Genome-wide mapping of the heterochromatin mark H3K9me2 in loss-of-function mutant worms

Histone modification marks such as H3K4me1 and H3K27ac have been used to map putative enhancers, but these marks represent only a fraction of the full repertoire of histone modifications decorating these regulatory regions. In mammals, methylation of histone H3 on lysine 79 (H3K79me), deposited by the methyltransferase DOT1L, is present at enhancers. In C. elegans, putative enhancers have been predicted based on chromatin modifications and regions of chromatin accessibility. Our own overlap analysis has revealed that both DOT-1.1, the C. elegans DOT1L homologue, and its major interacting partner, the chromatin-binding factor ZFP-1 (AF10 in mammals), are enriched at both distal and intragenic enhancers. Previous work has shown that Dot1 proteins prevent encroachment of heterochromatin to actively transcribed regions in yeast and in MLL-rearranged leukemias. Here we find that, in C. elegans, loss of DOT-1.1 is accompanied by ectopic H3K9me2, a hallmark of heterochromatin, at distal and intragenic enhancers. This observation suggests a new role for DOT1L in the control of heterochromatin formation at enhancers, which may partake on the control of developmentally regulated genes. Overall design: Profiling of H3K9me2 in wild-type (WT) and mutant (zfp-1, ced-3, dot-1.1; ced-3, rde-1; dot-1.1, and rde-4; dot-1.1) third larval (L3) stage Caenorhabditis elegans

诸如H3K4me1与H3K27ac之类的组蛋白修饰标记，曾被用于定位潜在增强子，但这类标记仅能覆盖这类调控区域所携带的全套组蛋白修饰中的一小部分。在哺乳动物体内，由甲基转移酶DOT1L催化沉积的组蛋白H3赖氨酸79甲基化修饰（H3K79me），可在增强子区域被检测到。在秀丽隐杆线虫（C. elegans）中，研究人员曾基于染色质修饰与染色质开放区域预测潜在增强子。本研究的重叠分析结果显示，秀丽隐杆线虫的DOT1L同源蛋白DOT-1.1，与其主要互作伴侣、染色质结合因子ZFP-1（哺乳动物中对应AF10），均在远端增强子与基因内增强子区域富集。既往研究表明，在酵母与MLL重排白血病（MLL-rearranged leukemias）中，Dot1家族蛋白可阻止异染色质侵入活跃转录区域。本研究发现，在秀丽隐杆线虫中，DOT-1.1功能缺失会导致远端与基因内增强子区域出现异位的H3K9me2修饰——这是异染色质的标志性修饰。这一发现提示DOT1L在增强子区域的异染色质形成调控中具有全新功能，该功能可能参与发育调控基因的表达调控。整体实验设计：对野生型（WT）及多种突变型[包括zfp-1、ced-3、dot-1.1; ced-3、rde-1; dot-1.1以及rde-4; dot-1.1]的第三龄幼虫（L3）阶段秀丽隐杆线虫（Caenorhabditis elegans）进行H3K9me2的谱型分析。