Human overgrowth syndrome is a class of diseases characterized by systemic or regional excess growth compared to peers of the same age. Recent human genetic studies have revealed a series of missense mutations in epigenetic regulators including a de novo DNA methyltransferase enzyme DNMT3A. It is suggested that cells derived from affected patients would exhibit defects in epigenetic modifications such as DNA methylation and histone modifications, leading to the alteration of specific gene expression. However, the molecular mechanism underlying pathogenesis of overgrowth syndrome is largely unknown. We therefore propose to develop stem cell models that are better suited to understand the molecular basis of DNMT3A mutations in overgrowth syndrome with intellectual disability (coined Tatton-Brown-Rahman Syndrome in OMIM).
In Specific Aim 1, we will generate isogenic human and mouse ESCs (hESCs and mESCs) carrying either wild-type DNMT3 allele or a spectrum of specific DNMT3A point mutations via CRISPR/Cas9 mediated gene editing technology. DNMT3A mutant hESCs and mESCs will be extensively characterized for potential defects in cell cycle regulation, DNA methylation, H3K27me3 and RNA transcriptome in order to determine convergent molecular pathways associated different types of DNMT3A mutations. To directly examine the defects in cell lineages involved in craniofacial development, in Specific Aim 2, we will examine the impact of DNMT3A mutations on sequential differentiation of mutant ESCs into neural precursor cells (NPCs), cortical- like neurons, glial cells, as well as neural crest derivatives. By performing analysis of DNA methylation, H3K27me3, and RNA transcriptome at different stage of cell differentiation, we will define shared molecular changes and regulatory pathways in RNA transcriptome and DNA methylation that underlie pathogenesis of craniofacial and brain development in vitro.
In Aim 3, we will generate transgenic mice carrying DNMT3A mutations, and examine mouse mutant phenotypes relevant to overgrowth. Molecular characterization of brain neurogenesis and craniofacial development in vivo in mutant mice will lead to the identification of either novel or known signaling pathways (such as PTEN/mTOR/IGF signaling) associated with overgrowth phenotype. Moreover, we will determine whether the defects of cell growth, proliferation, and differentiation in vivo will be consistent with what we observed in vitro in both human and mouse stem cell differentiation model in Aim 2. Finally, we will also perform learning and memory behavioral tests to determine the association of DNMT3A mutations with potential learning and memory deficits. Our proposed research will provide a novel approach to understanding the molecular pathogenesis of human DNMT3A overgrowth syndrome, potentially leading to the development of a therapeutic approach to prevent or cure human overgrowth disorders.
The proposed research is studying human disease mechanism underlying overgrowth syndromes. Our research project will be beneficial to design novel therapeutic approach to treat human diseases associated with dysfunction of DNA methylation, histone modifications and gene regulation.
|King, Andrew D; Huang, Kevin; Rubbi, Liudmilla et al. (2016) Reversible Regulation of Promoter and Enhancer Histone Landscape by DNA Methylation in Mouse Embryonic Stem Cells. Cell Rep 17:289-302|