Genetic co-regulation by master transcription factors in Drosophila intestinal stem cells

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Project Summary Many of our organs remain healthy and functional because they replace cells that are lost to injury or disease with new ones, through divisions of adult stem cells contained within them. Adult stem cells divide asymmetrically, generating a new copy of themselves (self-renew) and another cell that will eventually “differentiate” into a cell with specialized function (e.g. absorb nutrients, fight infections, produce a hormone, etc). While there has been great progress in identifying some of the so-called “master regulator genes” that control a stem cell’s decision between self-renewal or differentiation, we know comparatively much less about the specific genetic programs triggered by master regulators in the process. This is in spite of the many efforts to identify targets of master regulators through genome-wide screening technologies, which frequently fall in disuse due to a lack of further validation. Here I propose a research project that simultaneously addresses both problems, by asking… “Can we more efficiently identify critical stem cell genes by integrating public lists of putative master regulator targets obtained in complementary but unrelated studies?” To address the above question, I propose to use the Drosophila melanogaster (fruit fly) midgut as an in vivo experimental model. Previous studies have generated independent lists of putative targets for three known master regulators of stem cells in the Drosophila intestine (Escargot, Capicua and Stat92E). We used bioinformatics to compare these lists, and identified genes that are putatively targeted by only one or all three master regulators, which we refer to as “mono-“ or “co-regulated”, respectively. In Aim 1 of this proposal, we will inhibit each of the master regulators using RNAi-mediated protein knockdown in intestinal progenitors, and validate mono- and co-regulated candidates based on changes to their expression as determined by reverse transcription quantitative PCR (RT-qPCR). In Aim 2, we will compare the effects on the maintenance, proliferation rate and differentiation potential of intestinal progenitors following the RNAi-mediated knockdown of mono- or co- regulated candidates validated in Aim 1. This approach will allow us to test the hypothesis that co-regulated genes are more frequently critical for the biology of the intestinal stem cells than mono-regulated counterparts. Finally, our preliminary observations have shown that inhibiting Esg function leads to a reduction in Stat92E activity, and here I propose to test bioinformatic predictions about regulatory connections between Esg and Stat92E generated by incorporating yet another database of genetic and protein interaction data in. If successful, this project will: a) exploit Drosophila as a prime genetics model system to generate a host of new research leads for understanding and treating gastrointestinal diseases; b) provide a proof of concept to encourage and guide the use of valuable information that is currently deposited in public databases and yet rarely used by the research community.