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Histone 4 Lys-20 Mono-Methylation by SET8 Mediates Wnt Target Gene Activation

ABSTRACT: Histone methylation has an important role in transcriptional regulation. However, unlike H3K4 and H3K9 methylation, the role of H4K20 mono-methylation (H4K20me-1) in transcriptional regulation remains unclear. Here we show that Wnt3a specifically stimulates H4K20 mono-methylation at TBE (TCF-binding element) via the histone methylase SET8. Additionally, SET8 is crucial for activation of the Wnt reporter gene and target genes in both mammalian cells and zebrafish. Furthermore, SET8 interacts with TCF4/LEF1 directly, and this interaction is regulated by Wnt3a. Therefore, we conclude that SET8 is a Wnt signaling mediator and is recruited by LEF1/TCF4 to regulate the transcription of Wnt-activated genes, possibly via H4K20 mono-methylation at the target gene promoters. Our findings also indicate that H4K20me-1 is a marker for gene transcription activation, at least in canonical Wnt signaling. This chip experiment was carried out to find out how many genes would be regulated by SET8 after Wnt3a CM stimulated HEK293 cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE24708 | GEO | 2010/12/29

SECONDARY ACCESSION(S): PRJNA132325

REPOSITORIES: GEO

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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

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Histone 4 Lys-20 Mono-Methylation by SET8 Mediates Wnt Target Gene Activation

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