Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo.
Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo.
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DOI：
10.1101/gr.227124.117
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发表时间：
2018-04
期刊：
影响因子：
7
通讯作者：
Kaji K
中科院分区：
文献类型：
--
作者：
Tosti L;Ashmore J;Tan BSN;Carbone B;Mistri TK;Wilson V;Tomlinson SR;Kaji K
基因组中转录因子（TF）结合位点的鉴定对于理解基因调控网络（grn）至关重要。虽然ChIP-seq通常用于识别TF靶点，但它需要特定的chip级抗体和高细胞数量，这通常限制了其适用性。DNA腺嘌呤甲基转移酶鉴定（DNA adenine methyltransferase identification, DamID）是一种独特的研究蛋白质- DNA相互作用的技术，在果蝇中得到了广泛的应用。与ChIP-seq不同，它不需要抗体、沉淀步骤或化学蛋白质- dna交联，但迄今为止，由于技术限制，它很少用于哺乳动物细胞。在这里，我们描述了一种优化的DamID方法，结合小鼠细胞的下一代测序（DamID-seq），并证明了两种tf的结合位点，POU5F1（也称为OCT4）和SOX2，分别在少至1000个胚胎干细胞（ESCs）和神经干细胞（NSCs）中鉴定。此外，我们还首次在哺乳动物体内应用了这种技术。在交配后7.5 d的原肠胚小鼠胚胎（dpc）中，POU5F1 DamID-seq成功鉴定出多个与胚胎发育、神经管形成和中胚层心脏组织发育相关的基因近端的POU5F1结合位点，这与该TF在着床后胚胎中的关键作用一致。这项技术为在哺乳动物（包括体内样本）有限的特定细胞类型中史无前例地研究TF-DNA相互作用和grn铺平了道路。
The identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein–DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps, or chemical protein–DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical limitations. Here we describe an optimized DamID method coupled with next-generation sequencing (DamID-seq) in mouse cells and demonstrate the identification of the binding sites of two TFs, POU5F1 (also known as OCT4) and SOX2, in as few as 1000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. POU5F1 DamID-seq in the gastrulating mouse embryo at 7.5 d post coitum (dpc) successfully identified multiple POU5F1 binding sites proximal to genes involved in embryo development, neural tube formation, and mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigation of TF–DNA interactions and GRNs in specific cell types of limited availability in mammals, including in vivo samples.
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影响因子：
48
作者：
Huber W;Carey VJ;Gentleman R;Anders S;Carlson M;Carvalho BS;Bravo HC;Davis S;Gatto L;Girke T;Gottardo R;Hahne F;Hansen KD;Irizarry RA;Lawrence M;Love MI;MacDonald J;Obenchain V;Oleś AK;Pagès H;Reyes A;Shannon P;Smyth GK;Tenenbaum D;Waldron L;Morgan M
通讯作者：
Morgan M
影响因子：
9.8
作者：
Church DM;Goodstadt L;Hillier LW;Zody MC;Goldstein S;She X;Bult CJ;Agarwala R;Cherry JL;DiCuccio M;Hlavina W;Kapustin Y;Meric P;Maglott D;Birtle Z;Marques AC;Graves T;Zhou S;Teague B;Potamousis K;Churas C;Place M;Herschleb J;Runnheim R;Forrest D;Amos-Landgraf J;Schwartz DC;Cheng Z;Lindblad-Toh K;Eichler EE;Ponting CP;Mouse Genome Sequencing Consortium
通讯作者：
Mouse Genome Sequencing Consortium
影响因子：
64.5
作者：
Kind J;Pagie L;de Vries SS;Nahidiazar L;Dey SS;Bienko M;Zhan Y;Lajoie B;de Graaf CA;Amendola M;Fudenberg G;Imakaev M;Mirny LA;Jalink K;Dekker J;van Oudenaarden A;van Steensel B
通讯作者：
van Steensel B
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
64.8
作者：
Guelen, Lars;Pagie, Ludo;van Steensel, Bas
通讯作者：
van Steensel, Bas