The mammalian intestine is an ideal model system to study the molecular events that control cell fate specification during development and adult homeostasis. During perinatal development of the intestinal epithelium, proliferative intervillus domains invaginate into the underlying mesenchyme to produce crypts of Lieberkhn. Concomitant with crypt formation is the emergence of mature Lgr5-expressing intestinal stem cells (ISCs), which situate at base of these crypts and divide daily during adult homeostasis to self-renew and produce committed progenitor cells. As progenitor cells migrate up along the crypt walls, they divide further and undergo progressive differentiation. Upon exiting the crypt and entering villus projections, progenitor cells complete terminal differentiation and produce mature absorptive and secretory cells. Despite advances in identifying the transcriptional and molecular mechanisms that facilitate ISC development and differentiation, the epigenetic mechanisms directing these processes remain poorly characterized. The proposed study will therefore examine the role of UTX and JMJD3, two histone demethylases, in the intestinal epithelium. UTX and JMJD3 demethylate trimethylated histone 3 lysine 27 (H3K27me3), a histone modification that is required for gene silencing. By removing this repressive histone mark, UTX and JMJD3 de-repress lineage defining transcription factors to promote differentiation during development. Furthermore, UTX and JMJD3 facilitate differentiation in other regenerative adult tissues and are therefore critical for maintaining adult tissue homeostasis. Whether UTX and JMJD3 are important epigenetic regulators of differentiation in the intestinal epithelium remains unknown.
Aim 1 will test the hypothesis that UTX- and JMJD3-mediated H3K27me3 demethylation activates a mature stem cell transcriptional program in fetal progenitor cells and is therefore required for perinatal intestinal development. A mouse model of constitutive, intestine-specific Utx and Jmjd3 ablation will be employed to determine whether UTX and JMJD3 promote perinatal crypt formation and ISC development. To validate and extend in vivo results, an in vitro organoid formation assay will identify whether UTX and JMJD3 are required for the maturation of fetal intestinal spheroids into mature budding organoids. Furthermore, single-cell RNA-Sequencing (scRNA-Seq) and ChIP-Seq will be used to identify transcriptional and epigenomic changes induced in the perinatal intestine following UTX and JMJD3 ablation.
Aim 2 will test the hypothesis that UTX- and JMJD3-mediated H3K27me3 demethylation is required to activate differentiation genes and maintain proper differentiation of adult intestinal epithelial cells. Utx and Jmjd3 expression will be conditionally ablated within the intestinal epithelium of adult mice. These mice will be analyzed by immunohistochemistry, scRNA-Seq, and ChIP-Seq to determine whether the differentiation program is maintained at both the cellular and genomic levels. Together, these studies will provide novel insights into an important epigenetic mechanism directing intestinal differentiation during development and homeostasis.
Failure of intestinal stem and progenitor cells to correctly differentiate during development or in adult homeostasis results in pathological conditions such as colorectal cancer. Though changes in the chromatin landscape are critical for specifying cell fate, our understanding of the precise epigenetic mechanisms that function in the developing and adult intestine remains limited. The proposed study will address this critical knowledge gap by elucidating the role of UTX and JMJD3, two key H3K27me3 demethylases, in determining cell fate in the perinatal and adult intestine.
