Genome stability is significantly influenced by the precise coordination of chromatin complexes that facilitate the loading and eviction of histones from chromatin during replication, transcription, and DNA repair processes. In this study, we investigate the role of the Arabidopsis H3 histone chaperones ANTI-SILENCING FUNCTION 1 (ASF1) and HISTONE REGULATOR A (HIRA) in the maintenance of telomeres and 45S rDNA loci, genomic sites that are particularly susceptible to changes in the chromatin structure. We find that both ASF1 and HIRA are essential for telomere length regulation, as telomeres are significantly shorter in asf1a1b and hira mutants. However, these shorter telomeres remain localized around the nucleolus and exhibit a comparable relative H3 occupancy to the wild type. In addition to regulating telomere length, ASF1 and HIRA contribute to silencing 45S rRNA genes and affect their copy number. Besides, ASF1 supports global heterochromatin maintenance. Our findings also indicate that ASF1 transiently binds to the TELOMERE REPEAT BINDING 1 protein and the N terminus of telomerase in vivo, suggesting a physical link between the ASF1 histone chaperone and the telomere maintenance machinery.
- Klíčová slova
- 45S rDNA, ASF1, Arabidopsis thaliana, HIRA, chromatin, histone chaperones, telomeres,
- MeSH
- Arabidopsis * genetika metabolismus MeSH
- heterochromatin metabolismus genetika MeSH
- histonové chaperony * metabolismus genetika MeSH
- histony metabolismus genetika MeSH
- homeostáza telomer MeSH
- molekulární chaperony metabolismus genetika MeSH
- proteiny buněčného cyklu metabolismus genetika MeSH
- proteiny huseníčku * metabolismus genetika MeSH
- ribozomální DNA * genetika metabolismus MeSH
- RNA ribozomální genetika metabolismus MeSH
- sestřihové faktory MeSH
- telomerasa genetika metabolismus MeSH
- telomery * metabolismus genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- At2g20020 protein, Arabidopsis MeSH Prohlížeč
- heterochromatin MeSH
- histonové chaperony * MeSH
- histony MeSH
- molekulární chaperony MeSH
- proteiny buněčného cyklu MeSH
- proteiny huseníčku * MeSH
- ribozomální DNA * MeSH
- RNA ribozomální MeSH
- RNA, ribosomal, 45S MeSH Prohlížeč
- sestřihové faktory MeSH
- telomerasa MeSH
At the molecular scale, adaptive advantages during plant growth and development rely on modulation of gene expression, primarily provided by epigenetic machinery. One crucial part of this machinery is histone posttranslational modifications, which form a flexible system, driving transient changes in chromatin, and defining particular epigenetic states. Posttranslational modifications work in concert with replication-independent histone variants further adapted for transcriptional regulation and chromatin repair. However, little is known about how such complex regulatory pathways are orchestrated and interconnected in cells. In this work, we demonstrate the utility of mass spectrometry-based approaches to explore how different epigenetic layers interact in Arabidopsis mutants lacking certain histone chaperones. We show that defects in histone chaperone function (e.g., chromatin assembly factor-1 or nucleosome assembly protein 1 mutations) translate into an altered epigenetic landscape, which aids the plant in mitigating internal instability. We observe changes in both the levels and distribution of H2A.W.7, altogether with partial repurposing of H3.3 and changes in the key repressive (H3K27me1/2) or euchromatic marks (H3K36me1/2). These shifts in the epigenetic profile serve as a compensatory mechanism in response to impaired integration of the H3.1 histone in the fas1 mutants. Altogether, our findings suggest that maintaining genome stability involves a two-tiered approach. The first relies on flexible adjustments in histone marks, while the second level requires the assistance of chaperones for histone variant replacement.
- Klíčová slova
- Arabidopsis, chromatin remodeling, histone chaperone complex, histone variants, immunochemistry, mass spectrometry, post-translational modifications,
- MeSH
- Arabidopsis * genetika metabolismus MeSH
- epigeneze genetická * MeSH
- faktor 1 pro uspořádání chromatinu metabolismus genetika MeSH
- histonové chaperony * metabolismus genetika MeSH
- histony * metabolismus MeSH
- mutace MeSH
- posttranslační úpravy proteinů MeSH
- proteiny huseníčku * metabolismus genetika MeSH
- regulace genové exprese u rostlin MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- faktor 1 pro uspořádání chromatinu MeSH
- histonové chaperony * MeSH
- histony * MeSH
- proteiny huseníčku * MeSH
Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The precise mechanism of nucleosome reassembly mediated by Spt6 remains unclear. In this study, we used a hybrid approach combining cryo-electron microscopy and small-angle X-ray scattering to visualize the architecture of Spt6 from Saccharomyces cerevisiae. The reconstructed overall architecture of Spt6 reveals not only the core of Spt6, but also its flexible N- and C-termini, which are critical for Spt6's function. We found that the acidic N-terminal region of Spt6 prevents the binding of Spt6 not only to the Pol II CTD and Pol II CTD-linker, but also to pre-formed intact nucleosomes and nucleosomal DNA. The N-terminal region of Spt6 self-associates with the tSH2 domain and the core of Spt6 and thus controls binding to Pol II and nucleosomes. Furthermore, we found that Spt6 promotes the assembly of nucleosomes in vitro. These data indicate that the cooperation between the intrinsically disordered and structured regions of Spt6 regulates nucleosome and Pol II CTD binding, and also nucleosome assembly.
