Biology subjects

Buchmuller, B.

Publications and source records attributed to Buchmuller, B..

3 recordsLinked to original sources

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↗

bioRxiv2022

A translational repression reporter assay for the analysis of RNA-binding protein consensus sites

RNA-binding proteins are essential regulators of RNA processing and function. Translational repression assays can be used to study how they interact with specific RNA sequences by insertion of such a consensus sequence into the 5 untranslated region of a reporter mRNA and measuring reporter protein translation. The straightforward set-up of these translational repression assays avoids the need for the isolation of the protein or the RNA providing speed, robustness, and a low-cost method. Here we report the optimization of the assay to function with linear RNA sequences instead of the previously reported hairpin type sequences to allow the study of a wider variety of RNA-binding proteins. Multiplication of a consensus sequence strongly improves the signal allowing analysis by both fluorescence intensity measurements and fluorescence activated cell sorting.

molecular biology↗

bioRxiv2018

CRISPR/Cas12a-assisted PCR tagging of mammalian genes

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g. GFP), a Cas12a CRISPR RNA for cleavage of the target locus and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artefacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein tagged genes

cell biology↗