The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism

The ubiquitin-like, containing PHD and RING finger domains protein 1 (UHRF1) is essential for maintenance DNA methylationby DNA methyltransferase 1 (DNMT1). UHRF1 has been shown to recruit DNMT1 to replicated DNA by the ability of its SET andRING-associated (SRA) domain to bind to hemimethylated DNA. Here, we demonstrate that UHRF1 also increases the activity ofDNMT1 by almost 5-fold. This stimulation is mediated by a direct interaction of both proteins through the SRA domain of UHRF1and the replication focus targeting sequence domain of DNMT1, and it does not require DNA binding by the SRA domain. Disruptionof the interaction between DNMT1 and UHRF1 by replacement of key residues in the replication focus targeting sequence domainled to a strong reduction of DNMT1 stimulation. Additionally, the interaction with UHRF1 increased the specificity of DNMT1for methylation of hemimethylated CpG sites. These findings show that apart from the targeting of DNMT1 to the replicatedDNA UHRF1 increases the activity and specificity of DNMT1, thus exerting a multifaceted influence on the maintenance of DNAmethylatio

H3K14ac is linked to methylation of H3K9 by the triple Tudor domain of SETDB1

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the subnuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substraterecognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals crucial and distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis

Regelung der katalytischen Aktivität und Spezifität der DNA Nukleotid Methyltransferase 1

