Understanding the role of the HDAC3-NCoR1/2 complex in embryogenesis using embryonic stem cells

Nuclear Receptor Corepressor 1 (NCoR1) and Nuclear Receptor Corepressor 2 (NCoR2) are large scaffolding proteins. As a scaffolding protein, they have multiple domains, including a deacetylase activating domain (DAD) that interacts with histone deacetylase 3 (HDAC3). HDAC3 is an enzyme that functions to deacetylate histone tails, leading to gene repression. NCoR1/2 also contains multiple nuclear receptor interacting domains to assist nuclear receptors in the downregulation of gene expression. Germline deletion of NCoR1, NCoR2 and HDAC3 in mice were lethal at e15.5, e16.5 and e9.5, respectively. This, therefore, demonstrates that they are crucial in embryogenesis. To study their roles in the embryo, we used Ncor1/2 double knockout (DKO) and HDAC3 KO mouse embryonic stem cells (mESCs) as model systems. Using RNA-seq, we discovered that Ncor1/2 DKO ESCs showed upregulated Hox genes, including Cdx1, Pbx1 and Meis2. This indicates that Ncor1/2 are essential in repressing Hox gene expression in ESCs, thus preventing uncontrolled differentiation. Additionally, the Cowley lab previously showed that HDAC3 KO embryoid bodies displayed an increased endodermal marker expression, such as in Gata6, Foxa2 and Sox17. In this thesis, we utilised a novel model of the developing embryo called gastruloids. Gastruloids are 3D organoid models containing the three germ layers (ectoderm, mesoderm, and endoderm). They also display organised anterior-posterior, left-right, and dorsal-ventral body axes. Using HDAC3 KO ESCs to generate gastruloids, we also observed increased endodermal gene expression. Moreover, many of the upregulated genes were also involved in heart development. Furthermore, Hox cluster genes were downregulated in the HDAC3 KO gastruloids, suggesting that HDAC3 modulates embryogenesis by regulating the expression of endoderm, Hox, and heart developmental genes. Hence, in this study, we have identified some of the causes of mouse embryonic lethality, which were contributed to by the absence of HDAC3 and NCoR1/2.