RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases

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Biophysical characterization of histone H3.3 K27M point mutation

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NODULIN HOMEOBOX is required for heterochromatin homeostasis in Arabidopsis

Arabidopsis NODULIN HOMEOBOX (NDX) is a nuclear protein described as a regulator of specific euchromatic genes within transcriptionally active chro- mosome arms. Here we show that NDX is primarily a heterochromatin reg- ulator that functions in pericentromeric regions to control siRNA production and non-CG methylation. Most NDX binding sites coincide with pericen- tromeric het-siRNA loci that mediate transposon silencing, and are antag- onistic with R-loop structures that are prevalent in euchromatic chromosomal arms. Inactivation of NDX leads to differential siRNA accumulation and DNA methylation, of which CHH/CHG hypomethylation colocalizes with NDX binding sites. Hi-C analysis shows significant chromatin structural changes in the ndx mutant, with decreased intrachromosomal interactions at pericen- tromeres where NDX is enriched in wild-type plants, and increased inter- chromosomal contacts between KNOT-forming regions, similar to those observed in DNA methylation mutants. We conclude that NDX is a key reg- ulator of heterochromatin that is functionally coupled to het-siRNA loci and non-CG DNA methylation pathways

Transzkripciós faktorok és DNS közti kapcsolat vizsgálati módszereinek áttekintése és a kromatin immunprecipitáció módszer gyakorlati bemutatása

Szakdolgozatom során a DNS-fehérje interakciók vizsgálatával foglalkoztam. Irodalmi adatok alapján bemutattam ezen vizsgálatokhoz szükséges módszerek működési elveit. Kromatin immunprecipitációt végeztünk hiszton poszttranszlációs módosítások és transzkripciós faktorok DNS-hez való kötődésének vizsgálatára. Ezen vizsgálatok és jövőbeli terveink okán megismerkedtem a Sox transzkripciós faktorokkal, a Sox trióval (Sox5, 6, 9), különösen a porc differenciációban betöltött szerepükre fektetve a hangsúlyt.BSc/BABiológia BScg

Lipidképződés vizsgálata emlőrákos sejtvonalakon

A mellrák a második leggyakrabban diagnosztizált daganattípus a világon. Az esetek túlnyomó részében a tumorok ösztrogén receptor pozitívak. A magas ösztrogénszint az ösztrogén receptorokon keresztül hatva a daganatos sejtek proliferációját serkenti. A szteroid hormonok (pl. ösztrogén) szintézisének forrása a koleszterin, többek között emiatt a lipidmetabolizmusnak jelentős hatása van a mellrák kialakulására. Diplomamunkám során adipogenetikus mix (dexametazon, 3-izobutil-1-metilxantin, rosiglitazon, inzulin, aszkorbinsav) ill. külön-külön a mix alkotóelemek lipidképződésre gyakorolt hatását vizsgáltam emlőrákos sejtvonalakon. Két jól elkülönülő mellrák típushoz tartozó sejtvonalat, MCF-7-t (ER+, PR+, HER2–) és MDAMB-231-et (tripla negatív: ER–, PR–, HER2–) használtam a vizsgálatokhoz. A lipid-akkumulálódást Oil Red O sejtfestéssel figyeltem meg. Továbbá két, az adipocita differenciálódásban illetve lipidmetabolizmusban szerepet játszó gén – a PPARγ és a DHRS3 – kifejeződését vizsgáltam.MSc/MAmolekuláris biológiamagyarBiokémia-genomikanappal

Drugging the R-loop interactome: RNA-DNA hybrid binding proteins as targets for cancer therapy

Unravelling the origin of genetic alterations from point mutations to chromosomal rearrangements was greatly enhanced by the discovery of RNA-DNA hybrids (R-loops) that behave as hotspots of genomic instability in a variety of organisms. Current models suggest that uncontrolled R-loops are a hazard to genome integrity, therefore, identifying proteins that are involved in recognising and signalling R-loop structures are of key importance. Herein we analysed key RNA-DNA hybrid binding proteins in humans taking advantage of large-scale gene expression, survival rate, and drug-sensitivity data from cancer genomics databases. We show that ex- pression of RNA-DNA hybrid binding proteins in various cancer types is associated with survival and may have contrasting outcomes in responding to therapeutic treatments. Based on the revealed pharmacogenomic land- scape of human RNA-DNA hybrid binding proteins, we propose that R-loops and R-loop binding proteins are potentially relevant new epigenetic markers and therapeutic targets in multiple cancers.L

Hmgb1 can facilitate activation of the matrilin-1 gene promoter by Sox9 and L-Sox5/Sox6 in early steps of chondrogenesis.

The architectural high mobility group box 1 (Hmgb1) protein acts as both a nuclear and an extracellular regulator of various biological processes, including skeletogenesis. Here we report its contribution to the evolutionarily conserved, distinctive regulation of the matrilin-1 gene (Matn1) expression in amniotes. We previously demonstrated that uniquely assembled proximal promoter elements restrict Matn1 expression to specific growth plate cartilage zones by allowing varying doses of L-Sox5/Sox6 and Nfi proteins to fine-tune their Sox9-mediated transactivation. Here, we dissected the regulatory mechanisms underlying the activity of a conserved distal promoter element 1. We show that this element carries three Sox-binding sites, works as an enhancer in vivo, and allows promoter activation by the Sox5/6/9 chondrogenic trio. In early steps of chondrogenesis, declining Hmgb1 expression overlaps with the onset of Sox9 expression. Unlike repression in late steps, Hmgb1 overexpression in early chondrogenesis increases Matn1 promoter activation by the Sox trio, and forced Hmgb1 expression in COS-7 cells facilitates induction of Matn1 expression by the Sox trio. The conserved Matn1 control elements bind Hmgb1 and SOX9 with opposite efficiency in vitro. They show higher HMGB1 than SOX trio occupancy in established chondrogenic cell lines, and HMGB1 silencing greatly increases MATN1 and COL2A1 expression. Together, these data thus suggest a model whereby Hmgb1 helps recruit the Sox trio to the Matn1 promoter and thereby facilitates activation of the gene in early chondrogenesis. We anticipate that Hmgb1 may similarly affect transcription of other cartilage-specific genes

Nuclear dynamics of the Set1C subunit Spp1 prepares meiotic recombination sites for break formation

International audienceSpp1 is the H3K4me3 reader subunit of the Set1 complex (COMPASS/Set1C) that contributes to the mechanism by which meiotic DNA break sites are mechanistically selected. We previously proposed a model in which Spp1 interacts with H3K4me3 and the chromosome axis protein Mer2 that leads to DSB formation. Here we show that spatial interactions of Spp1 and Mer2 occur independently of Set1C. Spp1 exhibits dynamic chromatin binding features during meiosis, with many de novo appearing and disappearing binding sites. Spp1 chromatin binding dynamics depends on its PHD finger and Mer2-interacting domain and on modifiable histone residues (H3R2/K4). Remarkably, association of Spp1 with Mer2 axial sites reduces the effective turnover rate and diffusion coefficient of Spp1 upon chromatin binding, compared with other Set1C subunits. Our results indicate that ``chromosomal turnover rate'' is a major molecular determinant of Spp1 function in the framework of meiotic chromatin structure that prepares recombination initiation sites for break formation