts and cardiomyocytes. In my thesis I have focused on using an in-vitro (immortalized epicardial cells where Wt1 can be deleted by adding tamoxifen), and an in-vivo approach (genome wide expression analyses of Wt1 control and Wt1 knock-out epicardial enriched cells), to identify novel targets of Wt1 in the epicardium during development. I found that the chemokines Cxcl10 and Ccl5 are up-regulated in tamoxifen induced immortalized Wt1 knock-out epicardial cells and ex-vivo in heart explants when Wt1 is down-regulated. Ccl5 was found to be able to inhibit cardiomyocyte proliferation and Cxcl10 also inhibited epicardial cell migration, which could further explain ventricular thinning in Wt1 mutant mouse hearts. Wt1 is able to bind directly to the promoter of a chemokine and interferon response regulator gene, Irf7, which is also up-regulated in our in-vivo model. This could provide a mechanism by which Wt1 can inhibit chemokine expression during development, and could link Wt1 with immunological responses, which recently have been shown to play a role in the physiology and development of cells outside immunity, as well as being involved in physiological roles during damage and repair in adult tissues. I have also identified two Wt1-GFP populations (Wt1GFP++ and Wt1GFP+) in the ventricles of Wt1-GFP knock-in mice. The Wt1GFP++ population is enriched for epicardial cells, and a genome wide transcriptome analysis of these cells from E11.5 to E16.5 demonstrates they have a very dynamic regulation of a wide variety of genes, and also it indicates the existence of an early, transient and late Wt1GFP++ gene expression programs. The transcriptome analysis of Wt1GFP++ control and Wt1GFP++ Wt1 knock-out cells, from Gata5-Cre Wt1loxP/gfp mice at E13.5, reveals that Wt1 could regulate a number of previously un-described Wt1 targets related to the early Wt1GFP++ program, and gene ontology analyses indicate that many targets are related to cell to cell signalling and interaction, cell to extracellular matrix interaction, tissue development and morphogenesis. The Wt1GFP+ cell population is positive for a number of cardiomyocyte specific markers and has a low or negative expression of endothelial, epithelial and mesenchymal markers according to my transcriptome analysis. The findings I have described here shed light on the variety of targets of Wt1 and further reveal the function of Wt1 during epicardial development, which could be used in finding a regenerative approach to heart disease

WT1 regulates the expression of inhibitory chemokines during heart development

The embryonic epicardium is an important source of cardiovascular precursor cells and paracrine factors that are required for adequate heart formation. Signaling pathways regulated by WT1 that promote heart development have started to be described; however, there is little information on signaling pathways regulated by WT1 that could act in a negative manner. Transcriptome analysis of Wt1KO epicardial cells reveals an unexpected role for WT1 in repressing the expression of interferon-regulated genes that could be involved in a negative regulation of heart morphogenesis. Here, we showed that WT1 is required to repress the expression of the chemokines Ccl5 and Cxcl10 in epicardial cells. We observed an inverse correlation of Wt1 and the expression of Cxcl10 and Ccl5 during epicardium development. Chemokine receptor analyses of hearts from Wt1(gfp/+) mice demonstrate the differential expression of their chemokine receptors in GFP(+) epicardial enriched cells and GFP(-) cells. Functional assays demonstrate that CXCL10 and CCL5 inhibit epicardial cells migration and the proliferation of cardiomyocytes respectively. WT1 regulates the expression levels of Cxcl10 and Ccl5 in epicardial cells directly and indirectly through increasing the levels of IRF7. As epicardial cell reactivation after a myocardial damage is linked with WT1 expression, the present work has potential implications in adult heart repair