Whole-genome gene expression level changes in P19.6 mouse embryonal carcinoma cells compared to P19.6 cells treated for 48 hours with all-trans retinoic acid

Enhancers are developmentally-controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. Here, we show by genome-wide mapping that the newly discovered DNA modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells as well as during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates like Meis1 in P19 cells and PPARgamma in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5mC hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes A 6-chip study aiming to characterize regulated genes in P19.6 mouse embryonal carcinoma cells following 48 hours treatment with 1µM all-trans retinoic acid. RNAs were prepared from three independent triplicate experiments.

增强子（enhancer）是受发育程序调控的转录调控区域，其活性可通过组蛋白修饰或组蛋白变体沉积进行调控。本研究通过全基因组图谱分析发现，新发现的DNA修饰产物5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC）在小鼠P19细胞神经分化及小鼠3T3-L1细胞脂肪分化过程中，与转录因子结合远端调控位点呈现动态关联。功能注释结果显示，获得5hmC修饰的区域，在P19细胞神经分化阶段与神经组织特异性表达基因相关联，在3T3-L1细胞脂肪分化阶段则与脂肪组织特异性表达基因相关联。此外，在P19细胞中同时获得5hmC修饰与H3K4me2（组蛋白H3赖氨酸4二甲基化）、H3K27ac（组蛋白H3赖氨酸27乙酰化）的远端区域，可作为依赖于分化进程的转录增强子发挥功能。所鉴定的区域富集了调控特定细胞命运的转录因子结合基序，例如P19细胞中的Meis1以及3T3-L1细胞中的PPARγ（过氧化物酶体增殖物激活受体γ, PPARgamma）。据此，部分携带羟甲基化修饰的Meis1结合位点与5mC羟化酶Tet1的动态招募相关联。此外，对选定增强子的胞嘧啶羟甲基化动力学研究表明，DNA羟甲基化是增强子激活的早期事件。因此，在细胞特异性远端调控区域中获得5hmC修饰，可能是增强子向激活状态成熟的关键事件，并参与组织特异性基因的选择性激活。本研究包含一项6芯片（6-chip）实验，旨在表征经1μM全反式维甲酸（all-trans retinoic acid）处理48小时后的小鼠P19.6胚胎癌细胞中的调控基因特征；实验所用RNA样本取自3组独立的重复实验（每组设置3次生物学重复）。