Fluorescent labeling of genomic loci in Drosophila imaginal discs with heterologous DNA-binding proteins.
Fluorescent labeling of genomic loci in Drosophila imaginal discs with heterologous DNA-binding proteins.
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用异源DNA结合蛋白荧光标记果蝇成虫盘基因组位点。
DOI：
10.1016/j.crmeth.2022.100175
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发表时间：
2022-03-28
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以果蝇 Hox 基因 Ultrabithorax (Ubx) 为例，我们展示了使用三种异源 DNA 结合蛋白系统——LacI/LacO、ParB1/ParS1 和 ParB2/ParS2——通过插入小 DNA 标签来标记成虫盘中的基因组位点。我们比较每个系统，考虑基因组区域中标记的影响（1）转录基因体内部与外部以及（2）不同染色质可及性。我们通过询问基因表达水平和增强子-启动子距离以及 Ubx 基因座的等位基因间距离之间的关系来证明该系统的价值。我们发现重要的内含子顺式调控元件前胸（abx）和启动子之间的距离不随表达水平变化。相反，等位基因间距离与 Ubx 表达水平相关。用三种荧光结合蛋白标记基因组位点的工具 在不同基因组背景下表征这些标记工具 Ubx 的等位基因间距离与基因表达水平相关 Ubx 的增强子-启动子距离与基因表达水平解耦 需要了解基因组区域如何以及何时相互作用，以剖析基因组结构和了解基因调控。为此，拥有能够有效标记活组织和固定组织中 DNA 元件的工具非常重要。我们试图扩展果蝇研究界可用的工具箱，并通过标记成虫盘细胞核中的特定 DNA 序列来评估每种工具的优点和缺点。用异源荧光蛋白 (FP) 标记感兴趣的基因组区域，可以在细胞核内的三个维度上相对于彼此以及附加的核特征定位这些区域。德尔克等人。测试并表征了三个 FP 在果蝇成虫盘的不同基因组环境中的使用，以及使用两个 FP 来询问 Hox 基因 Ultrabithorax 的基因组邻近性。
Using the Drosophila melanogaster Hox gene Ultrabithorax (Ubx) as an example, we demonstrate the use of three heterologous DNA-binding protein systems—LacI/LacO, ParB1/ParS1, and ParB2/ParS2—to label genomic loci in imaginal discs with the insertion of a small DNA tag. We compare each system, considering the impact of labeling in genomic regions (1) inside versus outside of a transcribed gene body and (2) with varying chromatin accessibility. We demonstrate the value of this system by interrogating the relationship between gene expression level and enhancer-promoter distance, as well as inter-allelic distance at the Ubx locus. We find that the distance between an essential intronic cis-regulatory element, anterobithorax (abx), and the promoter does not vary with expression level. In contrast, inter-allelic distance correlates with Ubx expression level. Tools for labeling genomic loci with three fluorescent binding proteins Characterization of these labeling tools at distinct genomic contexts Inter-allelic distance at Ubx correlates with gene expression levels Enhancer-promoter distance at Ubx is decoupled from gene expression levels Understanding how and when genomic regions interact with each other is needed to dissect genome architecture and understand gene regulation. Toward this end, it is important to have tools to efficiently label DNA elements in both live and fixed tissues. We sought to expand the toolbox available to the Drosophila research community and to assess the strengths and weaknesses of each tool by labeling specific DNA sequences in the nuclei of imaginal discs. Labeling genomic regions of interest with heterologous fluorescent proteins (FPs) allows the localization of these regions in three dimensions within nuclei relative to each other and to additional nuclear features. Delker et al. test and characterize the use of three FPs in distinct genomic contexts of Drosophila melanogaster imaginal discs, as well as use two FPs to interrogate genomic proximity at the Hox gene Ultrabithorax.