Cytokine-sensing primary enhancer cluster launches secondary enhancers to activate multi-gene locus [RNA-seq]

Enhancer clusters are platforms occupied at high-density by the transcription machinery to activate lineage-specific genes. However, it is not clear yet whether they are merely a collection of conventional enhancers or represent unique and new types of regulatory elements. Specifically, the question remains whether they activate additional enhancer programs in multigene loci. We addressed this issue in the mouse mammary gland, where genes are highly activated during pregnancy. We identify a 300kb multi-gene locus with twenty-one regulatory elements, including a cytokine-dependent primary enhancer cluster (PEC). Using genome editing in combination with ChIP-seq, RNA-seq and 3C-technologies, we show that the PEC initiates the activation of lineage-specific genes through contacting and activating secondary enhancers. This new framework of enhancer clusters indicates a hitherto unrecognized hierarchy of regulation by which a PEC activates genes via regulating secondary enhancers. Overall design: Total RNA-seq was done for mammary tissues at pregnancy day 6 and 18 for wild-type and mutant mice carrying enhancer deletions within the Casein locus. One mouse lines carries a deletion of the enhancer cluster identified next to the primordial Odam within (Csn_S1-4). The other two mouse lines carry deletions of single enhancer elements: delCsn3-PE - deletion of the proximal enhancer of Csn3 and delCsn1s2b-E - a deletion of the enhancer of Csn1s2b.

增强子簇（enhancer cluster）是转录机器高密度占据的调控平台，其功能为激活谱系特异性基因。然而目前尚不清楚，增强子簇究竟仅是常规增强子的集合，还是代表一类独特的新型调控元件。具体而言，仍有待解答的核心问题是：增强子簇是否会在多基因位点中激活额外的增强子调控程序。我们在妊娠期间基因表达被高度激活的小鼠乳腺组织中开展了相关研究，以解答这一科学问题。我们鉴定出一个长度为300kb的多基因位点，共包含21个调控元件，其中包括一个细胞因子依赖性初级增强子簇（cytokine-dependent primary enhancer cluster，缩写PEC）。我们结合基因组编辑技术与染色质免疫沉淀测序（ChIP-seq）、全转录组RNA测序（Total RNA-seq）及染色质构象捕获（3C）技术开展实验，结果表明：PEC可通过接触并激活次级增强子，启动谱系特异性基因的表达激活过程。这一增强子簇的全新调控框架揭示了一种此前未被认知的调控层级：PEC可通过调控次级增强子来实现基因激活。实验设计总览：针对妊娠第6天和第18天的小鼠乳腺组织开展了全转录组RNA测序，实验对象涵盖野生型小鼠，以及在酪蛋白基因座（Casein locus）内存在增强子缺失的突变型小鼠。其中一个小鼠品系存在已鉴定出的、位于Csn_S1-4区域内的原始Odam旁侧的增强子簇缺失。另外两个小鼠品系则携带单个增强子元件的缺失：delCsn3-PE指Csn3的近端增强子缺失，delCsn1s2b-E指Csn1s2b的增强子缺失。