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Arabidopsis thaliana

ABSTRACT: The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status (mRNA-seq)

PROVIDER: PRJNA326338 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR3707330.fastq.gz	Fastqsanger.gz
	SRR3707340.fastq.gz	Fastqsanger.gz
	SRR3707331.fastq.gz	Fastqsanger.gz
	SRR3707341.fastq.gz	Fastqsanger.gz
	SRR3707332.fastq.gz	Fastqsanger.gz

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Single-nucleus methylcytosine and transcriptome sequencing in pollen reveals antagonism between MBD5/6 and MBD7 in transcriptional regulation [RNA-seq]

Project description:MBD5, MBD6, and MBD7 are CG-specific methyl-readers with opposite functions: MBD5 and MBD6 (MBD5/6) redundantly repress methylated loci in pollen vegetative nuclei (VN), while MBD7 prevents transgene silencing, possibly by promoting DNA demethylation. Here we show that transcriptional derepression in mbd5/6 pollen is rescued by loss of MBD7, suggesting that MBD5/6 counteract MBD7 activity. By simultaneously profiling DNA methylation and gene expression in single pollen nuclei, we found that CG methylation is lost at MBD5/6 targets specifically in the early post-mitotic VN of mbd5/6. This loss precedes mbd5/6 transcriptional derepression and is largely restored in mbd5/6/7 triple mutants. Altering MBD6 to recruit the MBD7 complex instead of its normal interactors caused demethylation and upregulation of MBD5/6 targets, turning MBD6 from a repressor into an activator. We propose that in VN, where chromatin is decondensed, MBD5/6 are required to protect accessible DNA from demethylation and derepression by the MBD7 complex.

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MethlyC-Seq of wild type (with 35S-SUC2 transgene) and idm3-1, mbd7-1 mutants (same 35S-SUC2 background)

Project description:In eukaryotes, heterochromatin is characterized by numerous epigenetic marks, including DNA methylation. Spreading of these marks into nearby active genes must be avoided in order to maintain appropriate gene expression. Here, we uncover Arabidopsis Methyl-CpG-Binding Domain 7 (MBD7) and Increased DNA Methylation 3 (IDM3) as anti-silencing factors that prevent transgene repression and genome-wide DNA hypermethylation. MBD7 preferentially binds to highly methylated, CG-dense regions associated with non-CG methylation and physically associates with other anti-silencing factors, including the histone acetyltransferase IDM1, IDM2, and IDM3. IDM1 and IDM2 were previously shown to facilitate active DNA demethylation by the 5-methylcytosine DNA glycosylase/lyase ROS1. Thus, MBD7 tethers the IDM proteins to methylated DNA, which enables the function of DNA demethylases that in turn establish chromatin boundaries and limit DNA methylation Using MethylC-Seq to provide single-base resolution of DNA methylation status in WT and idm3-1, mbd7-1 mutants Whole genome methylation maps of mbd7-1, idm3-1 and WT (all three are from 35S-SUC transgene background) were generated using BS-seq

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An antagonistic epigenetic mechanism regulating gene expression in pollen revealed through single-nucleus multiomics [ChIP-Seq]

Project description:Arabidopsis MBD5, MBD6, and MBD7 are CG-specific methyl-readers with opposite functions: MBD5 and MBD6 (MBD5/6) repress methylated loci in pollen vegetative nuclei (VN), while MBD7 prevents transgene silencing, possibly by promoting DNA demethylation. Here we show that loss of MBD7 rescues transcriptional defects at a large subset of MBD5/6-bound loci. Using simultaneous profiling of DNA methylation and transcription in single pollen nuclei, we found that MBD5/6-bound loci that are actively demethylated in the early post-mitotic VN lose additional methylation in mbd5/6, prior to transcriptional derepression. A subset of these loci is also bound by MBD7, correlating with demethylation and transcriptional derepression in mbd5/6 that are both reversed by loss of MBD7. Conversely, ectopically recruiting the MBD7 complex to MBD5/6 targets causes partial demethylation and upregulation. We propose that MBD5/6 maintain silencing in VN in part by preventing the MBD7 complex from enhancing the active demethylation that occurs during VN maturation.

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