DOI: 10.5061/dryad.00000008h
title: Imaging files for: ACD15, ACD21, and SLN regulate accumulation and mobility of MBD6 to silence genes and transposable elements

description: 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.
description: All of this data was obtained using confocal, fluorescence
 microscopy with 40xwater objective. 
description: # Imaging files for "ACD15, ACD21, and SLN regulate accumulation and
 mobility of MBD6 to silence genes and transposable elements."
 [https://doi.org/10.5061/dryad.00000008h](https://doi.org/10.5061/dryad.00000008h) This microscopy data was used for figures throughout the manuscript and is available for others to view. ## Description of the data and file structure These files are all image files from the Zeiss confocal microscope. To open these files use Zen software and/or ImageJ. The names of the files follow the general nomenclature: genotype background\_Protein(s) Imaged\_possible second protein imaged\_Date and/or replicate of the Image\_type of experiment (i.e. Z stack or FRAP). All MBD6 alleles are RFP tagged, all ACD15 are YFP tagged, all ACD21 are CFP tagged, and all SLN are CFP tagged. Example: "acd\_MBD6\_08202022" * MBD6 RFP in acd double mutant imaged in 08202022. "acd\_MBD6 RFP\_ACD15 YFP\_08202022" * MBD6 RFP and ACD15 YFP expressed in acd double mutant imaged on 08202022. General Naming guidelines: genotypes: * ACD21=*acd21* mutant * ACD15=*acd15* mutant * SLN=*sln* mutant * "acd"=*acd15 acd21* double mutant * "triple"= *acd15 acd21 sln* triple mutant * "MBD56"=*mbd5 mbd6* double mutant * MBD6 C term or MBD6 C deletion=MBD6 C terminus deletion RFP * MBD6 plus StkyC or MBD6 1.2 or MBD6 StkyC= MBD6 C terminal deletion with StkyC domain added back RFP * MBD6 N term or MBD6 N term deletion=MBD6 with the C terminus of the protein deleted RFP. ## Sharing/Access information Here is a link to download the Zen software: * https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html Here is a link to download the ImageJ Software: * https://imagej.net/ij/download.html ## Code/Software To analyze FRAP data, we used the following website: https://easyfrap.vmnet.upatras.gr/GettingStarted/?AspxAutoDetectCookieSupport=1 To count any nuclear foci use the 3D object counter function in the ImageJ software.