Characterization of the meiosis-specific zinc-finger protein PRDM9

PRDM9 (PR-domain containing protein 9) is a multi-domain protein, expressed in male and female germ cells at the beginning of meiosis. Using its long DNA-binding zinc finger domain, it controls the sites of the genetic exchange between maternal and paternal chromosomes, so called "recombination hotspots". This protein plays an important role for the protection of genome integrity, since it directs hotspots away from regions with gene expression. This work presents first insights into the kinetics of the PRDM9-DNA interaction. Using state-of-the-art, biophysical techniques such as switchSENSE (Dynamic Biosensor GmbH, Munich) and gel-shift assays (EMSA), we observed that PRDM9 forms a highly stable complex with specific hotspot DNA lasting many hours. Such a long-lived interaction may be required for a successful translocation of the hotspot DNA to the “recombination initiation machinery” that targets the DNA for a double strand break necessary for the start of meiotic recombination. Furthermore, we analyzed the effect of single nucleotide polymorphisms in the DNA on the affinity of PRDM9. Moreover, this thesis tested several cell systems for the expression of recombinant murine PRDM9Cst addressing their advantages and disadvantages. The large size of PRDM9 and the repetitive nature of its zinc finger domain poses difficulties to express this protein in multiple hosts, which is further complicated by its insolubility and degradation. Furthermore, several attempts of purifying PRDM9 led to a loss of function. Here we present an optimized protocol for PRDM9Cst lysate preparation, yielding a sufficient level of purity to be compatible with switchSENSE and EMSA, without the need for an affinity purification.PRDM9 („PR-domain containing protein 9“) ist ein Protein mit verschiedenen funktionellen Domänen, das zu Beginn der Meiose in weiblichen und männlichen Keimzellen exprimiert wird. Mit seiner langen Zink-Finger Domäne bindet es an DNS und reguliert die Regionen im Genom wo ein Austausch zwischen mütterlichen und väterlichen Chromosomen, den so genannten „Hotspots“ der Rekombination, stattfindet. Dieses Protein dient dem Schutz des Genoms, indem es die meiotische Rekombination von Regionen abwendet, die für die Genexpression wichtig sind. In dieser Arbeit werden erste Einblicke in die Bindungskinetik von PRDM9 an DNS präsentiert. Mit Hilfe von switchSENSE und Gel-Shift Experimenten (EMSA), haben wir herausgefunden, dass PRDM9 einen sehr stabilen Komplex mit spezifischer Hotspot-DNA bildet, der über einen Zeitraum von mehreren Stunden bestehen bleibt. Eine derart langlebige Interaktion könnte nötig sein, um die Hotspot-DNA mit der „Rekombinations-Initiations-Maschinerie“ in Kontakt zu bringen, welche die nächsten Schritte in der Rekombination einleitet. Außerdem wurde die Auswirkung von Einzelnukleotid-Polymorphismen in der DNA auf die Affinität von PRDM9 untersucht. Des Weiteren wurden in dieser Dissertation verschiedenste Expressionssysteme zur Produktion der, in der Maus vorkommenden, rekombinanten PRDM9Cst Variante getestet und deren Vor- und Nachteile gegeneinander abgewogen. Die beträchtliche Größe von PRDM9 sowie der repetitive Aufbau der Zink Finger Domäne verursachten in mehreren Expressionssystemen beträchtliche Probleme. Eine weitere Herausforderung stellten die Fragmentierung von PRDM9 und dessen Unlöslichkeit dar. Sämtliche Versuche zur Aufreinigung von PRDM9 zeigten letztendlich einen Funktionsverlust des Proteins. In dieser Arbeit stellen wir ein optimiertes Protokoll zur Herstellung eines PRDM9Cst Lysats vor, das eine ausreichende Reinheit zum Zwecke der switchSENSE und EMSA Messungen aufweist, ohne der Notwendigkeit für eine weitere Affinitätsreinigung.Author Mag.a Yasmin StriednerZusammenfassung in deutscher SpracheUniversität Linz, Dissertation, 2017OeBB(VLID)191701

The long zinc finger domain of PRDM9 forms a highly stable and long-lived complex with its DNA recognition sequence

PR domain containing protein 9 (PRDM9) is a meiosis-specific, multi-domain protein that regulates the location of recombination hotspots by targeting its DNA recognition sequence for double-strand breaks (DSBs). PRDM9 specifically recognizes DNA via its tandem array of zinc fingers (ZnFs), epigenetically marks the local chromatin by its histone methyltransferase activity, and is an important tether that brings the DNA into contact with the recombination initiation machinery. A strong correlation between PRDM9-ZnF variants and specific DNA motifs at recombination hotspots has been reported; however, the binding specificity and kinetics of the ZnF domain are still obscure. Using two in vitro methods, gel mobility shift assays and switchSENSE, a quantitative biophysical approach that measures binding rates in real time, we determined that the PRDM9-ZnF domain forms a highly stable and long-lived complex with its recognition sequence, with a dissociation halftime of many hours. The ZnF domain exhibits an equilibrium dissociation constant (K D) in the nanomolar (nM) range, with polymorphisms in the recognition sequence directly affecting the binding affinity. We also determined that alternative sequences (1516 nucleotides in length) can be specifically bound by different subsets of the ZnF domain, explaining the binding plasticity of PRDM9 for different sequences. Finally, longer binding targets are preferred than predicted from the numbers of ZnFs contacting the DNA. Functionally, a long-lived complex translates into an enzymatically active PRDM9 at specific DNA-binding sites throughout meiotic prophase I that might be relevant in stabilizing the components of the recombination machinery to a specific DNA target until DSBs are initiated by Spo11.(VLID)342326

Exploring the Micro-Mosaic Landscape of FGFR3 Mutations in the Ageing Male Germline and Their Potential Implications in Meiotic Differentiation.

Peer reviewed: TrueAcknowledgements: We would like to thank Philipp Hermann for the advice on statistical testing. We would also like to thank the Greenwood Genetic Center, South Carolina, for kindly providing a heterozygous DNA with the c.1948A>G mutation. Open Access Funding by the University of Linz.Publication status: PublishedFunder: Johannes Kepler UniversityAdvanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants-c.1138G>A (p.G380R) and c.1948A>G (p.K650E)-which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor's age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline

Exploring the Micro-Mosaic Landscape of <i>FGFR3</i> Mutations in the Ageing Male Germline and Their Potential Implications in Meiotic Differentiation

Advanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants—c.1138G>A (p.G380R) and c.1948A>G (p.K650E)—which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor’s age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline

Exploring the micro-mosaic landscape of FGFR3 mutations in the ageing male germline and their potential implications in meiotic differentiation

Advanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants—c.1138G>A (p.G380R) and c.1948A>G (p.K650E)—which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor’s age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline