Preventing excess replication origin activation to ensure genome stability.

Cells activate distinctive regulatory pathways that prevent excessive initiation of DNA replication to achieve timely and accurate genome duplication. Excess DNA synthesis is constrained by protein-DNA interactions that inhibit initiation at dormant origins. In parallel, specific modifications of pre-replication complexes prohibit post-replicative origin relicensing. Replication stress ensues when the controls that prevent excess replication are missing in cancer cells, which often harbor extrachromosomal DNA that can be further amplified by recombination-mediated processes to generate chromosomal translocations. The genomic instability that accompanies excess replication origin activation can provide a promising target for therapeutic intervention. Here we review molecular pathways that modulate replication origin dormancy, prevent excess origin activation, and detect, encapsulate, and eliminate persistent excess DNA.

Deacetylation of H4 lysine16 affects acetylation of lysine residues in histone H3 and H4 and promotes transcription of constitutive genes.

Histone modification map of H4 N-terminal tail residues in Saccharomyces cerevisiae reveals the prominence of lysine acetylation. Previous reports have indicated the importance of lysine acetylation in maintaining chromatin structure and function. H4K16, a residue with highly regulated acetylation dynamics has unique functions not overlapping with the other H4 N- terminal acetylable residues. The present work unravels the role of H4K16 acetylation in regulating expression of constitutive genes. H4K16 gets distinctly deacetylated over the coding region of constitutively expressed genes. Deacetylation of H4K16 reduces H3K9 acetylation at the cellular and gene level. Reduced H3K9 acetylation however did not negatively correlate with active gene transcription. Significantly, H4K16 deacetylation was found to be associated with hypoacetylated H4K12 throughout the locus of constitutive genes. H4K16 and K12 deacetylation is known to favour active transcription. Sas2, the HAT mutant showed similar patterns of hypoacetylated H3K9 and H4K12 at the active loci, clearly implying that the modifications were associated with deacetylation state of H4K16. Deacetylation of H4K16 was also concurrent with increased H3K56 acetylation in the promoter region and ORF of the constitutive genes. Combination of all these histone modifications significantly reduced H3 occupancy, increased promoter accessibility and enhanced RNAPII recruitment at the constitutively active loci. Consequently, we found that expression of active genes was higher in H4K16R mutant which mimic deacetylated state, but not in H4K16Q mimicking constitutive acetylation. To summarize, H4K16 deacetylation linked with H4K12 and H3K9 hypoacetylation along with H3K56 hyperacetylation generate a chromatin landscape that is conducive for transcription of constitutive genes.

ABI3 mediated repression of RAV1 gene expression promotes efficient dehydration stress response in Arabidopsis thaliana.

Dehydration stress response is a complex mechanism in plants involving several factors and hormone signalling pathways. RAV1 is a member of the AP2/ERF family of transcription factors that works in various developmental pathways. Here we show that downregulation of RAV1 gene expression is important for efficient dehydration stress response. Interestingly, the B3-domain transcription factor ABI3 negatively regulates RAV1 expression. In absence of ABI3, RAV1 expression increases during dehydration stress compared to control. As a part of stress response, ABI3 occupancy increases in the RAV1 promoter region. Such regulation of RAV1 gene expression seems vital as absence of RAV1 leads to reduced water loss during dehydration stress and consequently faster recovery compared to wild type. rav1 mutant seedlings show more abundant root growth under control condition and higher primary root elongation compared to wild type when subjected to dehydration stress. Mutants also exhibit enhanced ABA sensitivity compared to wild type. At the transcript level, rooting genes like NAC1, ARF16, SLR and SLR-downstream genes like ARF7, PLT3, SHR show differential expression in rav1 mutant, compared to wild type. Additionally, ethylene-responsive genes ETR1, EIN2 and ERF1 also get differentially expressed in presence and absence of RAV1 under control and stress conditions. This indicates an altered ethylene response in the rav1 mutant. All these features render rav1seedlings better equipped for responding to dehydration stress. It thus becomes evident that ABI3 mediated regulation of RAV1 gene expression is a significant part of dehydration stress signalling for efficient stress management at the molecular and morphological level.

Regulated acetylation and deacetylation of H4 K16 is essential for efficient NER in Saccharomyces cerevisiae.

Acetylation status of H4 K16, a residue in the histone H4 N-terminal tail plays a unique role in regulating chromatin structure and function. Here we show that, during UV-induced nucleotide excision repair H4 K16 gets hyperacetylated following an initial phase of hypoacetylation. Disrupting H4 K16 acetylation-deacetylation by mutating H4 K16 to R (deacetylated state) or Q (acetylated state) leads to compromised chromatin functions. In the silenced mating locus and telomere region H4 K16 mutants show higher recruitment of Sir proteins and spreading beyond the designated boundaries. More significantly, chromatin of both the H4 K16 mutants has reduced accessibility in the silenced regions and genome wide. On UV irradiation, the mutants showed higher UV sensitivity, reduced NER rate and altered H3 N-terminal tail acetylation, compared to wild type. NER efficiency is affected by reduced or delayed recruitment of early NER proteins and chromatin remodeller Swi/Snf along with lack of nucleosome rearrangement during repair. Additionally UV-induced expression of RAD and SNF5 genes was reduced in the mutants. Hindered chromatin accessibility in the H4 K16 mutants is thus non-conducive for gene expression as well as recruitment of NER and chromatin remodeller proteins. Subsequently, inadequate nucleosomal rearrangement during early phases of repair impeded accessibility of the NER complex to DNA lesions, in the H4 K16 mutants. Effectively, NER efficiency was found to be compromised in the mutants. Interestingly, in the transcriptionally active chromatin region, both the H4 K16 mutants showed reduced NER rate during early repair time points. However, with progression of repair H4 K16R repaired faster than K16Q mutants and rate of CPD removal became differential between the two mutants during later NER phases. To summarize, our results establish the essentiality of regulated acetylation and deacetylation of H4 K16 residue in maintaining chromatin accessibility and efficiency of functions like NER and gene expression.

ABI3 mediates dehydration stress recovery response in Arabidopsis thaliana by regulating expression of downstream genes.

ABI3, originally discovered as a seed-specific transcription factor is now implicated to act beyond seed physiology, especially during abiotic stress. In non-seed plants, ABI3 is known to act in desiccation stress signaling. Here we show that ABI3 plays a role in dehydration stress response in Arabidopsis. ABI3 gene was upregulated during dehydration stress and its expression was maintained during subsequent stress recovery phases. Comparative gene expression studies in response to dehydration stress and stress recovery were done with genes which had potential ABI3 binding sites in their upstream regulatory regions. Such studies showed that several genes including known seed-specific factors like CRUCIFERIN1, CRUCIFERIN3 and LEA-group of genes like LEA76, LEA6, DEHYDRIN LEA and LEA-LIKE got upregulated in an ABI3-dependent manner, especially during the stress recovery phase. ABI3 got recruited to regions upstream to the transcription start site of these genes during dehydration stress response through direct or indirect DNA binding. Interestingly, ABI3 also binds to its own promoter region during such stress signaling. Nucleosomes covering potential ABI3 binding sites in the upstream sequences of the above-mentioned genes alter positions, and show increased H3 K9 acetylation during stress-induced transcription. ABI3 thus mediates dehydration stress signaling in Arabidopsis through regulation of a group of genes that play a role primarily during stress recovery phase.