The spatial organization of the mammalian genome relies upon the formation of chromatin domains of various scales. At the level of gene regulation in cis, collections of enhancer sequences define large regulatory landscapes that usually match with the presence of topologically associating domains (TADs). These domains often contain ranges of enhancers displaying similar or related tissue specificity, suggesting that in some cases, such domains may act as coherent regulatory units, with a global on or off state. By using the HoxD gene cluster, which specifies the topology of the developing limbs via highly orchestrated regulation of gene expression, as a paradigm, we investigated how the arrangement of regulatory domains determines their activity and function. Proximal and distal cells in the developing limb express different levels of Hoxd genes, regulated by flanking 3′ and 5′ TADs, respectively. We characterized the effect of large genomic rearrangements affecting these two TADs, including their fusion into a single chromatin domain. We show that, within a single hybrid TAD, the activation of both proximal and distal limb enhancers globally occurred as when both TADs are intact. However, the activity of the 3′ TAD in distal cells is generally increased in the fused TAD, when compared to wild type where it is silenced. Also, target gene activity in distal cells depends on whether or not these genes had previously responded to proximal enhancers, which determines the presence or absence of H3K27me3 marks. We also show that the polycomb repressive complex 2 is mainly recruited at the Hox gene cluster and can extend its coverage to far-cis regulatory sequences as long as confined to the neighboring TAD structure. We conclude that antagonistic limb proximal and distal enhancers can exert their specific effects when positioned into the same TAD and in the absence of their genuine target genes. We also conclude that removing these target genes reduced the coverage of a regulatory landscape by chromatin marks associated with silencing, which correlates with its prolonged activity in time.

中文翻译：

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基因组结构对 Hox 调控景观功能激活和抑制的影响


哺乳动物基因组的空间组织依赖于各种尺度染色质结构域的形成。在顺式基因调控水平上，增强子序列的集合定义了通常与拓扑关联域（TAD）的存在相匹配的大调控景观。这些结构域通常包含一系列表现出相似或相关组织特异性的增强子，这表明在某些情况下，这些结构域可能充当连贯的调节单元，具有全局开启或关闭状态。 HoxD 基因簇通过高度协调的基因表达调节来指定发育中肢体的拓扑结构，通过使用 HoxD 基因簇作为范例，我们研究了调节域的排列如何决定其活性和功能。发育中肢体的近端和远端细胞表达不同水平的 Hoxd 基因，分别受侧翼 3' 和 5' TAD 调节。我们描述了影响这两个 TAD 的大型基因组重排的影响，包括它们融合成单个染色质结构域。我们发现，在单个混合 TAD 中，当两个 TAD 都完好无损时，近端和远端肢体增强子都会被全局激活。然而，与沉默的野生型相比，融合 TAD 中远端细胞中 3' TAD 的活性通常会增加。此外，远端细胞中的靶基因活性取决于这些基因之前是否对近端增强子有反应，这决定了 H3K27me3 标记的存在与否。我们还表明，多梳抑制复合物 2 主要在 Hox 基因簇处招募，并且只要限制在邻近的 TAD 结构内，就可以将其覆盖范围扩展到远顺式调控序列。 我们得出结论，拮抗性肢体近端和远端增强子在定位到相同 TAD 且缺乏其真正靶基因的情况下可以发挥其特定作用。我们还得出结论，去除这些靶基因减少了与沉默相关的染色质标记对调控景观的覆盖范围，这与其时间上的延长活性相关。