Engineering of effector domains for targeted DNA methylation with reduced off-target effects
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ABSTRACT:
PROVIDER: PRJNA596181 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10720185.fastq.gz | Fastqsanger.gz | |||
| SRR10720186.fastq.gz | Fastqsanger.gz | |||
| SRR10720187.fastq.gz | Fastqsanger.gz | |||
| SRR10720188.fastq.gz | Fastqsanger.gz | |||
| SRR10720189.fastq.gz | Fastqsanger.gz |
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Project description:Epigenome editing is a promising technology, potentially allowing the stable reprogramming of gene expression profiles without alteration of the DNA sequence. Targeted DNA methylation has been successfully documented by many groups for silencing selected genes, but recent publications have raised concerns regarding its specificity. In the current work, we developed new EpiEditors for programmable DNA methylation in cells with a high efficiency and improved specificity. First, we demonstrated that the catalytically deactivated Cas9 protein (dCas9)-SunTag scaffold, which has been used earlier for signal amplification, can be combined with the DNMT3A-DNMT3L single-chain effector domain, allowing for a strong methylation at the target genomic locus. We demonstrated that off-target activity of this system is mainly due to untargeted freely diffusing DNMT3A-DNMT3L subunits. Therefore, we generated several DNMT3A-DNMT3L variants containing mutations in the DNMT3A part, which reduced their endogenous DNA binding. We analyzed the genome-wide DNA methylation of selected variants and confirmed a striking reduction of untargeted methylation, most pronounced for the R887E mutant. For all potential applications of targeted DNA methylation, the efficiency and specificity of the treatment are the key factors. By developing highly active targeted methylation systems with strongly improved specificity, our work contributes to future applications of this approach.
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Project description:The transcription factors MEF2C, GATA4, and TBX5 (MGT) are well established for converting fibroblasts into induced cardiomyocytes (iCMs). However, these reprogramming factors contain effector domains, yet their functional roles in cardiac reprogramming remain largely uncharacterized. Furthermore, whether internal deletions of these domains improve or compromise reprogramming efficiency has not been elucidated. Here we found that the combination of MEF2C and GATA4 mutants (MΔGΔT) elevated reprogramming efficiency by 7-fold to ~21%, achieving robust induction of iCMs with well-defined sarcomeric structures.
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Project description:MARTX toxins are large single polypeptide bacterial toxins that translocate multiple cytotoxic and functionally independent effector domains into the cytosol of a target eukaryotic cell. Pandemic Vibrio cholerae El Tor O1 strains secrete a MARTX toxin with three effector domains — the actin crosslinking domain (ACD), the Rho inactivation domain (RID), and the alpha/beta-hydrolase domain (ABH) — to regulate innate immunity and enhance colonization. The goal of this study was to compare changes in the transcriptome of human intestinal epithelial cells (IECs) treated with V. cholerae modified to secret a toxin with only one effector domain to the transcriptome of cells treated with V. cholerae secreting the wild type MARTX toxin that delivers all three effector domains simultaneously. We demonstrate that when all three effectors are delivered there is no change in transcriptional response of IECs compared to untreated cells. However, when only ACD is delivered, transcriptional profiling revealed a significant proinflammatory response is activated. These data suggests that V. cholerae may utilize co-delivery of RID and/or ABH to silence the intestinal immune response to ACD activity. These data provide insight into how the V. cholerae MARTX toxin effector domains function together to alter the innate immune response of IECs during bacterial infection.
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