Hubrecht Institute and University Medical Centre Utrecht, Utrecht, The Netherlands.
PLoS Biol. 2010 Nov 16;8(11):e1000539. doi: 10.1371/journal.pbio.1000539.
Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/β-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/β-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and β-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. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/β-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a β-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 to a large extent dedicated to Wnt target gene regulation. In contrast, previously published β-catenin coactivators p300 and BRG1 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. We conclude that Mllt10/Af10-Dot1l are essential, largely 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.
Wnt 信号通过诱导核 TCF4/β-连环蛋白复合物的形成来维持肠隐窝祖细胞的未分化状态。在结直肠癌中，Wnt 通路成分的激活突变导致 TCF4/β-连环蛋白驱动的转录的不适当激活。尽管在发现 TCF4 和 β-连环蛋白作为 Wnt 信号的分子效应物之后已经过去了十年，但很少发现对这种转录程序的调节必不可少且独特的转录激活因子。使用蛋白质组学，我们在小鼠小肠隐窝中鉴定出与白血病相关的 Mllt10/Af10 和甲基转移酶 Dot1l 作为 Tcf4/β-连环蛋白的相互作用物。对于转录延伸至关重要的 Mllt10/Af10-Dot1l 以 β-连环蛋白依赖性方式被募集到 Wnt 靶基因，导致体内在增殖性隐窝中在其编码区域上的 H3K79 甲基化小鼠小肠中的结直肠癌和 Wnt 诱导的 HEK293T 细胞。在结直肠癌和 Wnt 诱导的 HEK293T 细胞中耗尽 MLLT10/AF10 后进行表达谱分析，确定 MLLT10/AF10 和 DOT1L 在很大程度上是调节 Wnt 靶基因所必需的激活因子。相比之下，以前发表的 β-连环蛋白共激活因子 p300 和 BRG1 显示出更具多效性的靶基因表达谱，可控制 Wnt 和其他途径。tcf4、mllt10/af10 和 dot1l 在斑马鱼的 Wnt 驱动组织中共同表达，并且对 Wnt 报告基因活性是必需的。在 APC 突变的斑马鱼中，肠道分化缺陷可以通过耗尽 Mllt10 和 Dot1l 来挽救，这表明这些基因在体内 APC 下游是 Wnt 靶基因激活中的激活因子。在斑马鱼中使用 morpholino 耗尽 mllt10/af10-dot1l 会导致肠道内稳态缺陷，体内直接 Wnt 靶基因的表达和增殖性肠道上皮细胞的数量显著减少。我们得出结论，Mllt10/Af10-Dot1l 是 Wnt 依赖性转录的必需、主要专用激活因子，对于维持肠道增殖和内稳态至关重要。甲基转移酶 DOT1L 可能是结直肠癌药物靶向的有吸引力的候选物。
