Homeostatic control of intestinal stem cell renewal by two transcriptional coactivators

PROJECT SUMMARY Mechanisms that integrate local and systemic cues to precisely regulate stem-cell based tissue renewal remain unclear, particularly in the rapidly cycling gastrointestinal (GI) epithelium. A pair of transcriptional coactivator proteins that promote growth and intestinal stem cell renewal – the Hippo pathway protein YAP1 and the nuclear

receptor (NR) interactor NCOA3 – are each implicated in the progression of colorectal cancers. Both of these factors have orthologs in the common fruit fly, D. melanogaster, which combines robust fertility, rapid generation time, a relatively simple genome, and an extensive genetic toolbox to make this system a powerful model to

investigate gut renewal mechanisms. Components of the mammalian Hippo and NR pathways have homologs in D. melanogaster, including the nuclear coactivator proteins Yorkie (Yki; YAP1 in humans) and Taiman (Tai; nuclear receptor coactivator-3, NCOA3 in humans). Prior studies established that (i) Yki and Tai physically

interact through respective WW (tryptophan-tryptophan) domains and PPxY (proline-proline-x-tyrosine) motifs, (ii) Tai modulates Yki-driven overgrowth of fly larval epithelia, and (iii) Tai and Yki each play a role in intestinal stem cell (ISC) proliferation. Based on this and other evidence, we hypothesize that the cooperativity of the

Tai/NR and Yki/Hippo complex regulates ISC renewal by activating a unique set of target genes distinct from canonical Tai/NR or Yki/Hippo targets. We generated a critical tool to test this hypothesis: a CRISPR mutant fly stock in which the PPxY motifs of endogenous Tai are converted to PPxA, blocking Tai’s binding to Yki. These

viable homozygous mutants (taiPPxA/taiPPxA) allow precise and selective decoupling of the Tai/NR and Yki/Hippo pathways in vivo. This proposal will investigate how Hippo and NR signaling converge on the Yki-Tai interaction to activate transcription of genes required for gut renewal using D. melanogaster midgut as a model via the

following specific aims: Aim 1 will define the distribution of intestinal cell types and mitotic activity in taiPPxA/taiPPxA mutant and control flies following acute intestinal injury; Aim 2 will define effects of taiPPxA on gene expression and chromatin accessibility during intestinal renewal to identify Yki-Tai target genes that are activated during

injury induced ISC renewal. The findings of this study will provide insight into how two systemic signaling pathways, Hippo and NR, converge on their downstream transcriptional targets, Yki/YAP1 and Tai/NCOA3, to regulate local ISC-dependent tissue renewal.