Methylation of histone H3 at lysine 36 (H3K36me3) marks active chromatin. The mark is interpreted by epigenetic readers that assist transcription and safeguard chromatin fiber integrity. In Drosophila, the chromodomain protein MSL3 binds H3K36me3 at X-chromosomal genes to implement dosage compensation. The PWWP-domain protein JASPer recruits the JIL1 kinase to active chromatin on all chromosomes. Because depletion of K36me3 had variable, locus-specific effects on the interactions of those readers, we systematically studied K36 methylation in a defined cellular model. Contrasting prevailing models, we found that K36me1, K36me2, and K36me3 each contribute to distinct chromatin states. Monitoring the changing K36 methylation landscape upon depletion of the three methyltransferases Set2, NSD, and Ash1 revealed local, context-specific methylation signatures. Each methyltransferase governs K36 methylation in dedicated genomic regions, with minor overlaps. Set2 catalyzes K36me3 predominantly at transcriptionally active euchromatin. NSD places K36me2/3 at defined loci within pericentric heterochromatin and on weakly transcribed euchromatic genes. Ash1 deposits K36me1 at putative enhancers. The mapping of MSL3 and JASPer suggested that they bind K36me2 in addition to K36me3, which was confirmed by direct affinity measurement. This dual specificity attracts the readers to a broader range of chromosomal locations and increases the robustness of their actions.
- MeSH
- chromatin * metabolismus MeSH
- DNA vazebné proteiny metabolismus genetika MeSH
- Drosophila melanogaster genetika metabolismus MeSH
- euchromatin metabolismus genetika MeSH
- heterochromatin metabolismus genetika MeSH
- histonlysin-N-methyltransferasa * metabolismus genetika MeSH
- histony * metabolismus MeSH
- lysin metabolismus MeSH
- metylace MeSH
- protein-serin-threoninkinasy MeSH
- proteiny Drosophily * metabolismus genetika MeSH
- transkripční faktory metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- ASH1 protein, Drosophila MeSH Prohlížeč
- chromatin * MeSH
- DNA vazebné proteiny MeSH
- euchromatin MeSH
- heterochromatin MeSH
- histonlysin-N-methyltransferasa * MeSH
- histony * MeSH
- JIL-1 protein, Drosophila MeSH Prohlížeč
- lysin MeSH
- protein-serin-threoninkinasy MeSH
- proteiny Drosophily * MeSH
- transkripční faktory MeSH
The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA * chemie metabolismus MeSH
- flavinmononukleotid * chemie metabolismus MeSH
- flaviny chemie metabolismus MeSH
- kinetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- oxidace-redukce * MeSH
- simulace molekulární dynamiky MeSH
- světlo * MeSH
- Thermosynechococcus metabolismus MeSH
- transkripční faktory metabolismus chemie MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny MeSH
- DNA vazebné proteiny MeSH
- DNA * MeSH
- flavinmononukleotid * MeSH
- flaviny MeSH
- transkripční faktory MeSH
Replication stress, particularly in hard-to-replicate regions such as telomeres and centromeres, leads to the accumulation of replication intermediates that must be processed to ensure proper chromosome segregation. In this study, we identify a critical role for the interaction between RECQ4 and MUS81 in managing such stress. We show that RECQ4 physically interacts with MUS81, targeting it to specific DNA substrates and enhancing its endonuclease activity. Loss of this interaction, results in significant chromosomal segregation defects, including the accumulation of micronuclei, anaphase bridges, and ultrafine bridges (UFBs). Our data further demonstrate that the RECQ4-MUS81 interaction plays an important role in ALT-positive cells, where MUS81 foci primarily colocalise with telomeres, highlighting its role in telomere maintenance. We also observe that a mutation associated with Rothmund-Thomson syndrome, which produces a truncated RECQ4 unable to interact with MUS81, recapitulates these chromosome instability phenotypes. This underscores the importance of RECQ4-MUS81 in safeguarding genome integrity and suggests potential implications for human disease. Our findings demonstrate the RECQ4-MUS81 interaction as a key mechanism in alleviating replication stress at hard-to-replicate regions and highlight its relevance in pathological conditions such as RTS.
