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
Deficiency in chromatin assembly factor-1 (CAF-1) in plants through dysfunction of its components, FASCIATA1 and 2 (FAS1, FAS2), leads to the specific and progressive loss of rDNA and telomere repeats in plants. This loss is attributed to defective repair mechanisms for the increased DNA breaks encountered during replication, a consequence of impaired replication-dependent chromatin assembly. In this study, we explore the role of KU70 in these processes. Our findings reveal that, although the rDNA copy number is reduced in ku70 mutants when compared with wild-type plants, it is not markedly affected by diverse KU70 status in fas1 mutants. This is consistent with our previous characterisation of rDNA loss in fas mutants as a consequence part of the single-strand annealing pathway of homology-dependent repair. In stark contrast to rDNA, KU70 dysfunction fully suppresses the loss of telomeres in fas1 plants and converts telomeres to their elongated and heterogeneous state typical for ku70 plants. We conclude that the alternative telomere lengthening pathway, known to be activated in the absence of KU70, overrides progressive telomere loss due to CAF-1 dysfunction.
- Klíčová slova
- 45S rDNA, ALT, Arabidopsis thaliana, CAF‐1, FAS1, KU70, genome instability, telomeres,
- MeSH
- Arabidopsis * genetika metabolismus fyziologie MeSH
- chromozomy rostlin metabolismus MeSH
- DNA vazebné proteiny * MeSH
- faktor 1 pro uspořádání chromatinu * metabolismus genetika MeSH
- homeostáza telomer * MeSH
- mutace MeSH
- proteiny huseníčku * genetika metabolismus MeSH
- ribozomální DNA genetika metabolismus MeSH
- telomery metabolismus genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- At2g20020 protein, Arabidopsis MeSH Prohlížeč
- DNA vazebné proteiny * MeSH
- faktor 1 pro uspořádání chromatinu * MeSH
- FAS protein, Arabidopsis MeSH Prohlížeč
- KU70 protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku * MeSH
- ribozomální DNA MeSH
Chromatin Assembly Factor 1 (CAF-1) is a major nucleosome assembly complex which functions particularly during DNA replication and repair. Here we studied how the nucleosome landscape changes in a CAF-1 mutant in the model plant Arabidopsis thaliana. Globally, most nucleosomes were not affected by loss of CAF-1, indicating the presence of efficient alternative nucleosome assemblers. Nucleosomes that we found depleted in the CAF-1 mutant were enriched in non-transcribed regions, consistent with the notion that CAF-1-independent nucleosome assembly can compensate for loss of CAF-1 mainly in transcribed regions. Depleted nucleosomes were particularly enriched in proximal promoters, suggesting that CAF-1-independent nucleosome assembly mechanisms are often not efficient upstream of transcription start sites. Genes related to plant defense were particularly prone to lose nucleosomes in their promoters upon CAF-1 depletion. Reduced nucleosome occupancy at promoters of many defense-related genes is associated with a primed gene expression state that may considerably increase plant fitness by facilitating plant defense. Together, our results establish that the nucleosome landscape in Arabidopsis is surprisingly robust even in the absence of the dedicated nucleosome assembly machinery CAF-1 and that CAF-1-independent nucleosome assembly mechanisms are less efficient in particular genome regions.
- Klíčová slova
- Arabidopsis thaliana, CAF-1, GSE87421, chromatin, plant defense,
- MeSH
- Arabidopsis genetika imunologie metabolismus MeSH
- chromatin genetika MeSH
- faktor 1 pro uspořádání chromatinu genetika metabolismus MeSH
- imunita rostlin genetika MeSH
- mutace MeSH
- nukleozomy genetika metabolismus MeSH
- oprava DNA genetika MeSH
- počátek transkripce MeSH
- promotorové oblasti (genetika) genetika MeSH
- replikace DNA genetika MeSH
- restrukturace chromatinu genetika MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chromatin MeSH
- faktor 1 pro uspořádání chromatinu MeSH
- nukleozomy MeSH
Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability.
