Nucleic Acids Research最新文献

Heterochromatin is characterized by an inaccessibility to the transcriptional machinery and is associated with the histone mark H3K9me3. However, studying the functional consequences of heterochromatin loss in human cells has been challenging. Here, we used CRISPRi-mediated silencing of the histone methyltransferase SETDB1 to remove H3K9me3 heterochromatin in human neural progenitor cells. Despite a major loss of H3K9me3 peaks resulting in genome-wide reorganization of heterochromatin domains, silencing of SETDB1 had a limited effect on cell viability. Cells remained proliferative and expressed appropriate marker genes. We found that a key event following the loss of SETDB1-mediated H3K9me3 was the expression of evolutionarily young L1 retrotransposons. Derepression of L1s was associated with a loss of CpG DNA methylation at their promoters, suggesting that deposition of H3K9me3 at the L1 promoter is required to maintain DNA methylation. In conclusion, these results demonstrate that loss of H3K9me3 in human neural somatic cells transcriptionally activates evolutionary young L1 retrotransposons.

Deep learning has successfully been applied to design cis-regulatory elements (CREs) for a few species, but a broadly applicable platform for generating functional promoters for thousands of prokaryotes remains lacking. In this study, we introduce a language model for prokaryotic CREs, referred to as PromoGen2, to design CREs without prior experimental data. PromoGen2 was pretrained on CREs derived from 17 000 prokaryotic genomes. It achieved the highest zero-shot prediction correlation of promoter strength across species, improving the average Spearman correlation from 0.27 to 0.50 compared to the best baseline, while reducing the number of parameters by 103. Artificial CREs designed with PromoGen2 demonstrated a 100% success rate in Escherichia coli, Bacillus subtilis, Bacillus licheniformis, and Agrobacterium tumefaciens. Based on PromoGen2, we developed the Promoter-Factory framework to design promoters from unannotated genomes. Experimental validation showed that most of the promoters designed for Jejubacter sp. L23, a newly isolated halophilic bacterium with no available CREs, were active and capable of driving lycopene overproduction. Additionally, we introduced PromoGen2-proka, a taxonomy-aware model for CRE design based on prokaryotic genera. Experimental validation confirmed its reliable success rate. The combined use of PromoGen2-proka and Promoter-Factory offers a broadly applicable tool for designing CREs for prokaryotes, fulfilling the needs of synthetic biology and microbiology research.

Eukaryotic gene expression is dynamically regulated through the interplay between histone modifications and chromatin remodeling, yet how these processes are coordinated remains incompletely understood. Here, we uncover IBD1 as a critical adaptor that bridges histone acetylation and SWR-mediated H2A.Z deposition. Mechanistically, IBD1's bromodomain recognizes histone acetylation, specifically H3K9/K14 di-acetylation, to recruit the SWR complex subunit ARP6, ensuring precise H2A.Z incorporation into chromatin. H3K9Q mutation and genetic disruption of IBD1, either by deletion or bromodomain mutation, significantly reduce H2A.Z occupancy at target loci. In contrast, disruption of IBD1 has little effect on H3K9/K14 acetylation levels, confirming the directional hierarchy of the acetylation-IBD1-H2A.Z regulatory axis. Intriguingly, perturbation of this axis, through IBD1 loss or bromodomain impairment, leads to widespread transcriptional upregulation, particularly at genes co-enriched for IBD1, H3K9/K14ac, and H2A.Z, with the strongest effects at hyperacetylated loci. This transcriptional imbalance coincides with reduced growth rates, underscoring the functional significance of IBD1-mediated H2A.Z deposition. Given that H2A.Z enrichment is classically correlated with transcriptional levels, this observation highlights a dual role for H2A.Z: sustaining basal transcription and constraining overactivation at highly active genes. Together, our findings define a novel regulatory mechanism in which IBD1 bridges acetyl-mark decoding with SWR-dependent H2A.Z deposition, establishing transcriptional homeostasis.

DNA polymerase eta (Pol η) is a Y-family translesion polymerase responsible for synthesizing new DNA across UV-damaged templates. It is recruited to replication forks following mono-ubiquitination of the PCNA DNA clamp. This interaction is mediated by PCNA-interacting protein motifs within Pol η, as well as by its C-terminal ubiquitin-binding zinc finger (UBZ) domain. Previous work has suggested that Pol η itself is mono-ubiquitinated at four C-terminal lysine residues, which is dependent on prior ubiquitin-binding by its UBZ domain. Here, we show that Pol η can be modified at the same lysine residues by the ubiquitin-like protein, NEDD8. Like ubiquitination, this modification is driven by non-covalent interactions between NEDD8 and the UBZ domain. While only a small proportion of Pol η is mono-NEDDylated under normal conditions, these levels rapidly increase following inhibition of the COP9 signalosome, revealing that mono-NEDDylation is maintained under strong negative regulation. Finally, we demonstrate that mono-NEDDylation prevents Pol η foci formation in UV-C irradiated cells, suggesting that this modification prevents Pol η from participating in translesion DNA synthesis. These results thereby reveal a new mechanism by which human Pol η is regulated by ubiquitin-like proteins.

