Biology subjects

Tiemann-Boege, I.

Publications and source records attributed to Tiemann-Boege, I..

11 recordsLinked to original sources

bioRxiv2026

G-quadruplex structures act as a novel recognition motif for the meiosis-specific histone methyltransferase PRDM9

The histone methyltransferase PR domain containing protein 9 (PRDM9) is a key determinant of meiotic recombination in humans. It deposits activating histone marks thereby promoting recruitment of the meiotic recombination machinery. It recognizes DNA through a repetitive zinc-finger array that binds specific sequence motifs whose complementary G-rich strands can form DNA secondary structures, particularly G-quadruplexes (G4s). These may present an additional binding platform for PRDM9 and contribute to the formation of a chromatin environment permissive for meiotic recombination. We investigated the relationship between PRDM9 binding sites and G4 motifs using computational analyses of predicted and experimentally validated G4s and found that G4 motifs are among the most prevalent features at PRDM9 binding sites, with the strongest enrichment observed for highly stable G4s, largely independent of loop length. Using electrophoretic mobility shift assays, we further examined whether PRDM9 can bind short, single-stranded G4-forming oligonucleotides in addition to its canonical double-stranded DNA targets. PRDM9 directly bound folded G4 structures, and binding increased with G4 stability. This relationship was observed across different G4 motifs and following stabilization of the same G4 by increasing the potassium concentration or adding a G4-stabilizing ligand. PRDM9 also bound an artificial G4-forming sequence absent from the human genome, which was abolished when mutating the G4 motif to avoid structure formation. Together, these results support a model in which stable G4 structures facilitate PRDM9 recruitment by creating and discrete increased local chromatin accessibility, thereby contributing to the initiation of meiotic recombination. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/742039v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@428bdorg.highwire.dtl.DTLVardef@10ead06org.highwire.dtl.DTLVardef@8171c4org.highwire.dtl.DTLVardef@540459_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

bioRxiv2024

Mitochondrial DNA mutations in human oocytes undergo frequency-dependent selection but do not increase with age

Mitochondria, cellular powerhouses, harbor DNA (mtDNA) inherited from the mothers. MtDNA mutations can cause diseases, yet whether they increase with age in human germline cells--oocytes--remains understudied. Here, using highly accurate duplex sequencing of full-length mtDNA, we detected de novo mutations in single oocytes, blood, and saliva in women between 20 and 42 years of age. We found that, with age, mutations increased in blood and saliva but not in oocytes. In oocytes, mutations with high allele frequencies ([≥]1%) were less prevalent in coding than non-coding regions, whereas mutations with low allele frequencies (<1%) were more uniformly distributed along mtDNA, suggesting frequency-dependent purifying selection. In somatic tissues, mutations caused elevated amino acid changes in protein-coding regions, suggesting positive or destructive selection. Thus, mtDNA in human oocytes is protected against accumulation of mutations having functional consequences and with aging. These findings are particularly timely as humans tend to reproduce later in life. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/627454v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@18242f6org.highwire.dtl.DTLVardef@fa5a97org.highwire.dtl.DTLVardef@51a452org.highwire.dtl.DTLVardef@1fb2374_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Mutations in ErbB2 accumulating in the male germline measured by error-corrected sequencing

Mutations in the male germline are a driving force behind rare genetic diseases. Driver mutations enjoying a selective advantage expand to mutant clusters within the aged testis and are thus overrepresented in sperm with age. Other kinds of driver mutations, occurring prepubescently, have been the focus of recent attention given their high occurrence rate independent of age. Here, we investigated the ErbB2 gene via error-corrected sequencing and detected a high percentage of missense mutations, including recurrent mutations, which were observed mainly in the tyrosine kinase domain with likely functional consequences, as we verified for a subset via biophysical methods. While these mutations increased with age, we found no evidence that they originated from mutational clusters in the aged testis, and young donors also showed an accumulation of driver mutations, suggesting that mutational enrichment is not exclusive to the sexually mature germline but can occur earlier during germline development, and are likely evenly distributed in the testis and stable in size.

genetics↗

bioRxiv2022

Activating mutations in FGFR3 are associated with clonal expansion events and high de novo rates in the male germline

