The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status
- Institute of Integrative Genome Biology, University of California, United States
- Lanzhou University, China
- Hebei Collaboration Innovation Center for Cell Signaling, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China
- Salk Institute for Biological Studies, United States
- Guangdong Provincial Key Laboratory for Plant Epigenetics, Shenzhen University, China
- College of Life Sciences, Peking University, China
- David Geffen School of Medicine at UCLA, United States
- Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, China
- Howard Hughes Medical Institute, University of California, United States
Figures
Figure 1 with 3 supplements
LIL promotes expression of methylated LUC reporters.
(A) Luciferase (LUC) luminescence in YJ, YJ lil-1, LUCH and LUCH lil-2 seedlings as diagramed on the left. (B) Quantification of LUC transcript levels by RT-qPCR. Transcript levels were normalized …
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Figure 1—source data 1
Alignment of the YJ and LUCH transgenes.
Clustal alignment numbered relative to YJ. Perfectly matched residues are marked with an asterisk (*) below the alignment.
- https://doi.org/10.7554/eLife.19893.003
Figure 1—figure supplement 1
Characterization of the LUCH and YJ luciferase (LUC) reporter lines.
(A) LUC luminescence in LUCH and YJ backgrounds as diagrammed on top. (B) Quantification of LUC transcript levels by RT-qPCR. LUC transcript levels were normalized to UBIQUITIN5 and LUCH expression …
Figure 1—figure supplement 2
Investigation of transgene copy number.
(A) Diagram of the At1g02740 gene showing the location of the YJ reporter insertion site based on the MethylC-seq reads. Chimeric reads between the YJ reporter, in the vicinity of the RB and LB …
Figure 1—figure supplement 3
Sequence alignment of LIL and its paralogs.
LIL paralogs were identified using the Arabidopsis thaliana WU-BLAST2 Search function using the TAIR10 Proteins dataset and the full-length amino-acid sequence of LIL (At1g20870) as a query (http://w…
Figure 2 with 3 supplements
mbd7, ros4, and ros5 mutants phenocopy lil-1 at the YJ transgene.
(A) Luciferase (LUC) luminescence in 10-day-old seedlings as diagramed on the left. (B) Quantification of LUC or (C) NPTII transcript levels by RT-qPCR. Transcript levels were normalized to UBIQUITIN…
Figure 2—figure supplement 1
Yeast two-hybrid and affinity purification analyses.
(A) Diagrams of the yeast two-hybrid constructs used in (B). Full-length or partial fragments of LIL or MBD proteins were fused to the GAL4 DNA binding domain (BD) or the GAL4 activation domain (AD) …
Figure 2—figure supplement 2
Isolation and characterization of ros4, mbd5, mbd7, and ros5 mutants.
(A) Schematic diagram showing the positions of the two mutations in the ROS4 gene isolated in the YJ screen. Both the ros4-2 and ros4-3 alleles contain G-to-A mutations that result in the generation …
Figure 2—figure supplement 3
mbd5 mutants do not exhibit reduced LUC expression.
(A) Luciferase (LUC) luminescence in 10-day-old seedlings as diagramed on the left. (B) Quantification of LUC transcript levels by RT-qPCR. LUC transcript levels were normalized to UBIQUITIN5 with …
Figure 3
MBD7 and LIL regulate LUC expression in a DNA methylation-dependent manner.
(A) Luciferase luminescence of mock or 5-aza-2’-deoxycytidine (5-Aza-dC)-treated seedlings as diagramed on the left. (B) Quantification of LUC transcript levels by RT-qPCR. LUC transcript levels …
Figure 4 with 1 supplement
Disruption of the MBD7 complex results in subtle, but reproducible hyper-methylation at the d35S promoter.
Cytosine methylation analysis by McrBC-PCR (A) and McrBC-qPCR (B) using mock or McrBC treated genomic DNA from the indicated genotypes. In (A), two tandem copies of the 35S sequence result in two …
Figure 4—figure supplement 1
Complementation of the mbd7-5 phenotype with MBD7-3xHA.
(A) Luciferase luminescence in YJ, YJ mbd7-5, and YJ mbd7-5 MBD7-3xHA seedlings as diagramed on the left. Ins # indicates independent, single-locus insertion lines tested. (B) Quantification of LUC …
Figure 5 with 1 supplement
Genetic uncoupling of the DNA methylation and LUC expression phenotypes at the YJ reporter.
