Identification of cis-regulatory modules in mouse embryonic limb buds and heart using endogenous epitope-tagged transcription factors

The genes that control mouse embryonic development are tightly regulated spatially and temporally by the integration of various signaling pathways. The interaction of downstream transcription factors with cis-regulatory modules located in the genomic landscapes of their target genes is crucial to control gene expression and enable the growth and patterning of embryonic tissues. However, in most of the cases, the genome wide range of target regions bound by a given trans-acting factor in a tissue- specific manner is not known. In this thesis, I have taken advantage of epitope- tagging essential transcriptional regulators such as SMAD4 and HAND2 to gain mechanistic insight into their functions during limb and heart development.
In particular, the morphogenesis of the limb bud depends on a self-regulatory system of signaling feedback loops in the core of which the BMP antagonist GREMLIN1 plays a critical role. We analyzed the cis-regulatory landscape of Gremlin1 and identified a conserved region that recapitulates several aspects of the dynamic expression of Gremlin1 during limb bud development and requires the BMP signal transducer SMAD4 to be expanded in the distal anterior part of the limb bud. To investigate the Gremlin1 cis-regulatory modules that directly interact with SMAD4- containing chromatin complexes, I have generated a new mouse line that expresses a 3xFLAG epitope-tagged version of SMAD4 from its endogenous locus, with which we will perform ChIP-Seq analysis from developing embryonic tissues, including limb buds.
Following a similar rationale, I have used ChIP-Seq data from a mouse line expressing a Hand23xFLAG allele to provide the first identification of the HAND2 target gene networks that function during cardiogenesis, which is severely impaired in the absence of HAND2. This analysis indicates that HAND2 directly controls the expression of the paralogous genes Tbx2 and Tbx3 in the progenitors of the second heart field that give rise to the outflow tract and right ventricle. This regulation is mediated by the interaction of HAND2 with tissue-specific cis-regulatory modules located in Tbx2 and Tbx3 genomic landscapes. Furthermore, we observed in Hand2-deficient embryos that the cells of the atrioventricular canal do not undergo the endothelial-mesenchymal transition that normally initiates the formation of the cardiac cushions, which are the precursors of the cardiac valves. We established that the expression of Snai1, Twist1 and Has2, which are key genes in this process, are directly controlled by HAND2-containing chromatin complexes. Epitope-tagging of transcription factors is thus a powerful tool to identify the range of cis-regulatory modules they bind to, which allows to uncover the transcriptional networks they control during embryonic development.