- MeSH
- elektronová kryomikroskopie MeSH
- genetická transkripce MeSH
- histonové chaperony genetika metabolismus MeSH
- nukleozomy * genetika metabolismus MeSH
- RNA-polymerasa II metabolismus MeSH
- Saccharomyces cerevisiae - proteiny * metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- transkripční elongační faktory metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- histonové chaperony MeSH
- nukleozomy * MeSH
- RNA-polymerasa II MeSH
- Saccharomyces cerevisiae - proteiny * MeSH
- SPT6 protein, S cerevisiae MeSH Prohlížeč
- transkripční elongační faktory MeSH
Successful reproduction requires an oocyte competent to sustain early embryo development. By the end of oogenesis, the oocyte has entered a transcriptionally silenced state, the mechanisms and significance of which remain poorly understood. Histone H3.3, a histone H3 variant, has unique cell cycle-independent functions in chromatin structure and gene expression. Here, we have characterised the H3.3 chaperone Hira/Cabin1/Ubn1 complex, showing that loss of function of any of these subunits causes early embryogenesis failure in mouse. Transcriptome and nascent RNA analyses revealed that transcription is aberrantly silenced in mutant oocytes. Histone marks, including H3K4me3 and H3K9me3, are reduced and chromatin accessibility is impaired in Hira/Cabin1 mutants. Misregulated genes in mutant oocytes include Zscan4d, a two-cell specific gene involved in zygote genome activation. Overexpression of Zscan4 in the oocyte partially recapitulates the phenotypes of Hira mutants and Zscan4 knockdown in Cabin1 mutant oocytes partially restored their developmental potential, illustrating that temporal and spatial expression of Zscan4 is fine-tuned at the oocyte-to-embryo transition. Thus, the H3.3 chaperone Hira complex has a maternal effect function in oocyte developmental competence and embryogenesis, through modulating chromatin condensation and transcriptional quiescence.
- Klíčová slova
- Competent oocyte, Hira complex, Histone H3.3, Oocyte-to-embryo transition, Zygotic genome activation,
- MeSH
- adaptorové proteiny signální transdukční metabolismus MeSH
- chromatin metabolismus MeSH
- embryonální vývoj genetika MeSH
- genový knockdown MeSH
- histonové chaperony genetika metabolismus MeSH
- histony metabolismus MeSH
- myši inbrední C57BL MeSH
- myši transgenní MeSH
- myši MeSH
- oocyty růst a vývoj metabolismus MeSH
- oogeneze genetika MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- signální transdukce genetika MeSH
- transkripční faktory genetika metabolismus MeSH
- zvířata MeSH
- zygota metabolismus MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- adaptorové proteiny signální transdukční MeSH
- Cabin1 protein, mouse MeSH Prohlížeč
- chromatin MeSH
- Hira protein, mouse MeSH Prohlížeč
- histonové chaperony MeSH
- histony MeSH
- proteiny buněčného cyklu MeSH
- transkripční faktory MeSH
- Zscan4d protein, mouse MeSH Prohlížeč
Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.
- Klíčová slova
- DNA repair, chromatin, histone chaperones, histone variants,
- MeSH
- adenosintrifosfatasy genetika metabolismus MeSH
- Arabidopsis genetika metabolismus MeSH
- chromatin genetika metabolismus MeSH
- histonové chaperony genetika metabolismus MeSH
- mutace genetika MeSH
- nestabilita genomu genetika fyziologie MeSH
- proteiny huseníčku genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adenosintrifosfatasy MeSH
- chromatin MeSH
- FAS protein, Arabidopsis MeSH Prohlížeč
- histonové chaperony MeSH
- NAP1 protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku MeSH
Transcription elongation factor Spt6 associates with RNA polymerase II (RNAP II) via a tandem SH2 (tSH2) domain. The mechanism and significance of the RNAP II-Spt6 interaction is still unclear. Recently, it was proposed that Spt6-tSH2 is recruited via a newly described phosphorylated linker between the Rpb1 core and its C-terminal domain (CTD). Here, we report binding studies with isolated tSH2 of Spt6 (Spt6-tSH2) and Spt6 lacking the first unstructured 297 residues (Spt6ΔN) with a minimal CTD substrate of two repetitive heptads phosphorylated at different sites. The data demonstrate that Spt6 also binds the phosphorylated CTD, a site that was originally proposed as a recognition epitope. We also show that an extended CTD substrate harboring 13 repetitive heptads of the tyrosine-phosphorylated CTD binds Spt6-tSH2 and Spt6ΔN with tighter affinity than the minimal CTD substrate. The enhanced binding is achieved by avidity originating from multiple phosphorylation marks present in the CTD. Interestingly, we found that the steric effects of additional domains in the Spt6ΔN construct partially obscure the binding of the tSH2 domain to the multivalent ligand. We show that Spt6-tSH2 binds various phosphorylation patterns in the CTD and found that the studied combinations of phospho-CTD marks (1,2; 1,5; 2,4; and 2,7) all facilitate the interaction of CTD with Spt6. Our structural studies reveal a plasticity of the tSH2 binding pockets that enables the accommodation of CTDs with phosphorylation marks in different registers.
- Klíčová slova
- CTD, RNA polymerase II, Spt6, NMR structure, phosphorylation,
- MeSH
- epitopy genetika MeSH
- fosforylace genetika MeSH
- genetická transkripce * MeSH
- histonové chaperony genetika MeSH
- RNA-polymerasa II genetika MeSH
- Saccharomyces cerevisiae - proteiny genetika MeSH
- Saccharomyces cerevisiae genetika MeSH
- sekvence aminokyselin genetika MeSH
- src homologní domény genetika MeSH
- transkripční elongační faktory genetika MeSH
- vazba proteinů genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- epitopy MeSH
- histonové chaperony MeSH
- RNA-polymerasa II MeSH
- Saccharomyces cerevisiae - proteiny MeSH
- SPT6 protein, S cerevisiae MeSH Prohlížeč
- transkripční elongační faktory MeSH