Delayed fatherhood results in a higher risk to inherit a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early or late-onset effects in the offspring. Here, we investigated the mutation frequency in the DNA of human testis and sperm and the activation state of the expressed mutant protein of eight different FGFR3 variants categorized by ClinVar as deleterious, benign, or not reported. Overall, the ligand-independent activation of the mutant protein resulted in a increased number of mutant sperm; although, strong activating mutations did not necessarily result in the highest frequencies. Moreover, only two mutants c.952G>A and c.1620C>A showed an increase with the donors age; the latter also forming larger clonal expansions in the testis. We also showed that the prediction of deleteriousness of a mutation is not always accurate, and similar in silico scores can reflect either a gain-of-function or loss-of-function. Our approach led to the discovery of two novel variants c.1261G>A and c.952G>A to have promiscuous FGFR3 activation and increased mutation frequencies in the male germline. The large fraction of donors with mutations suggests a high de novo rate potentially explained by a selective advantage before the maturation of the male germline. This sequence-function study provides important data for the evaluation and interpretation of variants with relevant clinical implications.

genetics↗

bioRxiv2022

Quantitation of FGFR3 signaling via GRB2 recruitment on micropatterned surfaces

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/MAPK pathway by their tyrosine-kinase activation known to determine cell-growth, tissue differentiation and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some of these mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (mGFP-FGFR3) in certain areas of the plasma membrane of living cells. Receptor activation was then quantified via the recruitment of the downstream signal transducer GRB2 tagged with mScarlet (GRB2-mScarlet) to FGFR3 patterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) in the wildtype (WT), as well as in different FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, K650E). Our data showed that the addition of ligands increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared to previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This may be explained by the different receptor populations probed, since the micropatterning method specifically reports on signaling events at the plasma membrane. Graphical AbstractSpecifications: The maximum size of the image should be 200 x 500 pixels with a minimum resolution of 300 dpi, using Arial font with a size of 10-16 points; Preferred file types: TIFF, EPS, PDF or MS Office files O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/487861v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@4a279eorg.highwire.dtl.DTLVardef@6f35d1org.highwire.dtl.DTLVardef@13661e7org.highwire.dtl.DTLVardef@155b480_HPS_FORMAT_FIGEXP M_FIG C_FIG Research HighlightsO_LIQuantification of FGFR3 signaling in live cells on micropatterned surfaces C_LIO_LIAnalysis of GRB2 recruitment to the mature receptor at the plasma membrane C_LIO_LILigand-independent kinase activation of FGFR3 mutants C_LIO_LIActivation of FGFR3 at the cell surface can be different than in bulk cell extracts C_LI

molecular biology↗

bioRxiv2021

Discovery of an unusual high number of de novo mutations in sperm of older men using duplex sequencing

De novo mutations (DNMs) are an important player in heritable diseases and evolution. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultra-deep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined [~]4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified sites with variant frequencies of 10-4 to 10-5, with an overall mutation frequency of the region of [~]6x10-7. Some of the substitutions were re-current and were found at a higher variant frequency in older donors than in younger ones, or exclusively, in older donors. Also, older donors harbored more mutations associated with congenital disorders. Other mutations were present in both age groups suggesting that these might result from a different mechanism (e.g., post-zygotic mosaicism). We also observed that independent of age, the frequency and deleteriousness of the mutational spectra was more similar to COSMIC than to gnomAD variants. Our approach is an important strategy to identify mutations that could be associated with a gain-of-function of the receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

genetics↗

bioRxiv2019

Increased yields of duplex sequencing data by a series of quality control tools

Duplex sequencing is currently the most reliable method to identify ultra-low frequency DNA variants by grouping sequence reads derived from the same DNA molecule into families with information on the forward and reverse strand. However, only a small proportion of reads are assembled into duplex consensus sequences, and reads with potentially valuable information are discarded at different steps of the bioinformatics pipeline, especially reads without a family. We developed a bioinformatics tool-set that analyses the tag and family composition with the purpose to understand data loss and implement modifications to maximize the data output for the variant calling. Specifically, our tools show that tags contain PCR and sequencing errors that contribute to data loss and lower DCS yields. Our tools also identified chimeras, which result in unpaired families that do not form DCS. Finally, we also developed a tool called Variant Analyzer that re-examines variant calls from raw reads and provides different summary data that categorizes the confidence level of a variant call by a tier-based system. We demonstrate that this tool identified false positive variants tagged by the tier-based classification. Furthermore, with this tool we can include reads without a family and check the reliability of the call, which increases substantially the sequencing depth for variant calling, a particular important advantage for low-input samples or low-coverage regions.