DNA nucleotide methyltransferase 1 (Dnmt1) is mainly responsible for the maintenance of DNA methylation in mammals and plays a crucial role in the epigenetic control of gene expression. Dnmt1 recognizes and methylates hemimethylated CpG sites formed during DNA replication. In the present work, the mechanistic details of the substrate recognition by the catalytic domain of Dnmt1, the possible role of the CXXC and RFTS domains of Dnmt1 in the regulation of specificity and activity of Dnmt1, and the influence of the Ubiquitin-like PHD and RING finger domain-containing 1 (Uhrf1) protein on the enzymatic properties of Dnmt1 was investigated. Using modified substrates, the functional roles of individual contacts of the Dnmt1 catalytic domain with the CpG site of the DNA substrate were analysed. The data show that the interaction with the 5-methylcytosine:guanine pair is required for the catalytic activity of Dnmt1, whereas the contacts to the non-target strand guanine are not important, since its replacement with adenine increased the activity of Dnmt1. It was proposed that the CXXC domain binding to unmethylated CpG sites increases the specificity of Dnmt1 for hemimethylated DNA. Our data showed that the CXXC domain does not influence the enzyme’s specificity in the full-length Dnmt1. In contrast, mutagenesis in the catalytic domain introducing an M1235S exchange resulted in a significant reduction in specificity. Therefore, the readout for the hemimethylated DNA occurs within its catalytic domain. It was observed in a crystal structure that the RFTS domain of Dnmt1 inhibits the activity of the enzyme by binding to the catalytic domain and blocking the entry of the DNA. By amino acid substitution in the RFTS domain its positioning within the catalytic domain was destabilized and a corresponding increase in the catalytic rate was observed, which supports this concept and suggests a possible mechanism to allosterically regulate the activity of Dnmt1 in cells. Uhrf1 has been shown to target Dnmt1 to replicated DNA, which is essential for DNA methylation. Here it is demonstrated that Uhrf1 as well as its isolated SRA domain increase the activity and specificity of Dnmt1 in an allosteric mechanism. The stimulatory effect was independent of the SRA domain’s ability to bind hemimethylated DNA. The RFTS domain of Dnmt1 is required for the stimulation, since its deletion or blocking of its interaction with the SRA domain, significantly reduced the ability of Uhrf1 to increase the activity and specificity of Dnmt1. Uhrf1, therefore, plays multiple roles that support DNA methylation including targeting of Dnmt1, its stimulation and an increase of its specificity.Die DNA-Methyltransferase 1 (Dnmt1) ist hauptverantwortlich für die Konservierung der DNA-Methylierung bei Säugetieren und spielt eine entscheidende Rolle in der epigenetischen Kontrolle der Genexpression. Dnmt1 erkennt und methyliert hemimethylierte CpG-Stellen, die während der DNA-Replikation gebildet werden. In der vorliegenden Arbeit wurden mechanistische Details der Substraterkennung durch die katalytische Domäne von Dnmt1, die mögliche Rolle der CXXC- und RFTS-Domänen in der Regulation der Spezifität und Aktivität von Dnmt1 sowie der Einfluss des Ubiquitin-like PHD- und RING-Finger-Domänen enthaltenden 1 (Uhrf1) Proteins auf die enzymatischen Eigenschaften von Dnmt1 untersucht. Mit verschiedenen modifizierten Substraten wurde die funktionelle Rolle einzelner Kontakte der katalytischen Domäne von Dnmt1 mit der CpG-Stelle der Substrat-DNA untersucht. Unsere Daten zeigen, dass die Interaktion mit dem 5-Methylcytosin:Guanin-Paar für die katalytische Aktivität von Dnmt1 notwendig ist, während die Kontakte zum im Gegenstrang liegenden Guanin offenbar nicht von Bedeutung sind, da der Austausch dieses Guanins gegen Adenin zu einer erhöhten Aktivität von Dnmt1 führte. In der Literatur wurde vorgeschlagen, dass die CXXC-Domäne durch die Bindung an unmethylierte DNA die Spezifität von Dnmt1 für hemimethylierte DNA erhöhen kann. Wir konnten allerdings zeigen, dass die CXXC-Domäne von Dnmt1 die Spezifität des Enzyms nicht beeinflusst. Im Gegensatz dazu führte der Austausch M1235S in der katalytischen Domäne von Dnmt1 zu einer signifikanten Reduktion der Spezifität. Daher muss die Erkennung hemimethylierter DNA innerhalb der katalytischen Domäne von Dnmt1 stattfinden. Die Untersuchung einer Kristallstruktur ergab, dass die RFTS-Domäne die Aktivität von Dnmt1 durch Bindung an die katalytische Domäne und die Blockierung der Eintrittsstelle der DNA hemmt. Durch Aminosäuresubstitutionen in der RFTS-Domäne konnte deren Positionierung innerhalb der katalytischen Domäne destabilisiert werden, was zu einer entsprechenden Erhöhung der katalytischen Rate führte. Unsere Beobachtung unterstützt dieses Konzept und zeigt einen möglichen Mechanismus auf, mit dem die Aktivität von Dnmt1 in Zellen allosterisch reguliert werden kann. Uhrf1 rekrutiert Dnmt1 an kürzlich replizierte DNA. Außerdem konnten wir zeigen, dass Uhrf1 sowie seine isolierte SRA-Domäne die Aktivität und Spezifität von Dnmt1 nach einem allosterischen Mechanismus erhöht. Diese stimulierende Wirkung war unabhängig von der Fähigkeit der SRA-Domäne, hemimethylierte DNA zu binden. Die RFTS-Domäne war für die Stimulation erforderlich, da ihre Entfernung oder die Blockade der Wechselwirkung mit der SRA-Domäne die Fähigkeit von Uhrf1, die Aktivität und Spezifität von Dnmt1 zu steigern, deutlich reduziert. Unsere Daten zeigen, dass Uhrf1 bei der Unterstützung der DNA-Methylierung mehrere Aufgaben erfüllt, welche die Rekrutierung, Stimulation und Steigerung der Spezifität von Dnmt1 umfassen

Development of novel antineoplastic treatment approaches by targeted epigenetic silencing of the vascular endothelial growth factor A and its receptors

