SpyChIP identifies cell type-specific transcription factor occupancy from complex tissues.
SpyChIP identifies cell type-specific transcription factor occupancy from complex tissues.
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DOI：
10.1073/pnas.2122900119
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发表时间：
2022-06-21
影响因子：
11.1
通讯作者：
中科院分区：
文献类型：
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作者：
我们已经开发了SpyChIP，这是一种依赖于SpyTag和SpyCatcher两个肽之间形成共价异肽键的方法，可以在自然生理环境中识别细胞类型特定的转录因子占据的位置，而不需要组织解离或核分离。利用SpyChIP，我们在果蝇成像盘的两种不同细胞类型中表征了Hox蛋白Ultrabithorax(Ubx)的全基因组结合图谱，揭示了广泛的细胞类型特异性Ubx-DNA结合事件。将细胞类型特异性共价键的形成应用于染色质免疫沉淀，为在体内进行许多其他细胞类型特异性分析和遗传操作奠定了基础，这些分析和遗传操作以前是不切实际的。染色质免疫沉淀(ChIP)是研究体内蛋白质-DNA结合的一种重要技术。芯片技术的一个缺点是在分析复杂组织时缺乏细胞类型特异性。为了克服这一限制，我们开发了SpyChIP来在自然生理环境中识别细胞类型特定的转录因子(TF)结合位点，而不需要组织分离或核分离。SpyChIP利用15个氨基酸的SpyTag和17-kDa的SpyCatcher蛋白之间迅速形成的特定共价等肽键。在SpyChIP中，通过基因组工程将目标TF与SpyTag融合，并在目的细胞群体中表达标记为SpyCatcher的表位，在那里它与SpyTag-TF共价结合。细胞类型特异性芯片是通过免疫沉淀从整个组织中制备的染色质，使用针对表位标记的Spycatcher的抗体来获得的。利用SpyChIP，我们在果蝇成像盘的两种不同细胞类型中鉴定了Hox蛋白Ultrabithorax(UBx)的全基因组结合图谱。我们的结果揭示了广泛的区域特异性Ubx-DNA结合事件，突出了细胞类型特异性芯片的重要性和全组织芯片方法的局限性。对Ubx：：SpyChIP结果的分析为人们提供了对染色质可及性和Ubx-DNA结合之间的关系的见解，以及Ubx用来调节其下游顺式调节模块的不同机制。除了SpyChIP，我们建议SpyTag-Spycatcher技术，以及其他形成共价等肽键的蛋白质对，将促进许多以前不切实际的体内额外应用。
We have developed SpyChIP, a method that depends on covalent isopeptide bond formation between two peptides, SpyTag and SpyCatcher, to identify sites of cell type-specific transcription factor occupancy in native physiological contexts without tissue dissociation or nuclei sorting. Using SpyChIP, we characterized the genome-wide binding profiles of the Hox protein Ultrabithorax (Ubx) in two distinct cell types of the Drosophila haltere imaginal disc, revealing extensive cell type-specific Ubx–DNA binding events. The application of cell type-specific covalent bond formation to chromatin immunoprecipitation sets the stage for carrying out many other cell type-specific analyses and genetic manipulations in vivo that were previously impractical. Chromatin immunoprecipitation (ChIP) is an important technique for characterizing protein–DNA binding in vivo. One drawback of ChIP-based techniques is the lack of cell type-specificity when profiling complex tissues. To overcome this limitation, we developed SpyChIP to identify cell type-specific transcription factor (TF) binding sites in native physiological contexts without tissue dissociation or nuclei sorting. SpyChIP takes advantage of a specific covalent isopeptide bond that rapidly forms between the 15-amino acid SpyTag and the 17-kDa protein SpyCatcher. In SpyChIP, the target TF is fused with SpyTag by genome engineering, and an epitope tagged SpyCatcher is expressed in cell populations of interest, where it covalently binds to SpyTag-TF. Cell type-specific ChIP is obtained by immunoprecipitating chromatin prepared from whole tissues using antibodies directed against the epitope-tagged SpyCatcher. Using SpyChIP, we identified the genome-wide binding profiles of the Hox protein Ultrabithorax (Ubx) in two distinct cell types of the Drosophila haltere imaginal disc. Our results revealed extensive region-specific Ubx–DNA binding events, highlighting the significance of cell type-specific ChIP and the limitations of whole-tissue ChIP approaches. Analysis of Ubx::SpyChIP results provided insights into the relationship between chromatin accessibility and Ubx–DNA binding, as well as different mechanisms Ubx employs to regulate its downstream cis-regulatory modules. In addition to SpyChIP, we suggest that SpyTag–SpyCatcher technology, as well as other protein pairs that form covalent isopeptide bonds, will facilitate many additional in vivo applications that were previously impractical.
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鉴定雌激素受体结合与乳腺癌的临床结局有关。
影响因子：
64.8
作者：
Ross-Innes, Caryn S.;Stark, Rory;Teschendorff, Andrew E.;Holmes, Kelly A.;Ali, H. Raza;Dunning, Mark J.;Brown, Gordon D.;Gojis, Ondrej;Ellis, Ian O.;Green, Andrew R.;Ali, Simak;Chin, Suet-Feung;Palmieri, Carlo;Caldas, Carlos;Carroll, Jason S.
通讯作者：
Carroll, Jason S.
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
16
作者：
Heinz S;Benner C;Spann N;Bertolino E;Lin YC;Laslo P;Cheng JX;Murre C;Singh H;Glass CK
通讯作者：
Glass CK
DOI：
10.1016/j.tig.2016.03.004
发表时间：
2016-06
期刊：
Trends in genetics : TIG
影响因子：
--
作者：
Merabet S;Mann RS
通讯作者：
Mann RS
影响因子：
3.3
作者：
Feng, Siqian;Lu, Shan;Mann, Richard S.
通讯作者：
Mann, Richard S.