Critical Role of Mybl2 in Spontaneous and Short-Chain Fatty Acid-Induced Intestin

Montefiore Medical Center (Bronx, Ny), New York NY

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Abstract

DESCRIPTION (from the applicant): Colonic epithelial cell differentiation is driven by reprogramming of stem and/or progenitor cells as they migrate upward along the crypt axis. This process is recapitulated in immortalized cell lines derived from human colon adenocarcinomas that proliferate in an immature state in culture yet can be induced by the short chain fatty acid (SCFA) butyrate (a product of dietary fiber fermentation in the human colon), or contact inhibition of growth to differentiate into absorptive (Caco-2), goblet (HT29 Cl16E), or secretory (HT29 Cl19A) colonic epithelial cells. Our group has provided key insights into mechanisms that drive this maturation by characterizing the gene expression profiles of these cell lineages as they differentiate. To identify genes for which altered expression is fundamental to the reprogramming of intestinal epithelial cells as they undergo maturation induced by physiological regulators such as butyrate, we focused on sequences that are consistently downregulated in expression during growth arrest and lineage specific differentiation of colonic epithelial cells in vivo. Suppression of one such gene, Mybl2, a molecule that has been suggested to regulate critical proliferation and differentiation decisions in stem cells and other cell types, by siRNA in proliferating colon epithelial cells induces expression of a subset of differentiation-specific genes. The aims of this proposal focus on determining whether Mybl2 modulates colon epithelial cell reprogramming, including that induced by the SCFA butyrate, by exerting parallel control over proliferation and differentiation pathways and elucidating how Mybl2 is itself regulated during colon cell maturation. Specific aims 1 and 2 will determine whether Mybl2 suppression can promote, or its overexpression can prevent, colon cell maturation through the coordinate regulation of proliferation and differentiation-specific genes. Specific aim #1 pursues our identification of a subset of genes, involved in promoting or preventing colon cell maturation, that is altered upon Mybl2 knockdown or overexpression, and specific aim #2 will reveal which gene promoters are bound and regulated by Mybl2 in maturing colon cells. Although transcriptional activation and repression account for many differentiation-specific gene expression changes, post-transcriptional regulation by microRNAs (miRNAs) provides another critical level of gene modulation during colon cell differentiation. We have found that the Mybl2 promoter is not significantly downregulated in differentiating colon epithelial cells and have therefore hypothesized that miRNAs may regulate its expression during maturation. We have utilized novel bioinformatic techniques to identify potential miRNA targets in the Mybl2 3'untranslated region (UTR). These potential targets: 1) are predicted by computer analysis to bind one or more known miRNAs and 2) form significant RNA secondary structure as indicated by a predicted free energy <0. These candidate miRNA target regions therefore have the potential to bind miRNA's that regulate fundamental mechanisms of differentiation in all three colon cell lineages. This target-centered approach will be used to identify specific miRNAs and their targets that regulate Mybl2 expression during colon cell differentiation. This approach, rather than a screening of expression of known miRNAs, will isolate and identify known and as well as unidentified miRNAs of biological relevance to colonic cell differentiation. Therefore, aim 3 will first determine whether predicted miRNA targets in the Mybl2 3'UTR are functional by assaying their ability to suppress luciferase expression when inserted into the 3'UTR of luciferase in differentiating colonic epithelial cells, as already indicated by preliminary data for one such predicted miRNA target. Functional targets will then be immobilized on streptavidin/agarose to physically capture miRNAs (known or novel) from small RNAs of differentiating colonic epithelial cells. The proposed studies will therefore reveal fundamental mechanisms utilized by several colon epithelial cell lineages to regulate the decision to proliferate or differentiate.