Loss of Apc impairs Lgr5+ intestinal stem cell fate programs [RNA-seq]. Loss of Apc impairs Lgr5+ intestinal stem cell fate programs [RNA-seq]

Loss of APC is the main driving alteration associated with colorectal cancer (CRC) development. We investigate the immediate outcome of this genetic event on the transcriptomic and DNA methylation profiles of Lgr5+ intestinal stem cells (ISCs), considered as the cell-of-origin of CRC. RNA-seq analyses show that the sequential deletion of Apc alleles has a rapid impact on the transcriptional profiles of ISCs. Indeed, Apc loss-of-function dictates an altered cell fate program at transcriptional level, resuting in an impaired commitment of those cells to differentiation. Reduced-representation bisufite sequencing (RRBS) on the genomic DNA (gDNA) also shows that focal alterations occur in the DNA methylations profiles of ISCs at this stage, and functionally contribute to altered cell fate in those cells. This study sheds light on the earliest events in CRC development, proving evidences on the role exerted by DNA methylation changes associated with Apc inactivation in the homeostatic rupture at CRC initation. Overall design: Lgr5+-GFP+ Intestinal stem cells were FACS-sorted from 4 Apc+/+ : , 4 ApcFlox/+, : and 4 ApcFlox/Flox : Lgr5-CreERT2-ires-eGFP mice 15 days after the activation of the Cre recombinase by tamoxifen administration to study the impact of the mutation on gene expression and DNA methylation profiles.

腺瘤性结肠息肉病蛋白（APC）缺失是结直肠癌（CRC）发生的主要驱动性遗传改变。本研究聚焦该遗传事件对Lgr5阳性肠道干细胞（ISCs）的转录组与DNA甲基化谱的即时影响——这类细胞被认为是结直肠癌的起源细胞。RNA测序（RNA-seq）分析显示，Apc等位基因的序贯缺失会快速改变肠道干细胞的转录组谱。实际上，Apc功能缺失会在转录层面调控异常的细胞命运程序，导致这些干细胞的分化潜能受损。基因组DNA（gDNA）的简化代表性亚硫酸氢盐测序（RRBS）结果同样显示，该阶段肠道干细胞的DNA甲基化谱存在局灶性改变，且这些改变会功能性地导致细胞命运异常。本研究阐明了结直肠癌发生的早期事件，为Apc失活相关的DNA甲基化改变在结直肠癌起始阶段的稳态失衡中所发挥的作用提供了证据。实验整体设计：通过他莫昔芬诱导激活Cre重组酶15天后，从4只Apc+/+型、4只ApcFlox/+型以及4只ApcFlox/Flox型Lgr5-CreERT2-ires-eGFP小鼠中，通过荧光激活细胞分选（FACS）分离得到Lgr5阳性-绿色荧光蛋白阳性（Lgr5+-GFP+）肠道干细胞，以此研究该突变对基因表达与DNA甲基化谱的影响。