BPM1 regulates RdDM-mediated DNA methylation via a cullin 3 independent mechanism

AtBPM1 regulates DDR complex activity and RdDM mediated DNA methylation via a cullin 3 independent mechanism Mateja Jagić1§, Tamara Vuk1§, Andreja Škiljaica1, Lucija Markulin1, Vedrana Vičić Bočkor1, Mirta Tokić1, Karlo Miškec1, Genadij Razdorov2, Siniša Habazin2 Marko Šoštar3, Igor Weber3, Nataša Bauer1, Dunja Leljak Levanić1* 1Division of Molecular Biology, Department of Biology, Faculty of Science, University of 11 Zagreb, Zagreb, Croatia 2Genos Glycoscience Research Laboratory, Zagreb, Croatia 3Division of Molecular Biology,Institute Ruđer Bošković, Zagreb, Croatia * Correspondence: Dunja Leljak Levanić [e-mail] § Both authors contributed equally to this work Abstract The best-known function of MATH-BTB proteins, including those from Arabidopsis (the BPM proteins) is their role as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin proteasome pathway. In this process, BTB domain enables assembly with the CUL3, while less conserved MATH domain serves as an adaptor and binds substrates destined for degradation via 26S proteasome. Here, we report a new CUL3 independent role of AtBPM1 in RNA directed DNA methylation (RdDM). Proved by different interaction assays, nuclear fraction of BPM1 interacted with DMS3 and RDM1 proteins, the components of the chromatin remodeling DDR complex that is involved in positioning of RdDM methylation machinery. BTB domain itself had a strong interaction affnity for RDM1, while MATH domain itself, despite detected interactions with both partners, was not essential for the interactions, both suggesting that DMS3 and RDM1 are not substrates for proteasomal degradation. Moreover, in transgenic line lacking a functional CUL3-based E3 ligase complex, DMS3 remained ubiquitinated and the levels of DMS3 did not differ between BPM1-overexpresing plants and wild type. Finally, increased CHH methylation status of the two RdDM controled genes, FWA and CLM41 in BPM1-overexpressing plants confirmed that BPM1 does not participate in DMS3 and RDM1 degradation, instead it is involved in recruitment of the RdDM to specific chromatin positions destined for de novo DNA methylation.

AtBPM1通过不依赖于Cullin 3的机制调控DDR复合物活性与RdDM介导的DNA甲基化
Mateja Jagić1§, Tamara Vuk1§, Andreja Škiljaica1, Lucija Markulin1, Vedrana Vičić Bočkor1, Mirta Tokić1, Karlo Miškec1, Genadij Razdorov2, Siniša Habazin2, Marko Šoštar3, Igor Weber3, Nataša Bauer1, Dunja Leljak Levanić1*
1 克罗地亚萨格勒布大学理学院生物学系分子生物学分部，萨格勒布，克罗地亚
2 Genos糖科学研究实验室，萨格勒布，克罗地亚
3 鲁杰尔·博什科维奇研究所分子生物学分部，萨格勒布，克罗地亚
* 通讯作者：Dunja Leljak Levanić，邮箱：[e-mail]
§ 两位作者对本研究贡献均等

摘要：MATH-BTB蛋白最广为人知的功能是作为泛素-蛋白酶体通路中基于Cullin 3的E3泛素连接酶的底物特异性适配子，包括拟南芥中的BPM蛋白也属于此类蛋白。在此过程中，BTB结构域介导与Cullin 3的组装结合，而保守性较弱的MATH结构域则作为适配子，结合经26S蛋白酶体降解的靶底物。本研究报道了AtBPM1在RNA指导的DNA甲基化（RdDM）中一种不依赖于Cullin 3的新功能。通过多种互作实验证实，BPM1的核组分与染色质重塑DDR复合物的组成蛋白DMS3和RDM1存在相互作用，该复合物参与RdDM甲基化机器的定位过程。BTB结构域本身对RDM1具有较强的结合亲和力；尽管MATH结构域可与两种靶蛋白产生互作，但它并非该互作的必需结构域，这两点均表明DMS3和RDM1并非蛋白酶体降解的靶底物。此外，在缺失功能性基于Cullin 3的E3泛素连接酶复合物的转基因植株中，DMS3仍可发生泛素化，且过表达BPM1的植株与野生型植株的DMS3蛋白水平并无差异。最终，过表达BPM1的植株中两个受RdDM调控的基因FWA与CLM41的CHH甲基化水平显著升高，这证实BPM1并不参与DMS3和RDM1的降解，而是参与将RdDM机器招募至需要进行从头DNA甲基化的特定染色质位点。

搜集汇总

背景与挑战

背景概述

该数据集源自一项研究，揭示了拟南芥AtBPM1蛋白在RNA指导的DNA甲基化（RdDM）中的新功能，即通过不依赖cullin 3的机制与DDR复合物组分DMS3和RDM1相互作用，参与招募RdDM机制到特定染色质位置进行从头DNA甲基化，而非介导这些蛋白的降解。数据集包含蛋白质组学实验的质谱数据文件（如.mzML格式）和MaxQuant蛋白质组分析表格，适用于研究蛋白质相互作用和DNA甲基化调控机制。