Cubitt, R. L. | Search bioRxiv

bioRxiv2022

Integrative genome-scale analyses reveal post-transcriptional signatures of early human smallintestinal development in a directed differentiation organoid model

MicroRNAs (miRNAs) are important post-transcriptional gene regulators in organ development. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage. We leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Our analyses showed that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/499825v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ebf48eorg.highwire.dtl.DTLVardef@4a0d9org.highwire.dtl.DTLVardef@1dc3be9org.highwire.dtl.DTLVardef@1e14207_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2020

Single Cell Analysis Reveals Multi-faceted miR-375 Regulation of the Intestinal Crypt

The role of individual miRNAs in small intestinal (SI) epithelial homeostasis is under-explored. In this study, we discovered that miR-375 is among the most enriched miRNAs in intestinal crypts and stem cells (ISCs), especially facultative ISCs. We then showed by multiple manipulations, including CRISPR/Cas9 editing, that miR-375 is strongly suppressed by Wnt-signaling. Single-cell RNA-seq analysis of SI crypt-enriched cells from miR-375 knockout (375-KO) mice revealed elevated numbers of tuft cells and increased expression of pro-proliferative genes in ISCs. Accordingly, the genetic loss of miR-375 promoted resistance to helminth infection and enhanced the regenerative response to irradiation. The conserved effects of miR-375 were confirmed by gain-of-function studies in Drosophila midgut stem cells in vivo. Moreover, functional experiments in enteroids uncovered a regulatory relationship between miR-375 and Yap1 that controls cell survival. Finally, analysis of mouse model and clinical data revealed an inverse association between miR-375 levels and intestinal tumor development. HighlightsO_LImiR-375 is one of the most enriched miRNAs in ISCs, especially facultative ISCs. C_LIO_LImiR-375 modifies tuft cell abundance and pro-proliferative gene expression in ISCs. C_LIO_LILoss of miR-375 in mice enhances the host response to helminth infection and crypt regeneration. C_LIO_LIMouse and human intestinal cancer are associated with reduced miR-375 expression. C_LI eTOC BlurbSethupathy and colleagues show that miR-375 is a Wnt-responsive, ISC-enriched miRNA that serves as a break on intestinal crypt proliferation. They also show that miR-375 modulates tuft cell abundance and pro-proliferative gene expression in ISCs, that miR-375 loss enhances the host response to helminth infection as well as crypt regeneration post-irradiation, and its reduced expression is associated with intestinal cancer.

molecular biology↗