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| Status |
Public on Apr 26, 2017 |
| Title |
The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status |
| Organism |
Arabidopsis thaliana |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
DNA methylation is a chromatin modification that is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation have been identified, relatively little is understood about how DNA methylation influences gene expression. Using complementary genetic and biochemical approaches we identified a protein complex that antagonizes the transcriptional gene silencing of two LUCIFERASE (LUC) reporters in a manner that requires DNA methylation. At its core, this complex contains LOW IN LUCIFERASE EXPRESSION (LIL), an α-crystallin domain protein, and METHYL-CpG-BINDING DOMAIN 7 (MBD7), a protein previously associated with DNA methylation. At the LUC reporters, loss of MBD7 or LIL resulted in decreased LUC expression concomitant with modest, but reproducible increases in DNA methylation that can be phenocopied by DNA demethylase mutants. These findings are consistent with other reports and reveal a genetic connection between MBD7, LIL and DNA demethylation. However, we found that the hyper-methylation and gene expression phenotypes at a LUC reporter can be genetically uncoupled, demonstrating that changes in DNA methylation alone are not sufficient to silence LUC expression, and suggesting a role for the MBD7-LIL complex downstream of DNA methylation. Consistent with this hypothesis, our more extensive analysis of DNA methylation in mbd7 and lil mutants revealed only a small number of hyper-methylated loci genome wide. Furthermore, these loci displayed minimal overlap with demethylase targets, suggesting that, in general, the DNA demethylation machinery does not function in a manner dependent on the MBD7-LIL complex. Taken together, our findings place the MBD7-LIL complex amongst a small number of factors that regulate gene expression without causing significant changes in DNA methylation. This complex, however, is unique in that it functions to suppress, rather than enforce the silencing effects of DNA methylation, enabling gene expression of several transgene reporters despite high levels of promoter methylation.
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| Overall design |
A total 23 bisufite libraries (samples 1-23) were sequenced in several sets as detailed in Table S4, each set with its own controls including three replicates in the lil-1 background. In addition, traditional bisulfite sequencing (conversion, cloning, and sanger based sequencing) was conducted on 9 samples (24-32) and for these experiments the percent methylation at each cystosine was tabulated and is presented in wiggle format files.
Please note that [1] Ler_r6_Cat3.C*wig files contain no methylated reads mapping to the transgene as they are negative control samples.
[2] For the samples 19 and 23, the DMRs relative to both the Wt control and the mutant background that was complemented were called to study complementation of a genetic defect. In all the other cases the only comparison was to the Wt control (i.e. one comparison per mutant).
[3] The traditional bisulfite sequencing samples (samples 24-32) have their raw data available only in FASTA format (generated by 'ABI 3730XL DNA Analyzer'), and thus their raw data were not provided. Please contact the submitter for the sequence data.
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| Contributor(s) |
Li D, Palanca AS, Won SY, Gao L, Feng Y, Vashisht AA, Liu L, Zhao Y, Liu X, Wu X, Li S, Le B, Kim YJ, Yang G, Li S, Liu J, Wohlschlegel JA, Guo H, Mo B, Chen X, Law JA |
| Citation(s) |
28452714 |
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| Submission date |
Jun 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Julie Ann Law |
| E-mail(s) |
jlaw@salk.edu
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| Phone |
8584534100
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| Organization name |
The Salk Institute for Biological Studies
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| Department |
PLANT MOLECULAR AND CELLULAR BIOLOGY LABORATORY
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| Lab |
Law Lab
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| Street address |
10010 North Torrey Pines Rd
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platforms (3) |
| GPL13222 |
Illumina HiSeq 2000 (Arabidopsis thaliana) |
| GPL17639 |
Illumina HiSeq 2500 (Arabidopsis thaliana) |
| GPL22012 |
AB 5500 Genetic Analyzer (Arabidopsis thaliana) |
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| Samples (32)
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| Relations |
| BioProject |
PRJNA325663 |
| SRA |
SRP076555 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE83355_LUC_Transgene6.fa.gz |
1.4 Kb |
(ftp)(http) |
FA |
| GSE83355_LUC_Transgene7.fa.gz |
1.5 Kb |
(ftp)(http) |
FA |
| GSE83355_RAW.tar |
2.6 Gb |
(http)(custom) |
TAR (of BED, WIG) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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