Biology subjects

Striedner, Y.

Publications and source records attributed to Striedner, Y..

4 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↗

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↗

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↗