AtBPM1 regulates DDR complex activity and RdDM mediated DNA methylation via a cullin 3 independent mechanism

Published: 10 January 2022| Version 1 | DOI: 10.17632/66ynzwm63d.1
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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 dunja@zg.biol.pmf.hr § 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.

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