- MeSH
- chromozomální nestabilita MeSH
- DNA vazebné proteiny * metabolismus genetika MeSH
- endonukleasy * metabolismus genetika MeSH
- helikasy RecQ * metabolismus genetika MeSH
- homeostáza telomer MeSH
- lidé MeSH
- mutace MeSH
- replikace DNA * MeSH
- Rothmundův-Thomsonův syndrom * genetika metabolismus MeSH
- segregace chromozomů MeSH
- telomery * metabolismus genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA vazebné proteiny * MeSH
- endonukleasy * MeSH
- helikasy RecQ * MeSH
- MUS81 protein, human MeSH Prohlížeč
- RECQL4 protein, human MeSH Prohlížeč
Biological mechanisms related to cancer development can leave distinct molecular fingerprints in tumours. By leveraging multi-omics and epidemiological information, we can unveil relationships between carcinogenesis processes that would otherwise remain hidden. Our integrative analysis of DNA methylome, transcriptome, and somatic mutation profiles of kidney tumours linked ageing, epithelial-mesenchymal transition (EMT), and xenobiotic metabolism to kidney carcinogenesis. Ageing process was represented by associations with cellular mitotic clocks such as epiTOC2, SBS1, telomere length, and PBRM1 and SETD2 mutations, which ticked faster as tumours progressed. We identified a relationship between BAP1 driver mutations and the epigenetic upregulation of EMT genes (IL20RB and WT1), correlating with increased tumour immune infiltration, advanced stage, and poorer patient survival. We also observed an interaction between epigenetic silencing of the xenobiotic metabolism gene GSTP1 and tobacco use, suggesting a link to genotoxic effects and impaired xenobiotic metabolism. Our pan-cancer analysis showed these relationships in other tumour types. Our study enhances the understanding of kidney carcinogenesis and its relation to risk factors and progression, with implications for other tumour types.
- Klíčová slova
- Cancer Biology, Genomic Epidemiology, Integrative Multi-omics Analysis, Kidney Cancer, Tumour Microenvironment,
- MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- epigeneze genetická MeSH
- epitelo-mezenchymální tranzice * genetika MeSH
- glutathion-S-transferasa fí genetika metabolismus MeSH
- histonlysin-N-methyltransferasa genetika metabolismus MeSH
- karcinogeneze * genetika MeSH
- lidé MeSH
- metylace DNA * MeSH
- multiomika MeSH
- mutace * MeSH
- nádorové supresorové proteiny genetika metabolismus MeSH
- nádory ledvin * genetika patologie MeSH
- regulace genové exprese u nádorů MeSH
- stárnutí genetika MeSH
- thiolesterasa ubikvitinu MeSH
- transkripční faktory genetika metabolismus MeSH
- transkriptom MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- BAP1 protein, human MeSH Prohlížeč
- DNA vazebné proteiny MeSH
- glutathion-S-transferasa fí MeSH
- GSTP1 protein, human MeSH Prohlížeč
- histonlysin-N-methyltransferasa MeSH
- nádorové supresorové proteiny MeSH
- PBRM1 protein, human MeSH Prohlížeč
- SETD2 protein, human MeSH Prohlížeč
- thiolesterasa ubikvitinu MeSH
- transkripční faktory MeSH
DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs) and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin (SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids and efficient DNA repair.
- MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA-helikasy metabolismus genetika MeSH
- DNA * metabolismus chemie MeSH
- dvouřetězcové zlomy DNA * MeSH
- fosforylace MeSH
- homeostáza genetika MeSH
- lidé MeSH
- oprava DNA * MeSH
- proteinfosfatasa 2 metabolismus genetika MeSH
- R-smyčka MeSH
- RNA-helikasy metabolismus genetika MeSH
- RNA-polymerasa II * metabolismus MeSH
- RNA * metabolismus genetika chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA vazebné proteiny MeSH
- DNA-helikasy MeSH
- DNA * MeSH
- proteinfosfatasa 2 MeSH
- RNA-helikasy MeSH
- RNA-polymerasa II * MeSH
- RNA * MeSH
Stress responses play a vital role in cellular survival against environmental challenges, often exploited by cancer cells to proliferate, counteract genomic instability, and resist therapeutic stress. Heat shock factor protein 1 (HSF1), a central transcription factor in stress response pathways, exhibits markedly elevated activity in cancer. Despite extensive research into the transcriptional role of HSF1, the mechanisms underlying its activation remain elusive. Upon exposure to conditions that induce protein damage, monomeric HSF1 undergoes rapid conformational changes and assembles into trimers, a key step for DNA binding and transactivation of target genes. This study investigates the role of HSF1 as a sensor of proteotoxic stress conditions. Our findings reveal that purified HSF1 maintains a stable monomeric conformation independent of molecular chaperones in vitro. Moreover, while it is known that heat stress triggers HSF1 trimerization, a notable increase in trimerization and DNA binding was observed in the presence of protein-based crowders. Conditions inducing protein misfolding and increased protein crowding in cells directly trigger HSF1 trimerization. In contrast, proteosynthesis inhibition, by reducing denatured proteins in the cell, prevents HSF1 activation. Surprisingly, HSF1 remains activated under proteotoxic stress conditions even when bound to Hsp70 and Hsp90. This finding suggests that the negative feedback regulation between HSF1 and chaperones is not directly driven by their interaction but is realized indirectly through chaperone-mediated restoration of cytoplasmic proteostasis. In summary, our study suggests that HSF1 serves as a molecular crowding sensor, trimerizing to initiate protective responses that enhance chaperone activities to restore homeostasis.
- MeSH
- DNA vazebné proteiny metabolismus chemie genetika MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- proteiny tepelného šoku HSP70 metabolismus chemie MeSH
- reakce na tepelný šok MeSH
- sbalování proteinů * MeSH
- transkripční faktory tepelného šoku * metabolismus genetika chemie MeSH
- transkripční faktory metabolismus chemie MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA vazebné proteiny MeSH
- HSF1 protein, human MeSH Prohlížeč
- proteiny tepelného šoku HSP70 MeSH
- transkripční faktory tepelného šoku * MeSH
- transkripční faktory MeSH
Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair.
- MeSH
- DNA vazebné proteiny * metabolismus genetika MeSH
- jednovláknová DNA * metabolismus genetika MeSH
- lidé MeSH
- multiproteinové komplexy MeSH
- mutace MeSH
- rekombinasa Rad51 * metabolismus genetika MeSH
- replikace DNA * genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA vazebné proteiny * MeSH
- jednovláknová DNA * MeSH
- multiproteinové komplexy MeSH
- RAD51 protein, human MeSH Prohlížeč
- rekombinasa Rad51 * MeSH
In response to DNA damage, the histone PARylation factor 1 (HPF1) regulates PARP1/2 activity, facilitating serine ADP-ribosylation of chromatin-associated factors. While PARP1/2 are known for their role in DNA single-strand break repair (SSBR), the significance of HPF1 in this process remains unclear. Here, we investigated the impact of HPF1 deficiency on cellular survival and SSBR following exposure to various genotoxins. We found that HPF1 loss did not generally increase cellular sensitivity to agents that typically induce DNA single-strand breaks (SSBs) repaired by PARP1. SSBR kinetics in HPF1-deficient cells were largely unaffected, though its absence partially influenced the accumulation of SSB intermediates after exposure to specific genotoxins in certain cell lines, likely due to altered ADP-ribosylation of chromatin. Despite reduced serine mono-ADP-ribosylation, HPF1-deficient cells maintained robust poly-ADP-ribosylation at SSB sites, possibly reflecting PARP1 auto-poly-ADP-ribosylation at non-serine residues. Notably, poly-ADP-ribose chains were sufficient to recruit the DNA repair factor XRCC1, which may explain the relatively normal SSBR capacity in HPF1-deficient cells. These findings suggest that HPF1 and histone serine ADP-ribosylation are largely dispensable for PARP1-dependent SSBR in response to genotoxic stress, highlighting the complexity of mechanisms that maintain genomic stability and chromatin remodeling.
