Biology subjects

Engelhard, L.

Publications and source records attributed to Engelhard, L..

4 recordsLinked to original sources

bioRxiv2026

Proteomics-Based Discovery of Symmetry-Specific Readers and Antireaders of 5-Formylcytosine in Mammalian DNA

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 O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/717454v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@10773borg.highwire.dtl.DTLVardef@103ced4org.highwire.dtl.DTLVardef@fff20eorg.highwire.dtl.DTLVardef@16c9678_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

HM-DyadCap - Capture and Mapping of 5-Hydroxymethyl-cytosine/5-Methylcytosine CpG Dyads in Mammalian DNA

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 unravelling the function of this dyad in chromatin biology and cancer.

genomics↗

bioRxiv2025

Mammalian Proteome Profiling Reveals Readers and Antireaders of Strand-Symmetric and -Asymmetric 5-Hydroxymethylcytosine-Modifications in DNA

The cytosine (C) modifications 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are central regulatory elements of mammalian genomes. Both marks occur in dinucleotide contexts of 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 basis of potential symmetry-dependent regulation mechanisms. We report comparative enrichment/proteomics studies with promoter probes being strand-symmetrically or asymmetrically modified with C, mC and hmC, enabling a direct assessment of their reader profiles in the same sequence, tissue, and experimental contexts. We identify in human and mouse nuclear lysates 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 critically involved in primary MHC class II deficiency to discriminate between specific 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.

biochemistry↗

bioRxiv2021

Molecular architecture of black widow spider neurotoxins

Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (-latrotoxin (-LTX)), five specialized on insects (, {beta}, {gamma}, {delta}, {varepsilon}-latroinsectotoxins (LITs), and one on crustaceans (-latrocrustatoxin (-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the -LCT monomer and the {delta}-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel -helices. Both domains are flexibly linked via an N-terminal -helical domain and a small {beta}-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant -LCT and {delta}-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between -LCT and {delta}-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.

biophysics↗