(A) Diagram of the YJ transgene indicating the region examined by traditional bisulfite sequencing (631–1004). DNA methylation tracks show the DNA methylation level (0–1, where 1 = 100% methylated) …
Figure 5—figure supplement 1
Traditional bisulfite sequencing at the YJ reporter.
CyMATE (Hetzl et al., 2007) alignments showing the methylation patterns at the 3’ half of the dual 35S promoter driving LUC expression in the YJ reporter. For each sample set, the genotypes are …
Figure 6 with 4 supplements
Genomic DMRs identified in the lil and mbd7 mutants.
(A) Table summarizing DMRs that overlap among all the lil and mbd7 datasets ‘6-way Direct DMR overlaps’. (B) Screenshots showing the DNA methylation levels (0–1, where 1 = 100% methylated, with CG, …
Figure 6—figure supplement 1
Visualization and quantification of DNA methylation at the direct overlap DMRs.
Screenshots showing the DNA methylation levels (0–1, where 1 = 100% methylated with CG, CHG and CHH contexts in green, blue and red, respectively) at several DMRs in the genotypes indicated on the …
Figure 6—figure supplement 2
Identification of a more relaxed set of DMRs conserved amongst the mbd7 and lil datasets.
(A) A complete set of all DMRs called in each context (hyper or hypo CG, CHG and CHH) amongst all the mbd7 and lil alleles and biological replicates were compiled and the levels of DNA methylation …
Figure 6—figure supplement 3
Complementation of the lil-1 phenotype with 9xMyc-LIL.
(A) Luciferase (LUC) luminescence in YJ, YJ lil-1, and YJ lil-1 9xMyc-LIL seedlings as diagramed on the left. Two sibling, homozygous lines from the same single locus insertion event were analyzed …
Figure 6—figure supplement 4
Gene expression at previously characterized loci in mbd7 and lil mutants.
(A) Normalized expression values (FPKM; fragments per kilobase mapped) of control and mutant RNA-seq samples at genes previously shown to be associated with hyper DMRs and to display reduced …
Author response image 1
Expression of putative MBD7 targets in dry seeds.
Quantification of transcript levels by RT-qPCR using pooled dry seeds. Transcript levels were normalized to UBIQUITIN5 with the expression level of the target loci in the YJ and LUCH controls set to …
Author response image 2
Investigation into variation in DNA methylation.
Comparisons of the average number of DMRs observed between all pairwise combinations of three YJ replicates (YJ_r1, YJ_r5 and YJ_r7) or between all pairwise combinations of these controls and their …
Additional files
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Source code 1
Custom Perl script used to trim 3’ adapter sequences 839 from raw reads.
- https://doi.org/10.7554/eLife.19893.021
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Supplementary file 1
MethylC-seq and smRNAseq data processing
(A) LUC reporter MethylC-seq mapping information. (B) LUC reporter MethylC-seq coverage. (C) LUC reporter mapping and coverage of small RNA data. (D) List of supplemental materials for LUC Reporter Genomics. (E) TAIR10 genome mapping and coverage information. (F) List of supplemental materials for the genome-wide analyses (TAIR10).
- https://doi.org/10.7554/eLife.19893.022
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Supplementary file 2
DMRs and DMR overlaps
(A) Hyper and Hypo DMRs in the CG, CHG and CHH contexts. (B-F) Direct DMR overlaps in the various lil and mbd7 datasets corresponding to 2-way, 3-way, 4-way, 5-way, and 6-way overlaps, respectively. (G-J) six way DMR coordinates in the hyper CG, CHG, CHH and hypo CG contexts, respectively. (K-P) Master DMR coordinates in the hyper CG, CHG, CHH, and hypo CG, CHG, and CHH contexts, respectively. (Q-U) Relaxed DMR coordinates in the hyper CG, CHG, CHH, and hypo CG, CHG, and CHH contexts, respectively. (V) mbd7 and lil hyper DMRs, all mC contexts merged. (W) rdd hyper DMRs, all mC contexts merged.
- https://doi.org/10.7554/eLife.19893.023
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Supplementary file 3
Analysis of lil and mbd7 RNAseq experiments.
- https://doi.org/10.7554/eLife.19893.024
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Supplementary file 4
Primers.
- https://doi.org/10.7554/eLife.19893.025