- MeSH
- buněčné linie MeSH
- chromatin metabolismus MeSH
- DNA vazebné proteiny metabolismus genetika MeSH
- histony metabolismus MeSH
- jaderné proteiny metabolismus genetika MeSH
- jednořetězcové zlomy DNA * MeSH
- lidé MeSH
- oprava DNA * MeSH
- poly-ADP-ribosylace MeSH
- poly(ADP-ribosa)polymerasa 1 * metabolismus genetika MeSH
- poly(ADP-ribosa)polymerasy metabolismus genetika MeSH
- protein XRCC1 metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chromatin MeSH
- DNA vazebné proteiny MeSH
- histony MeSH
- HPF1 protein, human MeSH Prohlížeč
- jaderné proteiny MeSH
- PARP1 protein, human MeSH Prohlížeč
- poly(ADP-ribosa)polymerasa 1 * MeSH
- poly(ADP-ribosa)polymerasy MeSH
- protein XRCC1 MeSH
- XRCC1 protein, human MeSH Prohlížeč
Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed.
- MeSH
- "flap" endonukleasy metabolismus genetika MeSH
- DEAD-box RNA-helikasy * metabolismus genetika MeSH
- DNA vazebné proteiny * metabolismus genetika MeSH
- DNA-helikasy * metabolismus genetika MeSH
- DNA-ligasa ATP metabolismus genetika MeSH
- DNA metabolismus genetika MeSH
- endonukleasy * metabolismus genetika MeSH
- genetická transkripce MeSH
- lidé MeSH
- multifunkční enzymy * metabolismus genetika MeSH
- R-smyčka * MeSH
- replikace DNA * MeSH
- RNA-helikasy * metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- "flap" endonukleasy MeSH
- DEAD-box RNA-helikasy * MeSH
- DNA vazebné proteiny * MeSH
- DNA-helikasy * MeSH
- DNA-ligasa ATP MeSH
- DNA MeSH
- endonukleasy * MeSH
- multifunkční enzymy * MeSH
- MUS81 protein, human MeSH Prohlížeč
- RNA-helikasy * MeSH
- SETX protein, human MeSH Prohlížeč
RAD18 is an E3 ubiquitin ligase that prevents replication fork collapse by promoting DNA translesion synthesis and template switching. Besides this classical role, RAD18 has been implicated in homologous recombination; however, this function is incompletely understood. Here, we show that RAD18 is recruited to DNA lesions by monoubiquitination of histone H2A at K15 and counteracts accumulation of 53BP1. Super-resolution microscopy revealed that RAD18 localizes to the proximity of DNA double strand breaks and limits the distribution of 53BP1 to the peripheral chromatin nanodomains. Whereas auto-ubiquitination of RAD18 mediated by RAD6 inhibits its recruitment to DNA breaks, interaction with SLF1 promotes RAD18 accumulation at DNA breaks in the post-replicative chromatin by recognition of histone H4K20me0. Surprisingly, suppression of 53BP1 function by RAD18 is not involved in homologous recombination and rather leads to reduction of non-homologous end joining. Instead, we provide evidence that RAD18 promotes HR repair by recruiting the SMC5/6 complex to DNA breaks. Finally, we identified several new loss-of-function mutations in RAD18 in cancer patients suggesting that RAD18 could be involved in cancer development.
- MeSH
- 53BP1 * metabolismus genetika MeSH
- chromatin * metabolismus genetika MeSH
- DNA vazebné proteiny * metabolismus genetika MeSH
- dvouřetězcové zlomy DNA * MeSH
- histony * metabolismus MeSH
- homologní rekombinace genetika MeSH
- lidé MeSH
- oprava DNA spojením konců MeSH
- oprava DNA MeSH
- proteiny buněčného cyklu metabolismus genetika MeSH
- rekombinační oprava DNA MeSH
- replikace DNA MeSH
- ubikvitinace * MeSH
- ubikvitinligasy * metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 53BP1 * MeSH
- chromatin * MeSH
- DNA vazebné proteiny * MeSH
- histony * MeSH
- proteiny buněčného cyklu MeSH
- RAD18 protein, human MeSH Prohlížeč
- TP53BP1 protein, human MeSH Prohlížeč
- ubikvitinligasy * MeSH
