Aurigemma, Ilaria (2023) Identifying critical regulatory factors during cardiopharyngeal mesoderm (CPM) differentiation into endothelial cells (ECs). [Tesi di dottorato]

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Abstract

The development of the cardiovascular system requires the coordinated differentiation of several cell types including endothelial cells (EC), smooth muscle cells and cardiomyocytes. This process involves the differentiation of cardiopharyngeal mesoderm (CPM) from which these cardiac cell types derive. The overall aim of my doctoral work is to understand better the genetic and epigenetic mechanisms responsible for cell fate transitions in multipotent cardiac progenitors to differentiate into endothelial cells (ECs). Notably, I have developed a model for differentiation of CPM into ECs starting from engineered mouse embryonic stem cells (mESCs), using a serum-free protocol with the addition of specific growth factors that induce cardiac and endothelial differentiation. The results showed that this procedure allows rapid vascular differentiation with high efficiency. I obtained approximately 91% CD144+ (VE-Cadherin) cells within 8 days. Then I performed RNA-seq and ATAC-seq on the early phases of CPM differentiation to define the transcriptomic and chromatin accessibility profile. First, I demonstrated that mESCs differentiation promoted the expression of EC-specific markers at day 4 of differentiation (d4), including Pecam1, VE-Cadherin (Cdh5), Eng, Kdr, Gata2, Gata6, Ets1, Flt1 and others. RNAseq performed between d2 and d4 identified 1735 differentially expressed genes, many of which are involved in angiogenesis, indicating the activation of an EC transcription program. ATAC-seq revealed 6348 Differential Accessible Regions (DARs) that changed their chromatin accessibility during this time window. Most of them were located in intra- and inter-genic regions. Thanks to the integration of these two methods, I identified, at the first, 2 putative enhancers defined as regions of increased accessibility, associated with endothelial-specific genes: Pecam1 and Notch1, both of which are critical for vascular development. Subsequently, I have extended the search of putative regulatory elements, identifying other 8 open chromatin regions, associated with Kdr (Vegfr2), Cdh5 (VE-Cadherin), CD34, Eng, Flt1 (Vegfr1), Tal1 (Scl1), Dusp5 and Gata6 endothelial genes. To validate the putative regulatory regions, I followed two strategies: DNA editing (putative enhancers deletion) and epigenetic decommissioning. For the first approach, I generated mESCs with deletion of Pecam1-enh.int2 and Notch1-enh.int15 (by CRISPR-Cas9 technology), which I then differentiated towards CM-EC lineages. Two Notch1-∆ enh.int15. independent mESC clones showed a significant reduction of Notch1 expression during the later stages of EC differentiation (d6 and d8). Similarly, Pecam1 expression was also downregulated in two independent Pecam1-∆ enh.int2. mESC clones at the same time points. These results indicated that the regions deleted are required for appropriate expression of the respective genes during EC differentiation process. The second validation strategy was based on epigenetic reprogramming by nuclease-deficient dCas9 fused with histone demethylase LSD1 (dCas9-LSD1). It removes mono and di-methylation of histone H3 lisyne 4 (H3K4me1 and me2) to promote the change of chromatin shape into a repressive configuration. I generated mESC clones constitutively expressing dCas9-LSD1 and transfected these cells with gRNAs targeting the putative enhancers and then differentiated into endothelial cells (ECs). In particular, I have analyzed so far only five putative enhancer regions: Notch1-enh.intr15; Kdr-enh.intr10; VE-Cadh.-enh.intr1; Eng-enh.intr2; Flt1-enh.intr10. The targeted five loci resulted affected by dCas9-LSD1 epigenetically repression, giving rise to relative reduction of gene-related expression, specifically at day8 of differentiation. Overall, 6 tested out of 10 identified putative enhancers seems to be regulatory elements and could be involved during later stages of EC differentiation. Moreover, to predict, computationally, transcription factor motifs in EC enhancers, I performed a preliminary motif analysis of DARs regions related to endothelial cell fate specification. Sequence analyses of regions opened at d4 identified Gata1, Gata2 and JunB transcription factors. They could regulate the differentiation of cardiopharyngeal mesoderm progenitors in derivative tissues, including EC. In conclusion, the experimental model and methods used for differentiation of CPM into ECs allowed me to efficiently identify novel putative endothelial enhancers. Thanks to genetic and epigenetic manipulation of these sequences, I established their requirement for the transcription process during differentiation from cardiopharyngeal mesoderm to ECs.

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