The ribotoxic stress response drives UV-mediated cell death.

While ultraviolet (UV) radiation damages DNA, eliciting the DNA damage response (DDR), it also damages RNA, triggering transcriptome-wide ribosomal collisions and eliciting a ribotoxic stress response (RSR). However, the relative contributions, timing, and regulation of these pathways in determining cell fate is unclear. Here we use time-resolved phosphoproteomic, chemical-genetic, single-cell imaging, and biochemical approaches to create a chronological atlas of signaling events activated in cells responding to UV damage. We discover that UV-induced apoptosis is mediated by the RSR kinase ZAK and not through the DDR. We identify two negative-feedback modules that regulate ZAK-mediated apoptosis: (1) GCN2 activation limits ribosomal collisions and attenuates ZAK-mediated RSR and (2) ZAK activity leads to phosphodegron autophosphorylation and its subsequent degradation. These events tune ZAK's activity to collision levels to establish regimes of homeostasis, tolerance, and death, revealing its key role as the cellular sentinel for nucleic acid damage.

SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion.

Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.

Single-cell atlas reveals correlates of high cognitive function, dementia, and resilience to Alzheimer's disease pathology.

Alzheimer's disease (AD) is the most common cause of dementia worldwide, but the molecular and cellular mechanisms underlying cognitive impairment remain poorly understood. To address this, we generated a single-cell transcriptomic atlas of the aged human prefrontal cortex covering 2.3 million cells from postmortem human brain samples of 427 individuals with varying degrees of AD pathology and cognitive impairment. Our analyses identified AD-pathology-associated alterations shared between excitatory neuron subtypes, revealed a coordinated increase of the cohesin complex and DNA damage response factors in excitatory neurons and in oligodendrocytes, and uncovered genes and pathways associated with high cognitive function, dementia, and resilience to AD pathology. Furthermore, we identified selectively vulnerable somatostatin inhibitory neuron subtypes depleted in AD, discovered two distinct groups of inhibitory neurons that were more abundant in individuals with preserved high cognitive function late in life, and uncovered a link between inhibitory neurons and resilience to AD pathology.

Pan-cancer analysis of post-translational modifications reveals shared patterns of protein regulation.

Post-translational modifications (PTMs) play key roles in regulating cell signaling and physiology in both normal and cancer cells. Advances in mass spectrometry enable high-throughput, accurate, and sensitive measurement of PTM levels to better understand their role, prevalence, and crosstalk. Here, we analyze the largest collection of proteogenomics data from 1,110 patients with PTM profiles across 11 cancer types (10 from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium [CPTAC]). Our study reveals pan-cancer patterns of changes in protein acetylation and phosphorylation involved in hallmark cancer processes. These patterns revealed subsets of tumors, from different cancer types, including those with dysregulated DNA repair driven by phosphorylation, altered metabolic regulation associated with immune response driven by acetylation, affected kinase specificity by crosstalk between acetylation and phosphorylation, and modified histone regulation. Overall, this resource highlights the rich biology governed by PTMs and exposes potential new therapeutic avenues.

Functional interrogation of DNA damage response variants with base editing screens.

Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.

Bacteria-to-Human Protein Networks Reveal Origins of Endogenous DNA Damage.

DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.

Nuclear Genomic Instability and Aging.

The nuclear genome decays as organisms age. Numerous studies demonstrate that the burden of several classes of DNA lesions is greater in older mammals than in young mammals. More challenging is proving this is a cause rather than a consequence of aging. The DNA damage theory of aging, which argues that genomic instability plays a causal role in aging, has recently gained momentum. Support for this theory stems partly from progeroid syndromes in which inherited defects in DNA repair increase the burden of DNA damage leading to accelerated aging of one or more organs. Additionally, growing evidence shows that DNA damage accrual triggers cellular senescence and metabolic changes that promote a decline in tissue function and increased susceptibility to age-related diseases. Here, we examine multiple lines of evidence correlating nuclear DNA damage with aging. We then consider how, mechanistically, nuclear genotoxic stress could promote aging. We conclude that the evidence, in toto, supports a role for DNA damage as a nidus of aging.

A Non-catalytic Function of SETD1A Regulates Cyclin K and the DNA Damage Response.

