A TAD boundary at the HoxD locus segregates opposing limb regulatory landscapes and their target genes [Hi-C]

The mammalian HoxD cluster is positioned at the boundary between two topologically associating domains (TADs), each of them matching a distinct, enhancer-rich regulatory landscape. During limb development, the telomeric TAD controls the early phase of Hoxd gene transcription in future forearm cells, whereas the centromeric TAD subsequently regulates transcription of more posterior Hoxd genes in presumptive digit cells. The TAD boundary is essential as it prevents the terminal Hoxd13 gene to respond to potent forearm enhancers, thereby allowing the formation of proper proximal limb structures. Here we apply chromosome conformation capture onto embryonic proximal and distal limb bud cells micro-dissected from a set of nested deletions involving part- or all- of this boundary region to try to understand the nature and function of this CTCF- and cohesin-rich DNA region. We document a progressive release of the boundary effect, allowing for inter-TAD contacts to be established, which were favoured by the functional status of the newly accessed enhancers. However, the boundary was highly resilient and only a 400kb large deletion including the whole gene cluster was eventually able to merge the two neighbouring TADs into a single structure. We propose that the whole HoxD cluster is a dynamic transcriptional boundary, showing slight variations depending on both the transcriptional status and the ontogenetic context Overall design: Hi-C datasets of E12.5 limb tissues (distal or proximal forelimbs and hindlimbs) of Wt or serial HoxD-deleted alleles -delHoxd(1-13d9)lac and delHoxd(attP-Rel5)d9lac as well as the reanalysis of the CH12 dataset from Rao et al. 2014 (GSE63525, GSM15516333 to GSM15516347 = SRR1658716 to SRR1658730) and mESC dataset from Dixon et al. 2012 (GSE35156, GSM862720 = SRR443883 to SRR443885 and GSM862721 = SRR400251 to SRR400256) on mm10.

哺乳动物的HoxD基因簇定位于两个拓扑关联结构域（topologically associating domains, TADs）的边界处，每个TAD对应一套独特且富含增强子的调控格局。在肢体发育过程中，端粒侧TAD负责调控未来前臂细胞中Hoxd基因转录的早期阶段，而着丝粒侧TAD随后在预定指细胞中调控更靠后位置的Hoxd基因转录。TAD边界至关重要，它可阻止末端Hoxd13基因响应强效的前臂增强子，从而保障正确的肢体近端结构形成。本研究针对一系列携带有该边界区域部分或全部片段缺失的嵌套突变体，对其显微切割得到的胚胎肢体近端与远端肢芽细胞开展染色体构象捕获实验，以期阐明该富含CTCF与黏连蛋白（cohesin）的DNA区域的本质与功能。我们观测到边界效应呈渐进式解除，使得跨TAD接触得以建立，且此类接触的形成偏好于新接触到的增强子的功能状态。然而该TAD边界具有极强的弹性，仅当包含整个基因簇的400kb大片段缺失时，才最终将两个相邻TAD融合为单一结构域。我们提出，完整的HoxD基因簇属于动态转录边界，其功能会随转录状态与发育上下文发生细微变化。
整体实验设计：本研究使用的Hi-C数据集来自E12.5阶段的野生型（wild type, Wt）或系列HoxD缺失等位基因（包括delHoxd(1-13d9)lac与delHoxd(attP-Rel5)d9lac）的肢体组织（远端/近端前肢与后肢）；同时重新分析了Rao等于2014年发布的CH12数据集（GSE63525，GSM15516333至GSM15516347 = SRR1658716至SRR1658730），以及Dixon等于2012年发布的小鼠胚胎干细胞（mouse embryonic stem cell, mESC）数据集（GSE35156，GSM862720 = SRR443883至SRR443885，GSM862721 = SRR400251至SRR400256），所有分析均基于mm10参考基因组。