The cytoplasmic fate of messenger RNAs (mRNAs) is dictated by the balance of translation and mRNA degradation, governed in part by the 3' poly-adenosine tail and cytoplasmic poly(A)-binding proteins (PABPCs). Deadenylases remove poly(A) to initiate mRNA decay, while sequence-specific RNA-binding factors, including Pumilio proteins (PUM1 and PUM2), modulate these processes. We investigated how human PUM1&2 repress target mRNAs by accelerating their degradation. We found that the poly(A) tail plays a central role in PUM repression, dependent on the interplay of deadenylases and PABPCs. PUM-mediated repression requires the CCR4-NOT deadenylase but not the poly(A) nuclease. PUMs associate with and require PABPC1 and PABPC4 to repress. In the absence of PABPCs, both PUM targets and non-targets become unstable, bypassing PUM control. Increasing PABPC inhibits PUM activity in a concentration-dependent manner by stabilizing poly(A) mRNAs. The results support a Goldilocks principle, wherein PABPC abundance tunes the response of mRNAs to PUM-mediated repression through protection of poly(A) from deadenylation. We propose that this principle may apply to other poly(A) dependent regulatory factors. Variation of PABPC levels across tissues and development suggests physiological relevance for this mechanism.

Anchoring of a chromatin remodeler complex by long non-coding RNAs (lncRNAs) is a frequently utilized mechanism for lncRNAs to regulate gene expression. Hypoxia is a microenvironmental condition that plays a crucial role in promoting tumor progression. We previously identified a hypoxia-inducible lncRNA, RP11-390F4.3, that regulates epithelial-mesenchymal transition (EMT) without a delineated mechanism. Here, we show that the lncRNA RP11-390F4.3 (renamed MAHAC: MAintenance of Histone ACetylation) specifically induces histone H4 lysine 5 acetylation (H4K5ac) mark and promotes the deposition of H4K5ac mark on the promoters of EMT transcription factors. MAHAC scaffolds the ILF3/NF90-ILF2-CBP complex, which is co-localized with the members of the complex inside the nucleus under hypoxia. The minimal MAHAC region (nt 686-741) required for scaffolding the complex was mapped, and it induces allosteric activation of H4K5ac in in vitro histone acetyltransferase assay. This minimal MAHAC region is essential for hypoxia-induced EMT, migration, invasion, and H4K5ac activation. These findings demonstrate that hypoxia-induced MAHAC represents an unexplored allosteric regulator of H4K5ac that activates EMT and induces tumor progression.

Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate messenger RNA (mRNA) would be an advantageous approach to correct gene expression but has not been evaluated in an in vivo disease model. Here, we investigated whether a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identify and engineer human proteins capable of increasing mRNA translation using the CRISPR-Cas-inspired RNA-targeting system (CIRTS) platform to enable programmable, guide RNA-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3) that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold-key phenotypic indicators of Dravet syndrome. This work validates a strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential of targeted translational activation to address neurological haploinsufficiency.

Condensins are key regulators of chromosome architecture and have emerging functions in DNA repair that are understudied. Here, we show that combined depletion of Condensin I and II in cell lines of normal and tumor origin selectively impairs DNA double-strand break (DSB) repair and the checkpoint response (DDR) specifically in the G2 phase of the cell cycle, with no detectable effects in G1 or S phase. Condensin knockdown increased cellular radiosensitivity and delayed in G2-phase, but not in asynchronous cells, the resolution of γH2AX and 53BP1 foci, indicating G2-specific defects in DSB repair. Mechanistically, condensin loss suppressed DNA end-resection and resection-dependent repair pathways, including homologous recombination (HR), single-strand annealing (SSA), and alternative end-joining (alt-EJ), but failed to significantly alter classical non-homologous end-joining (c-NHEJ). Reduced RAD51 and RPA70 foci formation in G2 confirmed inhibition of HR and DNA end resection. The G2 checkpoint was also compromised. Cytogenetic analysis revealed inhibition of chromosome break repair and visible chromatin decondensation, suggesting that condensins function to maintain an appropriate chromatin state for efficient DSB repair in G2-phase. These results identify for the first time condensins as G2 phase-specific regulators of genome stability by fine-tuning HR and other resection-dependent DSB repair pathways.

Polyphosphate (polyP) is considered having regulatory functions in both procaryotic and eucaryotic cells. Under certain stress conditions, bacteria accumulate polyP, which results in liquid-liquid phase separation and polyP granules formation with not fully uncovered functions. We demonstrate that in starved Escherichia coli cells, replication initiator DnaA protein fails to form defined foci and does not bind to the origin of DNA replication (oriC), while to some extent interacts other sites on the chromosome. This is because polyP interacts with a long variant of CobB deacetylase and inhibits its activity, which results in an increased DnaA acetylation level preventing the DnaA interaction with oriC and consequently the initiation of DNA replication. This constitutes a polyP-dependent regulatory coupling targeting deacetylase for the inhibition of DNA replication initiation. Our experiments also demonstrate the importance of the multiplicity of regulatory mechanisms for the complete inhibition of initiation of DNA replication in stressed bacterial cells.

DNA superhelicity and transcription are intimately related because changes to DNA topology can influence gene expression and vice versa. Information is transferred through the modulation of local DNA torsional stress, where the expression of one gene may influence the superhelical level of neighbouring genes, either promoting or repressing their expression. In this work, we introduce a one-dimensional physical model that simulates supercoiling-mediated regulation. This TORCphysics model takes as input a genome architecture represented either by a plasmid or chromosomal DNA sequence with ends constrained under specific biological conditions and computes the molecule's output. Our findings demonstrate that the expression profiles of genes are directly influenced by the gene circuit design, including gene location, the positions of topological barriers, promoter sequences, and topoisomerase activity. The novelty that TORCphysics offers is versatility, where users can define distinct activity models for different types of proteins and protein-binding sites. The aim of this research is to establish a flexible framework for developing physical simulations of gene circuits to deepen our comprehension of the intricate mechanisms involved in gene regulation.