MLL/SET methyltransferases catalyze methylation of histone 3 lysine 4 and play critical roles in development and cancer. We assessed MLL/SET proteins and found that SETD1A is required for survival of acute myeloid leukemia (AML) cells. Mutagenesis studies and CRISPR-Cas9 domain screening show the enzymatic SET domain is not necessary for AML cell survival but that a newly identified region termed the "FLOS" (functional location on SETD1A) domain is indispensable. FLOS disruption suppresses DNA damage response genes and induces p53-dependent apoptosis. The FLOS domain acts as a cyclin-K-binding site that is required for chromosomal recruitment of cyclin K and for DNA-repair-associated gene expression in S phase. These data identify a connection between the chromatin regulator SETD1A and the DNA damage response that is independent of histone methylation and suggests that targeting SETD1A and cyclin K complexes may represent a therapeutic opportunity for AML and, potentially, for other cancers.

Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states.

UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene.

The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.

BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response.

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.

CCAR1 promotes DNA repair via alternative splicing.

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

Mechanisms of RNF168 nucleosome recognition and ubiquitylation.

RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear. Here, using cryoelectron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy, and functional assays, we provide a molecular description of the reaction cycle and dynamics of RNF168 as it modifies the nucleosome and recognizes its ubiquitylation products. We demonstrate an interaction of a canonical ubiquitin-binding domain within full-length RNF168, which not only engages ubiquitin but also the nucleosome surface, clarifying how such site-specific ubiquitin recognition propels a signal amplification loop. Beyond offering mechanistic insights into a key DDR protein, our study aids in understanding site specificity in both generating and interpreting chromatin ubiquitylation.

NEAT1 promotes genome stability via m6A methylation-dependent regulation of CHD4.

Long noncoding (lnc)RNAs emerge as regulators of genome stability. The nuclear-enriched abundant transcript 1 (NEAT1) is overexpressed in many tumors and is responsive to genotoxic stress. However, the mechanism that links NEAT1 to DNA damage response (DDR) is unclear. Here, we investigate the expression, modification, localization, and structure of NEAT1 in response to DNA double-strand breaks (DSBs). DNA damage increases the levels and N6-methyladenosine (m6A) marks on NEAT1, which promotes alterations in NEAT1 structure, accumulation of hypermethylated NEAT1 at promoter-associated DSBs, and DSB signaling. The depletion of NEAT1 impairs DSB focus formation and elevates DNA damage. The genome-protective role of NEAT1 is mediated by the RNA methyltransferase 3 (METTL3) and involves the release of the chromodomain helicase DNA binding protein 4 (CHD4) from NEAT1 to fine-tune histone acetylation at DSBs. Our data suggest a direct role for NEAT1 in DDR.

Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling.

PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling.

Genotoxic aldehyde stress prematurely ages hematopoietic stem cells in a p53-driven manner.

Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2-/-Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2-/-Fancd2-/- HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging.

Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

Genome Maintenance in Mammalian Stem Cells.

Various stem cells in the body are tasked with maintaining tissue homeostasis throughout the life of an organism and thus must be resilient to intrinsic and extrinsic challenges such as infection and injury. Crucial to these challenges is genome maintenance because a high mutational load and persistent DNA lesions impact the production of essential gene products at proper levels and compromise optimal stem cell renewal and differentiation. Genome maintenance requires a robust and well-regulated DNA damage response suited to maintaining specific niches and tissues. In this review, we explore the similarities and differences between diverse stem cell types derived from (or preceding) all germ layers, including extraembryonic tissues. These cells utilize different strategies, including implementation of robust repair mechanisms, modulation of cell cycle checkpoints best suited to eliminating compromised cells, minimization of cell divisions, and differentiation in response to excessive damage.

Stress-triggered hematopoietic stem cell proliferation relies on PrimPol-mediated repriming.

Stem cell division is linked to tumorigenesis by yet-elusive mechanisms. The hematopoietic system reacts to stress by triggering hematopoietic stem and progenitor cell (HSPC) proliferation, which can be accompanied by chromosomal breakage in activated hematopoietic stem cells (HSCs). However, whether these lesions persist in their downstream progeny and induce a canonical DNA damage response (DDR) remains unclear. Inducing HSPC proliferation by simulated viral infection, we report that the associated DNA damage is restricted to HSCs and that proliferating HSCs rewire their DDR upon endogenous and clastogen-induced damage. Combining transcriptomics, single-cell and single-molecule assays on murine bone marrow cells, we found accelerated fork progression in stimulated HSPCs, reflecting engagement of PrimPol-dependent repriming, at the expense of replication fork reversal. Ultimately, competitive bone marrow transplantation revealed the requirement of PrimPol for efficient HSC amplification and bone marrow reconstitution. Hence, fine-tuning replication fork plasticity is essential to support stem cell functionality upon proliferation stimuli.