Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing target epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset

Successful treatment of metastatic uveal melanoma with ipilimumab and nivolumab after severe progression under tebentafusp: a case report

Metastatic uveal melanoma (UM) is a rare form of melanoma differing from cutaneous melanoma by etiology, prognosis, driver mutations, pattern of metastases and poor response rate to immune checkpoint inhibitors (ICI). Recently, a bispecific gp100 peptide-HLA-directed CD3 T cell engager, tebentafusp, has been approved for the treatment of HLA-A*02:01 metastatic or unresectable UM. While the treatment regime is complex with weekly administrations and close monitoring, the response rate is limited. Only a few data exist on combined ICI in UM after previous progression on tebentafusp. In this case report, we present a patient with metastatic UM who first suffered extensive progression under treatment with tebentafusp but in the following had an excellent response to combined ICI. We discuss possible interactions that could explain responsiveness to ICI after pretreatment with tebentafusp in advanced UM

Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2

Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3( DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tailmodifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation

Molecular Processes Connecting DNA Methylation Patterns with DNA Methyltransferases and Histone Modifications in Mammalian Genomes

DNA methylation is an essential part of the epigenome chromatin modification network, which also comprises several covalent histone protein post-translational modifications. All these modifications are highly interconnected, because the writers and erasers of one mark, DNA methyltransferases (DNMTs) and ten eleven translocation enzymes (TETs) in the case of DNA methylation, are directly or indirectly targeted and regulated by other marks. Here, we have collected information about the genomic distribution and variability of DNA methylation in human and mouse DNA in different genomic elements. After summarizing the impact of DNA methylation on genome evolution including CpG depletion, we describe the connection of DNA methylation with several important histone post-translational modifications, including methylation of H3K4, H3K9, H3K27, and H3K36, but also with nucleosome remodeling. Moreover, we present the mechanistic features of mammalian DNA methyltransferases and their associated factors that mediate the crosstalk between DNA methylation and chromatin modifications. Finally, we describe recent advances regarding the methylation of non-CpG sites, methylation of adenine residues in human cells and methylation of mitochondrial DNA. At several places, we highlight controversial findings or open questions demanding future experimental work

Molecular Processes Connecting DNA Methylation Patterns with DNA Methyltransferases and Histone Modifications in Mammalian Genomes

The authors wish to make the following correction to their paper [...

Engineering of Effector Domains for Targeted DNA Methylation with Reduced Off-Target Effects

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