5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are the main epigenetic modifications of mammalian DNA, and play crucial roles in cell differentiation, development, and tumorigenesis. Both modifications co-exist with unmodified cytosine in palindromic CpG dyads in different symmetric and asymmetric combinations across the two DNA strands, each having unique regulatory potential. To facilitate investigating the individual functions of such dyad modifications, we report HM-DyadCap. This method employs an evolved methyl-CpG-binding domain (MECP2 HM) for the direct capture and sequencing of DNA fragments containing the CpG dyad hmC/mC. Binding studies reveal a high discrimination of MECP2 HM against off-target dinucleotides. We conduct comparative mapping experiments for mESC genomes with HM-DyadCap, standard MethylCap employing wild-type MECP2, as well as MeDIP and hMeDIP protocols. We find that MECP2 HM is blocked by hmC glucosylation and conduct control enrichments with glucosylated genomes that indicate highly selective enrichment of hmC/mC dyads by MECP2 HM. Metagene profiles correlate hmC/mC marks with actively transcribed genes and reveal global enrichment in gene bodies as well as depletion at transcription start sites. We anticipate that HM-DyadCap will enable effective enrichment and mapping of hmC/mC marks with broad applicability for unraveling the function of this dyad in chromatin biology and cancer.

Aminoacyl-tRNA synthetases mediate the activation and transfer of amino acids to their cognate tRNA, which constitutes one of the initial events in protein biosynthesis. Even though different mechanisms of action have been proposed for the catalysis of these enzymes, their entire catalytic cycle remains elusive. Here, we used electron paramagnetic resonance spectroscopy in vitro and in cells in combination with molecular dynamics simulations to study the role of amino acid interactions in the catalytic cycle of pyrrolysyl-tRNA synthetases (PylRS), a widely used tool for genetic code expansion. Experiments using the paramagnetic non-canonical amino acid SLK-1 revealed the presence and occupation of secondary amino acid binding sites in PylRS located at the intermonomer interface, distant from the catalytic binding site. Based on our results, we propose a model that assumes an alternating mode of action of the two PylRS monomers for the catalytic cycle of PylRS.

The cytosine (C) modifications 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are central regulatory elements of mammalian genomes. Both marks occur in double-stranded DNA in either strand-symmetric or -asymmetric fashion, but it is still poorly understood how this symmetry information is selectively read out by the nuclear proteome as the basis of potential symmetry-dependent regulation. We report enrichment/proteomics studies with promoter probes being strand-symmetrically or asymmetrically modified with C, mC, and hmC, enabling comparison of their reader profiles in the same sequence, tissue, and experimental contexts. We identify a high number of tissue-specific readers for hmC-modified sequences that fall into distinct, probe-specific sub-groups, including members of important transcription factor classes and chromatin regulators. Among them, we discover the master regulators MYC and MAX that play central roles in cell (de)differentiation and cancer progression to read hmC in a sequence-dependent manner. We also find RFX5, a transcription factor involved in primary MHC class II deficiency, to discriminate between hmC symmetries in CpG dyads. Our findings provide further support for the hypothesis that hmC symmetry information can provide distinct regulatory outputs and provide a resource for studying the molecular mechanisms triggered by symmetric and asymmetric hmC modifications in chromatin regulation during development and disease.

DNA aptamers are short, single-stranded DNA molecules that bind specifically to a range of targets such as proteins, cells, and small molecules. Typically, they are utilized in the development of therapeutic agents, diagnostics, drug delivery systems, and biosensors. Although aptamers perform well in controlled extracellular environments, their intracellular use has been less explored due to challenges of expressing them in vivo. In this study, we employed the bacterial retron system Eco2 to express a DNA light-up aptamer in Escherichia coli. Our data confirms that structure-guided insertion of the aptamer domain into the non-coding region of the retron enables reverse transcription and biosynthesis of functional aptamer constructs in bacteria. The purified DNA aptamer synthesized under intracellular conditions shows comparable activity to a chemically synthesized control. Our findings demonstrate that retrons can be used to express short DNA aptamers within living cells, potentially broadening and optimizing their application in intracellular settings.

ZUSAMMENFASSUNG Aminoacyl‐tRNA‐Synthetasen vermitteln die Aktivierung und Übertragung von Aminosäuren auf ihre jeweilige tRNA und stellen damit eines der initialen Ereignisse der Proteinbiosynthese dar. Obwohl verschiedene Wirkmechanismen für die Katalyse dieser Enzyme vorgeschlagen wurden, bleibt ihr vollständiger katalytischer Zyklus bislang unvollständig verstanden. Hier verwendeten wir Elektronenspinresonanz‐Spektroskopie (EPR) in vitro und in Zellen in Kombination mit Molekulardynamik‐Simulationen, um die Rolle von Aminosäure‐Interaktionen im katalytischen Zyklus der Pyrrolysyl‐tRNA‐Synthetasen (PylRS) zu untersuchen, einem weit verbreiteten System zur Erweiterung des genetischen Codes. Experimente mit der paramagnetischen nicht‐kanonischen Aminosäure SLK1 zeigten das Vorhandensein und die Belegung sekundärer Aminosäure‐Bindungsstellen in PylRS, die an der Intermonomer‐Grenzfläche liegen und sich vom katalytischen Bindungsort unterscheiden. Auf Grundlage unserer Ergebnisse schlagen wir ein Modell vor, das einen alternierenden Wirkmodus der beiden PylRS‐Monomere für den katalytischen Zyklus von PylRS annimmt.