genomics↗

bioRxiv2018

PRDM9 forms an active trimer mediated by its repetitive zinc finger array

PRDM9 has been identified as a meiosis-specific protein that plays a key role in determining the location of meiotic recombination hotspots. Although it is well-established that PRDM9 is a trans-acting factor directing the double strand break machinery necessary for recombination to its DNA binding site, the details of PRDM9 binding and complex formation are not well known. It has been suggested in several instances that PRDM9 acts as a multimer in vivo; however, there is little understanding about the protein stoichiometry or the components inducing PRDM9 multimerization. In this work, we used in vitro binding studies and mass spectrometry to characterize the size of the PRDM9 multimer within the active DNA-protein complex of two different murine PRDM9 alleles, PRDM9Cst and PRDM9Dom2. For this purpose, we developed a strategy to infer the molecular weight of the PRDM9-DNA complex from native gel electrophoresis based on gel shift assays (EMSAs). Our results show that PRDM9 binds as a trimer with the DNA. This multimerization is catalysed by the long ZnF array (ZnF) at the C-terminus of the protein and 11, 10, 7 or 5 ZnFs are already sufficient to form a functional trimer. Finally, we also show that only one ZnF-array within the PRDM9 trimer actively binds to the DNA, while the remaining two ZnF-arrays likely maintain the multimer by ZnF-ZnF interactions. Our results have important implications in terms of PRDM9 dosage, which determines the number of active hotspots in meiotic cells, and contribute to elucidate the molecular interactions of PRDM9 with other components of the meiotic initiation machinery.

molecular biology↗

bioRxiv2018

Family reunion via error correction: An efficient analysis of duplex sequencing data

Duplex sequencing is the most accurate approach for identification of sequence variants present at very low frequencies. Its power comes from pooling together multiple descendants of both strands of original DNA molecules, which allows distinguishing true nucleotide substitutions from PCR amplification and sequencing artifacts. This strategy comes at a cost--sequencing the same molecule multiple times increases dynamic range but significantly diminishes coverage, making whole genome duplex sequencing prohibitively expensive. Furthermore, every duplex experiment produces a substantial proportion of singleton reads that cannot be used in the analysis and are, technically, thrown away. In this paper we demonstrate that a significant fraction of these reads contains PCR or sequencing errors within duplex tags. Correction of such errors allows \"reuniting\" these reads with their respective families increasing the output of the method and making it more cost effective. Additionally, we combine error correction strategy with a number of algorithmic improvements in a new version of the duplex analysis software, Du Novo 2.0, readily available through Galaxy, Bioconda, and as the source code.

bioinformatics↗

bioRxiv2018

Length asymmetry and heterozygosity strongly influences the evolution of poly-A microsatellites at meiotic recombination hotspots

Meiotic recombination has strong, but poorly understood, effects on short tandem repeat (STR) instability. Here, we screened thousands of single recombinant products to characterize the transmission and evolution of polymorphic poly-A repeats at a human recombination hotspot. We show that length asymmetry between heterozygous poly-As plays a key role in the recombination outcome and their transmission. A difference of 10 As (9A/19A) elevates the frequency of non-crossovers, complex recombination products, and long conversion tracts. Moreover, asymmetry also influences STR transmission: the shorter allele is transmitted more frequently (deletion bias) at the asymmetric STR (9A/19A), while the longer allele is favored (insertion bias) at the site with a small STR length difference (6A/7A). Finally, potentially due to this opposing insertion/deletion driven evolution, we find that poly-As are enriched at human recombination hotspots predominantly with short poly-As, possibly influencing open chromatin regions that in turn can activate hotspots.

evolutionary biology↗

bioRxiv2017

LDJump: Estimating Variable Recombination Rates from Population Genetic Data

As recombination plays an important role in evolution, its estimation, as well as, the identification of hotspot positions is of considerable interest. We propose a novel approach for estimating historical recombination along a chromosome that involves a sequential multiscale change point estimator. Our method also permits to take demography into account. It uses a composite likelihood estimate and other summary statistics within a regression model fitted on suitable scenarios. Our proposed method is accurate, computationally fast, and provides a parsimonious solution by ensuring a type I error control against too many changes in the recombination rate. An application to human genome data suggests a good congruence between our estimated and experimentally identified hotspots. Our method is implemented in the R-package LDJump, which is freely available from https://github.com/PhHermann/LDJump.

bioinformatics↗