Unknown,Transcriptomics,Genomics,Proteomics

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Single cell RNA-sequencing experiment of human embryonic stem cells (hESCs) upon Wnt stimulation treatment at three time points (4 hours, 24 hours and 3 days)

ABSTRACT: Wnt/b-catenin signalling is an evolutionarily conserved mechanism for cell-cell communication implicated in both developmental processes and diseases such as cancer. A main part of this signalling pathway is the stabilization and nuclear translocation of b-catenin, driving the transcriptional regulation of target genes. However, while b-catenin target genes traditionally have been through to be collectively regulated upon Wnt pathway stimulation, this appears in contrast with the non-overlapping patterns of Wnt target gene expression in several contexts, including early mammalian embryogenesis. To address the question whether individual cells exhibits diverse responses to Wnt stimulation, we followed Wnt target gene activation in human embryonic stem cells (hESCs) via single cell RNA-sequencing (scRNAseq) after GSK3 inhibition at 4, 24 and 72 hours of stimulation.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Claudio Cantù

PROVIDER: E-MTAB-12598 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/beta-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/beta-catenin -driven transcription. Despite the passage of a decade after the discovery of TCF4 and beta-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Methodology/Principal Findings: Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l, as Tcf4/beta-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a beta-catenin -dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine, in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators dedicated to Wnt target gene regulation. In contrast, previously published b-catenin coactivators p300 and beta-catenin displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10 and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. Conclusions/Significance: We conclude that Mllt10/Af10-Dot1l are essential, dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase Dot1l may present an attractive candidate for drug targeting in colorectal cancer. 6 samples for Ls174T cells: si-b-catenin against si-control and dyeswap of it, si-control, si-MLLT10, si-BRG1 and si-P300 are hybridized against common reference RNA; 6 samples of HEK293T cells: Wnt3A or control medium (CM) induction for 9 hours, si-MLLT10, si-DOT1L, si-BRG1 and si-P300 upon 9 hour Wnt3A induction are all hybridized against common reference RNA

Dataset Information

Single cell RNA-sequencing experiment of human embryonic stem cells (hESCs) upon Wnt stimulation treatment at three time points (4 hours, 24 hours and 3 days)

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