ABSTRACT 5-methylcytosine (mC) and its oxidized derivatives are epigenetic modifications of mammalian DNA that play key roles in transcription, cell differentiation, and cancer. They predominantly occur within palindromic CpG dyads, creating multiple possible combinations across the two dyad strands, each representing a chemically distinct DNA major groove mark. Among the modifications, 5-formylcytosine (fC) interacts with a large number of proteins and has been associated with important roles in chromatin regulation. However, it is poorly understood whether nuclear proteins selectively recognize the symmetry of individual fC-containing CpG dyads. Here, we report the first proteome-wide interaction profiles of symmetric and asymmetric fC modifications in mammalian DNA. Our analysis spans several human and mouse cell lines and three DNA promoter probes containing CpG dyads with five distinct combinations of fC, C, and mC nucleobases. We identify a diverse set of fC reader proteins with distinct symmetry preferences, including transcription factors (e.g., MAX, HEY1, RFX5, SIX1, SIX2, and FOXJ3) and DNA repair proteins (MPG and TDG). Notably, some proteins act as fC readers or anti-readers depending on the sequence context, suggesting a role for fC in modulating their target selection. Our data reveal widespread symmetry-specific recognition of fC by mammalian proteins and provide a rich resource for studying its potential functions in chromatin regulation. GRAPHICAL ABSTRACT

5-Methylcytosine (mC) is a key regulatory element of mammalian genomes, and plays important roles in development and disease. mC is predominantly written onto CpG dyads by DNA methyltransferases, and can be further oxidized by ten-eleven translocation dioxygenases (TETs) to 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine. This process results in different symmetric and asymmetric combinations of cytosine forms across the two strands of CpGs, each of which represents a unique physicochemical signature in the major groove of DNA. A comprehensive understanding of the individual functions of oxidized mC modifications can therefore only be achieved by considering both strands of CpG dyads. Here, we provide a brief overview of the current state of knowledge on the sequencing and mapping of individual CpG dyad states, their influence on the intrinsic properties of DNA, and their interactions with chromatin proteins.

Ten-eleven translocation (TET) methylcytosine dioxygenases are part of the epigenetic regulatory machinery that erases DNA methylation. Aberrant TET activities are frequently found in hematopoietic malignancies, where loss of TET2 function leads to DNA hypermethylation. A comprehensive understanding of the biological role of TETs is essential to elucidate disease pathogenesis and identify novel therapeutic strategies. We present a robust pipeline integrating protein X-ray crystallography, molecular modeling, and pharmacophore analysis to advance the current TET inhibitor development. In addition, we have synthesized and evaluated a series of 8-hydroxyquinoline (8-HQ) derivatives, demonstrating their potential as chemical tools to explore TET function further. These findings lay the groundwork for a TET-centered therapeutic approach.

Ubiquitination is a dynamic post-translational modification governing protein abundance, function, and localization in eukaryotes. The Ubiquitin protein is conjugated to lysine residues of target proteins, but can also repeatedly be ubiquitinated itself, giving rise to a complex code of ubiquitin chains with different linkage types. To enable studying the cellular dynamics of linkage-specific ubiquitination, light-activatable polyubiquitin chain formation is reported here. By incorporating a photocaged lysine at specific sites within ubiquitin through amber codon suppression, light-dependent activation of ubiquitin chain extension is enabled for the monitoring of linkage-specific polyubiquitination. The studies reveal rapid, minute-scale ubiquitination kinetics for K11, K48, and K63 linkages. The role of individual components of the ubiquitin-proteasome system in K48-initiated chain synthesis is further studied by small molecule inhibition. The approach expands current perturbation strategies with the ability to control linkage-specific ubiquitination with high temporal resolution and should find broad application for studying ubiquitinome dynamics.

Mammalian genomes are regulated by epigenetic cytosine (C) modifications in palindromic CpG dyads. Including canonical cytosine 5-methylation (mC), a total of four different 5-modifications can theoretically co-exist in the two strands of a CpG, giving rise to a complex array of combinatorial marks with unique regulatory potentials. While tailored readers for individual marks could serve as versatile tools to study their functions, it has been unclear whether a natural protein scaffold would allow selective recognition of marks that vastly differ from canonical, symmetrically methylated CpGs. We conduct directed evolution experiments to generate readers of 5-carboxylcytosine (caC) dyads based on the methyl-CpG-binding domain (MBD), the widely conserved natural reader of mC. Despite the stark steric and chemical differences to mC, we discover highly selective, low nanomolar binders of symmetric and asymmetric caC-dyads. Together with mutational and modelling studies, our findings reveal a striking evolutionary flexibility of the MBD scaffold, allowing it to completely abandon its conserved mC recognition mode in favour of noncanonical dyad recognition, highlighting its potential for epigenetic reader design.

