Daniel Summerer

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.

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

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.

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.

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.

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.

Cytosine 5-modifications are central to human chromatin regulation. They occur in different combinations in the two DNA strands of CpG dyads, but the functions of individual combinations are poorly understood. We review recent technical developments and biological insights in this area.

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.

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.