Systematic identification and characterization of repressive domains in Drosophila transcription factors.
Systematic identification and characterization of repressive domains in Drosophila transcription factors.
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DOI：
10.15252/embj.2022112100
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发表时间：
2023-02-01
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影响因子：
--
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所有多细胞生命都依赖于差异基因表达，这是由调节DNA元件和DNA结合转录因子决定的，这些转录因子通过辅因子募集介导激活和抑制。虽然激活剂已被广泛表征，但阻遏物的研究较少：其阻遏结构域（RD）的身份和性质通常是未知的，并且它们招募的特定辅阻遏物（CoR）尚未确定。在这里，我们开发了一种高通量的，基于测序的下一代筛选方法，抑制结构域（RD）-seq，以系统地识别复杂DNA片段文库中的RD。筛选超过200，000个片段，涵盖了果蝇中所有转录相关蛋白的编码序列，我们在已知的阻遏物和以前与阻遏无关的蛋白质中鉴定出195个RD。许多RD含有重复的短肽基序，其在果蝇和人类之间是保守的，并且是RD功能所需的，如基序诱变所证明的。此外，我们发现，RD包含五个不同的抑制性基序之一的相互作用，并依赖于不同的CoR，如Groucho，CtBP，Sin 3A，或Smrter。这些发现推进了我们对阻遏物、它们的序列以及序列改变突变的功能影响的理解，并应为进一步的研究提供了宝贵的资源。在果蝇中基于NGS的RD-seq筛选方法揭示了已知转录阻遏物以及蛋白质中的195个阻遏结构域，这些结构域以前与基因阻遏无关。
All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA‐binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied: the identities and properties of their repressive domains (RDs) are typically unknown and the specific co‐repressors (CoRs) they recruit have not been determined. Here, we develop a high‐throughput, next‐generation sequencing‐based screening method, repressive‐domain (RD)‐seq, to systematically identify RDs in complex DNA‐fragment libraries. Screening more than 200,000 fragments covering the coding sequences of all transcription‐related proteins in Drosophila melanogaster, we identify 195 RDs in known repressors and in proteins not previously associated with repression. Many RDs contain recurrent short peptide motifs, which are conserved between fly and human and are required for RD function, as demonstrated by motif mutagenesis. Moreover, we show that RDs that contain one of five distinct repressive motifs interact with and depend on different CoRs, such as Groucho, CtBP, Sin3A, or Smrter. These findings advance our understanding of repressors, their sequences, and the functional impact of sequence‐altering mutations and should provide a valuable resource for further studies. NGS‐based RD‐seq screening method in flies reveals 195 repressive domains in known transcriptional repressors as well as in proteins, not previously linked to gene repression.
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DOI：
10.15252/embj.201798896
发表时间：
2018-08-15
期刊：
The EMBO journal
影响因子：
--
作者：
Arnold CD;Nemčko F;Woodfin AR;Wienerroither S;Vlasova A;Schleiffer A;Pagani M;Rath M;Stark A
通讯作者：
Stark A
影响因子：
64.5
作者：
Basu, Shaon;Mackowiak, Sebastian D.;Hnisz, Denes
通讯作者：
Hnisz, Denes
影响因子：
4.6
作者：
Chandru A;Bate N;Vuister GW;Cowley SM
通讯作者：
Cowley SM
影响因子：
14.9
作者：
Bailey TL;Johnson J;Grant CE;Noble WS
通讯作者：
Noble WS
影响因子：
3.7
作者：
Atanesyan, Lilit;Guenther, Viola;Schaffner, Walter
通讯作者：
Schaffner, Walter