5-Methylcytosine (5mC) is the central epigenetic mark of mammalian DNA, and plays fundamental roles in chromatin regulation. 5mC is dynamically read and translated into regulatory outputs by methyl-CpG-binding domain (MBD) proteins. These multidomain readers recognize 5mC via an MBD domain, and undergo additional domain-dependent interactions with multiple additional chromatin components. However, studying this dynamic process is limited by a lack of methods to conditionally control the 5mC affinity of MBD readers in cells. Light-control of MBD association to chromatin by genetically encoding a photocaged serine at the MBD-DNA interface is reported. The authors study the association of MBD1 to mouse pericentromeres, dependent on its CxxC3 and transcriptional repressor domains (TRD) which interact with unmethylated CpG and heterochromatin-associated complexes, respectively. Both domains significantly modulate association kinetics, arguing for a model in which the CxxC3 delays methylation responses of MBD1 by holding it at unmethylated loci, whereas the TRD promotes responses by aiding heterochromatin association is studied. Their approach offers otherwise inaccessible kinetic insights into the domain-specific regulation of a central MBD reader, and sets the basis for further unravelling how the integration of MBDs into complex heterochromatin interaction networks control the kinetics of 5mC reading and translation into altered chromatin states.

Correction for 'A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2' by Shubhendu Palei et al., RSC Med. Chem., 2022, 13, 1540-1548, https://doi.org/10.1039/D2MD00186A.

[This corrects the article DOI: 10.1039/D2MD00186A.].

Transcription-activator-like effectors (TALEs) are programmable DNA binding proteins that can be used for sequence-specific, imaging-based analysis of cellular 5-methylcytosine. However, this has so far been limited to highly repetitive satellite DNA. To expand this approach to the analysis of coding single gene loci, we here explore a number of signal amplification strategies for increasing imaging sensitivity with TALEs. We develop a straightforward amplification protocol and employ it to target the MUC4 gene, which features only a small cluster of repeat sequences. This offers high sensitivity imaging of MUC4, and in costaining experiments with pairs of one TALE selective for unmethylated cytosine and one universal control TALE enables analyzing methylation changes in the target independently of changes in target accessibility. These advancements offer prospects for 5-methylcytosine analysis at coding, nonrepetitive gene loci by the use of designed TALE probe collections.

5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel 'CpG duplex marks' could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.

Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent regulatory potential, and it initializes active demethylation by the base excision repair pathway. Potent small molecule inhibitors would be useful tools to study TET functions by conditional control. To facilitate the discovery of such tools, we here report a high-throughput screening pipeline and its application to screen and validate 31.5k compounds for inhibition of TET2. Using a homogenous fluorescence assay, we discover a novel quinoline-based scaffold that we further validate with an orthogonal semi-high throughput MALDI-MS assay for direct monitoring of substrate turnover. Structure-activity relationship (SAR) studies involving >20 derivatives of this scaffold led to the identification of optimized inhibitors, and together with computational studies suggested a plausible model for its mode of action.

5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC CpG, discriminates against all 14 other modified CpG dyads, and rivals the selectivity of wild-type MeCP2. Structural studies indicate that this protein has a conserved scaffold and recognizes hmC and mC with two dedicated sets of residues. The mutant allows us to selectively address and enrich hmC/mC-containing DNA fragments from genomic DNA backgrounds. We anticipate that this novel probe will be a versatile tool to unravel the function of hmC/mC marks in diverse aspects of chromatin biology.

Methyl-CpG binding domain (MBD) proteins and ten-eleven-translocation (TET) dioxygenases are the readers and erasers of 5-methylcytosine (5mC), the central epigenetic mark of mammalian DNA. We employ light-activatable human TET1 controlled by a genetically encoded photocaged serine to enable in vivo kinetic studies of their interplay at the common substrate methylated cytosine-guanine (mCpG). We identify the multidomain reader MBD1 to negatively regulate TET1-catalyzed 5mC oxidation kinetics via its mCpG-binding MBD domain. However, we also identify the third Cys-x-x-Cys (CXXC3) domain of MBD1 to promote oxidation kinetics by TET1, dependent on its ability to bind nonmethylated CpG, the final product of TET-mediated mCpG oxidation and active demethylation. In contrast, we do not observe differences in TET1 regulation for MBD1 variants with or without the transcriptional repressor domain. Our approach reveals a complex, domain-dependent interplay of these readers and erasers of 5mC with different domain-specific contributions of MBD1 to the overall kinetics of TET1-catalyzed global 5mC oxidation kinetics that contribute to a better understanding of dynamic methylome shaping.

The authors wish to make the following two corrections to this paper [...]