Exploring Six2 distal enhancer interactome in developing mouse kidney. Exploring Six2 distal enhancer interactome in developing mouse kidney

The Six2 distal enhancer regulates expression of the 60 kb-downstream gene Six2, but does not regulate the Six3 gene which is 70 kb further downstream. CTCF ChIP-Seq and Hi-C data points to a chromatin domain boundary between Six2 and Six3 which may intervene interaction between Six2-DE and Six3. The irradiation-induced Brachyrrhine (Br) mutant allele was found to carry a 320 kb genomic inversion that includes Six2 and Six3, but not Six2DE. This repositioned Six2 under Six3 enhancer control and Six3 under Six2 distal enhancer control. Consistent with this, Six3 is ectopically expressed in Br/+ kidneys and Six2 expression is reduced. To test whether there is a change of interaction between Six2 distal enhancer and Six2 or Six3 promoters, we performed 4C-Seq in E16.5 wildtyp or Br/+ mice nephron-genic zone cells. Overall design: The nephron genic zone cells from E16.5 wildtype or Br/+ kidneys were dissociated following Brown et al., 2015 and subject to 4C-Seq using Dpn II and NlaIII as primary and secondary digest enzyme, with Six2DE as view point.

Six2远端增强子（Six2 distal enhancer）可调控其下游60kb处的Six2基因表达，但不会调控再下游70kb处的Six3基因。CTCF染色质免疫共沉淀测序（ChIP-Seq）与Hi-C染色质构象捕获数据显示，Six2与Six3之间存在一处染色质结构域边界，该边界可能干扰Six2远端增强子与Six3之间的染色质相互作用。研究人员发现，辐射诱导的短鼻（Brachyrrhine, Br）突变等位基因携带一段长度为320kb的基因组倒位，该倒位涵盖Six2与Six3，但不包含Six2远端增强子。此次倒位将Six2置于Six3增强子的调控范围内，同时将Six3置于Six2远端增强子的调控范围内。与此一致的是，Br/+杂合子小鼠的肾脏中出现了Six3的异位表达，而Six2的表达水平则出现下调。为验证Six2远端增强子与Six2或Six3启动子之间的相互作用是否发生改变，研究人员在胚胎第16.5天（E16.5）的野生型或Br/+小鼠肾发生区细胞中开展了4C测序（4C-Seq）实验。实验设计概述：参照Brown等人2015年的方法，解离E16.5天野生型或Br/+小鼠肾脏中的肾发生区细胞，以Dpn II和NlaIII分别作为第一、第二轮限制性内切酶，并以Six2DE作为锚定视角位点开展4C测序。