- Klíčová slova
- 45S rDNA, Arabidopsis thaliana, DNA repair, FAS1, FAS2, RAD51B, chromatin assembly factor 1, genome instability,
- MeSH
- Arabidopsis genetika metabolismus MeSH
- faktor 1 pro uspořádání chromatinu genetika metabolismus MeSH
- nestabilita genomu genetika fyziologie MeSH
- oprava DNA genetika fyziologie MeSH
- proteiny huseníčku genetika metabolismus MeSH
- ribozomální DNA genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- faktor 1 pro uspořádání chromatinu MeSH
- FAS protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku MeSH
- RAD51B protein, Arabidopsis MeSH Prohlížeč
- ribozomální DNA MeSH
Dysfunction of chromatin assembly factor 1 in FASCIATA mutants (fas) of Arabidopsis thaliana results in progressive loss of telomeric DNA. Although replicative telomere shortening is typically associated with incomplete resynthesis of their ends by telomerase, no change in telomerase activity could be detected in vitro in extracts from fas mutants. Besides a possible telomerase malfunction, the telomere shortening in fas mutants could presumably be due to problems with conventional replication of telomeres. To distinguish between the possible contribution of suboptimal function of telomerase in fas mutants under in vivo conditions and problems in conventional telomere replication, we crossed fas and tert (telomerase reverse transcriptase) knockout mutants and analyzed telomere shortening in segregated fas mutants, tert mutants, and double fas tert mutants in parallel. We demonstrate that fas tert knockouts show greater replicative telomere shortening than that observed even in the complete absence of telomerase (tert mutants). While the effect of tert and fas mutations on telomere lengths in double mutants is additive, manifestations of telomere dysfunction in double fas tert mutants (frequency of anaphase bridges, onset of chromosome end fusions, and common involvement of 45S rDNA in chromosome fusion sites) are similar to those in tert mutants. We conclude that in addition to possible impairment of telomerase action, a further mechanism contributes to telomere shortening in fas mutants.
- MeSH
- Arabidopsis enzymologie genetika metabolismus MeSH
- chromozomy rostlin genetika metabolismus MeSH
- faktor 1 pro uspořádání chromatinu genetika metabolismus MeSH
- mutace * MeSH
- proteiny huseníčku genetika metabolismus MeSH
- sestřihové faktory MeSH
- telomerasa genetika metabolismus MeSH
- telomery genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- At2g20020 protein, Arabidopsis MeSH Prohlížeč
- faktor 1 pro uspořádání chromatinu MeSH
- FAS protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku MeSH
- sestřihové faktory MeSH
- telomerasa MeSH
- TERT protein, Arabidopsis MeSH Prohlížeč
Chromatin Assembly Factor 1 (CAF1) is a three-subunit H3/H4 histone chaperone responsible for replication-dependent nucleosome assembly. It is composed of CAC 1-3 in yeast; p155, p60, and p48 in humans; and FASCIATA1 (FAS1), FAS2, and MULTICOPY SUPPRESSOR OF IRA1 in Arabidopsis thaliana. We report that disruption of CAF1 function by fas mutations in Arabidopsis results in telomere shortening and loss of 45S rDNA, while other repetitive sequences (5S rDNA, centromeric 180-bp repeat, CACTA, and Athila) are unaffected. Substantial telomere shortening occurs immediately after the loss of functional CAF1 and slows down at telomeres shortened to median lengths around 1 to 1.5 kb. The 45S rDNA loss is progressive, leaving 10 to 15% of the original number of repeats in the 5th generation of mutants affecting CAF1, but the level of the 45S rRNA transcripts is not altered in these mutants. Increasing severity of the fas phenotype is accompanied by accumulation of anaphase bridges, reduced viability, and plant sterility. Our results show that appropriate replication-dependent chromatin assembly is specifically required for stable maintenance of telomeres and 45S rDNA.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- DNA rostlinná genetika metabolismus MeSH
- faktor 1 pro uspořádání chromatinu genetika metabolismus MeSH
- inzerční mutageneze MeSH
- mutace MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- restrukturace chromatinu MeSH
- ribozomální DNA genetika metabolismus MeSH
- RNA ribozomální genetika metabolismus MeSH
- telomery metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA rostlinná MeSH
- faktor 1 pro uspořádání chromatinu MeSH
- FAS protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku MeSH
- ribozomální DNA MeSH
- RNA ribozomální MeSH
- RNA, ribosomal, 45S MeSH Prohlížeč
