Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltansferase with CRISPR/dCas9 (ChIP-Seq)
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ABSTRACT:
PROVIDER: PRJNA729060 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14514838_1.fastq.gz | Fastqsanger.gz | |||
| SRR14514838_2.fastq.gz | Fastqsanger.gz | |||
| SRR14514839_1.fastq.gz | Fastqsanger.gz | |||
| SRR14514839_2.fastq.gz | Fastqsanger.gz | |||
| SRR14514840_1.fastq.gz | Fastqsanger.gz |
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Project description:Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltansferase with CRISPR/dCas9
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Project description:Although associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are prone to confounds and may fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA demethylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using this new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. We find no credible evidence of off-target DNA demethylation as a consequence of gRNA binding. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions.
2020-11-26 | GSE162138 | GEO
Project description:Although associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are prone to confounds and may fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA demethylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using this new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. We find no credible evidence of off-target DNA demethylation as a consequence of gRNA binding. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions.
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Project description:Despite four decades of research to support the association between DNA methylation and gene expression, the causality of this relationship remains unresolved. Here, we reaffirm that experimental confounds preclude resolution of this question with existing strategies, including recently developed CRISPR/dCas9 and TET-based epigenetic editors. Instead, we demonstrate a highly effective method using only nuclease-dead Cas9 and guide RNA to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzyme, thereby enabling the examination of the role of DNA demethylation per se in living cells, with no evidence of off-target activity. Using this method, we probe a small number of inducible promoters and find the effect of DNA demethylation to be small, while demethylation of CpG-rich FMR1 produces larger changes in gene expression. This method could be used to reveal the extent and nature of the contribution of DNA methylation to gene regulation.
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Project description:We report the generation of CRISPR-dCas9 DNA methyltransferases to mediate targeted DNA methylation. Using the dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B methyltransferases, we have demonstrated that these two methyltransferase can mediate targeted methylation in three human genes tested: uPA, TGFBR3, and CDKN2A in human HEK293T cells. We also showed that these methyltransferases could mediate gene inhibition.
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Project description:Histone monoaminylation is a new class of chromatin post-translational modification whereby glutamines are transamidated by tissue transglutaminase 2 (TGM2). Initial studies of these marks have identified serotonylation and dopaminylation on H3Q5, suggesting the modification is a marker of active chromatin. However, little work has been conducted to understand the regulation of TGM2-mediated monoaminylation of chromatin. Here, we employed a highly sensitive TGM2 assay using the small molecule biotin-cadaverine as an acyl-acceptor substrate. In doing so, we discovered that, in addition to H3Q5, H3Q19 and H2A.ZQ123 and Q124 are glutaminyl substrates for TGM2 in vitro. Modification of these residues occurs in a sequence independent manner, with the primary determining factor for substrate selectivity being steric accessibility. We demonstrated that moving substrate glutamines near the histone fold domain turns them into non-substrates. Furthermore, we found that steric accessibility of glutamines appears to be controlled by higher order chromatin structures. Using nucleosome arrays as substrates, we successfully showed that formation of chromatin fibers in vitro reduces levels of monoaminylation. We then successfully extended these findings to a cell based system, showing that TGM2 is not enriched in heterochromatin and that H3Q5 serotonylation and H3K9me3 are mutually exclusive marks. In total, our results strongly suggest that TGM2, and monoaminylation, are excluded from heterochromatin based on steric accessibility
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Project description:Epigenetic regulation of mutually exclusive transcription within the var gene family is important for infection and pathogenesis of the malaria parasite Plasmodium falciparum. var genes are kept transcriptionally silent via heterochromatic clusters located at the nuclear periphery; however, only a few proteins have been shown to play a direct role in var gene transcriptional regulation. Importantly, the chromatin components that contribute to var gene nuclear organization remain unknown. Here, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for de novo identification of factors associated with specific transcriptional regulatory sequences of var genes. Tagged, catalytically inactive Cas9 (“dCas9”) was targeted to var gene promoters or introns, cross-linked, and immunoprecipitated with all DNA, proteins, and RNA associated with the targeted locus. Chromatin immunoprecipitation followed by sequencing demonstrated that genome-wide dCas9 binding was specific and robust. Proteomics analysis of dCas9-immunoprecipitates identified specific proteins for each target region, including known and novel factors such as DNA binding proteins, chromatin remodelers, and structural proteins. We also demonstrate the ability to immunoprecipitate RNA that is closely associated to the targeted locus. Our CRISPR/dCas9 study establishes a new tool for targeted purification of specific genomic loci and advances understanding of virulence gene regulation in the human malaria parasite.
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