Kyrchanova Olga, Sokolov Vladimir, Tikhonov Maxim, Schedl Paul, Georgiev Pavel
Department of the Control of Genetic Processes, Institute of Gene Biology Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia.
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia.
bioRxiv. 2023 Feb 16:2023.02.16.528790. doi: 10.1101/2023.02.16.528790.
In higher eukaryotes enhancer-promoter interactions are known to be restricted by the chromatin insulators/boundaries that delimit topologically associated domains (TADs); however, there are instances in which enhancer-promoter interactions span one or more boundary elements/TADs. At present, the mechanisms that enable cross-TAD regulatory interaction are not known. In the studies reported here we have taken advantage of the well characterized Bithorax complex (BX-C) to study one potential mechanism for controlling boundary function and TAD organization. The regulatory domains of BX-C are flanked by boundaries which function to block crosstalk with their neighboring domains and also to support long distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, we asked whether transcriptional readthrough can impact boundary function. For this purpose, we took advantage of two BX-C boundary replacement platforms, and , in which the and boundaries, respectively, are deleted and replaced with an site. We introduced boundary elements, promoters and polyadenylation signals arranged in different combinations and then assayed for boundary function. Our results show that transcriptional readthrough can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is possible that many of them may function in the regulation of TAD organization.
Recent studies have shown that much genome in higher eukaryotes is transcribed into non-protein coding lncRNAs. It is though that lncRNAs may preform important regulatory functions, including the formation of protein complexes, organization of functional interactions between enhancers and promoters and the maintenance of open chromatin. Here we examined how transcription from promoters inserted into the Bithorax complex can impact the boundaries that are responsible for establishing independent regulatory domains. Surprisingly, we found that even a relatively low level of transcriptional readthrough can impair boundary function. Transcription also affects the activity of enhancers located in BX-C regulatory domains. Taken together, our results raise the possibility that transcriptional readthrough may be a widely used mechanism to alter chromosome structure and regulate gene expression.
在高等真核生物中，已知增强子与启动子之间的相互作用受到界定拓扑相关结构域（TADs）的染色质绝缘子/边界的限制；然而，也存在增强子与启动子之间的相互作用跨越一个或多个边界元件/TADs的情况。目前，促成跨TAD调控相互作用的机制尚不清楚。在本文报道的研究中，我们利用了特征明确的双胸复合体（BX-C）来研究一种控制边界功能和TAD组织的潜在机制。BX-C的调控结构域两侧是边界，这些边界的作用是阻止与其相邻结构域的串扰，同时支持调控结构域与其靶基因之间的长距离相互作用。由于在BX-C中发现了许多长链非编码RNA（lncRNA），我们不禁要问转录通读是否会影响边界功能。为此，我们利用了两个BX-C边界替换平台，即 和 ，其中 边界和 边界分别被删除，并用一个 位点取代。我们引入了以不同组合排列的边界元件、启动子和聚腺苷酸化信号，然后检测边界功能。我们的结果表明，转录通读会干扰边界活性。由于lncRNA在多细胞真核生物的RNA聚合酶II转录本中占很大比例，它们中的许多可能在TAD组织的调控中发挥作用。
最近的研究表明，高等真核生物中的许多基因组被转录为非蛋白质编码的lncRNA。人们认为lncRNA可能执行重要的调控功能，包括形成蛋白质复合物、组织增强子与启动子之间的功能相互作用以及维持开放染色质。在这里，我们研究了插入双胸复合体中的启动子转录如何影响负责建立独立调控结构域的边界。令人惊讶的是，我们发现即使相对较低水平的转录通读也会损害边界功能。转录还会影响位于BX-C调控结构域中的增强子的活性。综上所述，我们的结果提出了一种可能性，即转录通读可能是一种广泛用于改变染色体结构和调节基因表达的机制。