Decades of efforts of clinicians and scientists to fight against cancer resulted in the development of multiple targeted therapies, which improved clinical outcomes for many types of tumors. Nevertheless, cancer is still the second leading cause of death worldwide, thus more efficient therapeutic approaches are urgently needed. Inhibitors of the VEGFA/VEGFRs axis have shown efficiency against various solid malignancies via reduction of neoangiogenesis of tumors and inhibition of autocrine stimulation of proliferation, migration and invasion of tumor cells by VEGFA. Monoclonal inhibitory antibodies against VEGFA or its receptors efficiently block signaling along the VEGFA/VEGFRs axis, but are very expensive and require repetitive drug injections since they function at the posttranslational level. Targeted epigenome editing is a new emerging technology allowing to control the expression of selected genes at the transcriptional level. Regulation of gene expression is achieved via rewriting of chromatin marks at their cis-regulatory elements. For example, setting of DNA methylation at promoters may lead to stable silencing of corresponding genes. Epigenome editing can be achieved by EpiEditors, artificial chimeric proteins composed of a DNA-binding domain and a DNA methyltransferase, designed to set DNA methylation at target genomic loci. Previous studies demonstrated that methylation of the VEGFA promoter to approximately 50 % resulted in 70 % decrease of VEGFA gene expression in the ovarian cancer cell line SKOV3. This result was promising but only a moderate level of methylation was achieved. Additionally, off-target activity, epigenome editing at non-target genomic loci, has been reported in several studies, and has to be eliminated. This project aimed to develop the technology further to i) improve on-target editing efficiency at the VEGFA promoter to gain higher methylation level; ii) establish multiplex editing to methylate promoters of VEGFA and its receptors VEGFR1, VEGFR2 for simultaneous silencing of all three genes; iii) decrease off-target editing and iv) analyse established DNA methylation patterns. These goals were approached as follows: i) The DNA genomic targeting technique of EpiEditors was changed from ZPF- to the CRISPR/Cas9 technology. This allowed to employ the recently published EpiEditor composed of the dCas9-10xSunTag protein and the anti-SunTag antibody fused with the highly active DNMT3ACD-DNMT3LCD chimeric methyltransferase. The SunTag allows signal amplification by recruiting up to 10 effector domains, which lead to 40 % higher DNA methylation of the VEGFA promoter compared to the EpiEditors using a single effector domain published previously. ii) Multiplex methylation of the VEGFA, VEGFR1 and VEGFR2 promotors was established for the first time. Targeting of the dCas9-based EpiEditor to multiple loci was realized using vectors expressing several sgRNAs targeting these genes, which theoretically increases editing efficiency compared to co-transfection of vectors expressing single sgRNA used in previous reports. iii) Implementation of dCas9/sgRNA instead of ZFP used previously for targeting the VEGFA promoter significantly improved editing specificity by reducing of off-target effects originating from the DBD. In addition, use of more specific mutant version R887E of EpiEditor showed 5-fold lower off-target activity at a single representative locus. iv) An in-depth analysis of the introduced DNA methylation patterns revealed that the degree of methylation of individual CpG sites depends on several parameters, such as their distance from the sgRNA binding site and the flanking sequence preference of the effector domain. Furthermore, CpG sites can be in hemi- and fully methylated state and the predominance of one or the other state depends on the target locus. The current study led to the development of multiplex epigenome editing of VEGFA and its receptors with the efficiency and specificity superior to previous reports and revealed insights into established DNA patterns which will enhance future design to achieve stable genes silencing and desired antineoplastic therapeutic effects

HISAT2 indexes for mm39

HISAT2 indexes for mm39Kim, D., Paggi, J.M., Park, C. et al. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37, 907–915 (2019).</p

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 [...

RepEnTools - Cleaned RMSK.gtf for analysis

Cleaned RMSK.gtf for RepEnToolsOnly suitable for chm13v2Original bed12 file from {Hoyt, 2022}, made with repeat masker {www.repeatmasker.org}, downloaded from UCSC Table Browser {Karolchik, 2004}. Version: hub_3671779The repeat masker (RMSK) annotation file employed in RepEnTools is adjusted to correctly represent the annotated repeat element boundaries (e.g. red element). The UCSC Table Browser allows retrieval of the RMSK files generated specifically for chm13v2 {Hoyt, 2022; Karolchik, 2004 10.1093/nar/gkh103}. While the BigBed file displays the elements with correct annotations, visualising the unpacked version (bed12) reveals both the pHMM model used by RMSK (thin) and the actual annotated segment (thick). Notably, the UCSC gtf output reflects only the pHMM model, not the true repeat element (vertical red dashed line). To adjust the annotation file, we extracted the exons as gtf, sorted and merged overlapping annotations of identical name twice (in-house script), and performed table operations. Browser views generated using IGV v2.15 (Integrative Genomics Viewer) {Thorvaldsdottir, 2013}. pHMM – profile Hidden Markov Model.</p

RepEnTools - mm39 RMSK.gtf

mm39 RMSK.gtf for RepEnToolsOnly suitable for mm39Original bed12 file made with repeat masker (www.repeatmasker.org), downloaded from UCSC Table Browser (Karolchik, 2004). Last updated: 2020-07-30The repeat masker (RMSK) annotation file employed in RepEnTools is adjusted to correctly represent the information on the repeat element.</p

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