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Imaging files for: ACD15, ACD21, and SLN regulate accumulation and mobility of MBD6 to silence genes and transposable elements

Boone, Brandon1

Published Oct 12, 2023 on Dryad. https://doi.org/10.5061/dryad.00000008h

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Oct 12, 2023 version files 24.85 GB

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Abstract

It's well known that DNA methylation is linked to gene silencing but the mechanisms of how proteins that bind the DNA methylation cause gene silencing remains unclear. We demonstrated that the novel MBD5/6 complex contains three chaperone proteins, called ACD15, ACD21, and SLN, which specifically mediate the gene silencing function. ACD15 and ACD21 bridge the interaction of SLN to MBD5 and/or MBD6 while also functioning to drive the accumulation of the MBD5/6 complex at CG methylation sites. We further discovered that SLN also regulates the accumulation of the MBD5/6 complex and regulates the turnover of all protein members once accumulated at meCG sites. 

To demonstrate these results we primarily used fluorescence, confocal microscopy using RFP tagged MBD6, YFP tagged ACD15, and CFP tagg ACD21 and SLN imaging the roots of Arabidopsis thaliana. We expressed these constructs either alone or together in multiple mutant lines including mbd5 mbd6, acd15, acd21, acd15 acd21, sln, and acd15 acd21 sln mutant plants. We also truncated MBD6 to remove the C terminus, called the C term deletion, as well as a version of MBD6 lacking the C terminus but with the domain necessary for interaction with ACD15 added back (MBD6 plus StkyC). With these microscopy experiments, we were able to demonstrate the specificity of ACD15 for the StkyC domain, the function of the chaperone proteins, and linke protein accumulation with gene silencing.