Astrocyte-targeting therapy rescues cognitive impairment caused by neuroinflammation via the Nrf2 pathway.

Neuroinflammation is a common feature of neurodegenerative disorders such as Alzheimer's disease (AD). Neuroinflammation is induced by dysregulated glial activation, and astrocytes, the most abundant glial cells, become reactive upon neuroinflammatory cytokines released from microglia and actively contribute to neuronal loss. Therefore, blocking reactive astrocyte functions is a viable strategy to manage neurodegenerative disorders. However, factors or therapeutics directly regulating astrocyte subtypes remain unexplored. Here, we identified transcription factor NF-E2-related factor 2 (Nrf2) as a therapeutic target in neurotoxic reactive astrocytes upon neuroinflammation. We found that the absence of Nrf2 promoted the activation of reactive astrocytes in the brain tissue samples obtained from AD model 5xFAD mice, whereas enhanced Nrf2 expression blocked the induction of reactive astrocyte gene expression by counteracting NF-κB subunit p65 recruitment. Neuroinflammatory astrocytes robustly up-regulated genes associated with type I interferon and the antigen-presenting pathway, which were suppressed by Nrf2 pathway activation. Moreover, impaired cognitive behaviors observed in AD mice were rescued upon ALGERNON2 treatment, which potentiated the Nrf2 pathway and reduced the induction of neurotoxic reactive astrocytes. Thus, we highlight the potential of astrocyte-targeting therapy by promoting the Nrf2 pathway signaling for neuroinflammation-triggered neurodegeneration.

Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.

Neuronal enhancers are hotspots for DNA single-strand break repair.

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

END-seq: An Unbiased, High-Resolution, and Genome-Wide Approach to Map DNA Double-Strand Breaks and Resection in Human Cells.

DNA double-strand breaks (DSBs) represent the most toxic form of DNA damage and can arise in either physiological or pathological conditions. If left unrepaired, these DSBs can lead to genome instability which serves as a major driver to tumorigenesis and other pathologies. Consequently, localizing DSBs and understanding the dynamics of break formation and the repair process are of great interest for dissecting underlying mechanisms and in the development of targeted therapies. Here, we describe END-seq, a highly sensitive next-generation sequencing technique for quantitatively mapping DNA double-strand breaks (DSB) at nucleotide resolution across the genome in an unbiased manner. END-seq is based on the direct ligation of a sequencing adapter to the ends of DSBs and provides information about DNA processing (end resection) at DSBs, a critical determinant in the selection of repair pathways. The absence of cell fixation and the use of agarose for embedding cells and exonucleases for blunting the ends of DSBs are key advances that contribute to the technique's increased sensitivity and robustness over previously established methods. Overall, END-seq has provided a major technical advance for mapping DSBs and has also helped inform the biology of complex biological processes including genome organization, replication fork collapse and chromosome fragility, off-target identification of RAG recombinase and gene-editing nucleases, and DNA end resection at sites of DSBs.

53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.

Intra-Vκ Cluster Recombination Shapes the Ig Kappa Locus Repertoire.

During V(D)J recombination, RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) that contain either 12- or 23-nt spacer regions. Coordinated 12/23 cleavage predicts that DSBs at variable (V) gene segments should equal the level of breakage at joining (J) segments. Contrary to this, here we report abundant RAG-dependent DSBs at multiple Vκ gene segments independent of V-J rearrangement. We find that a large fraction of Vκ gene segments are flanked not only by a bone-fide 12 spacer but also an overlapping, 23-spacer flipped RSS. These compatible pairs of RSSs mediate recombination and deletion inside the Vκ cluster even in the complete absence of Jκ gene segments and support a V(D)J recombination center (RC) independent of the conventional Jκ-centered RC. We propose an improved model of Vκ-Jκ repertoire formation by incorporating these surprisingly frequent, evolutionarily conserved intra-Vκ cluster recombination events.

Topoisomerase II-Induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity.

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.

Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse.

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.

The Energetics and Physiological Impact of Cohesin Extrusion.

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.