Faculty Bibliography

The cellular networks that maintain genome stability encompass numerous pathways involved in all aspects of nucleic acid metabolism. Through bioinformatic analysis, we identified the Zinc Finger CCCH-Type Containing 4 protein (ZC3H4), a suppressor of noncoding RNA (ncRNA) production, as a pivotal player in this system. Experimentally, ZC3H4 deficiency led to increased DNA damage, abnormal mitosis, and cellular senescence. Biochemical analysis and super-resolution microscopy revealed that the loss of ZC3H4 increased replication stress (RS)-a major driver of genome instability-by inducing a hypertranscription state that promoted R loop formation and transcription-replication conflicts (TRCs), both of which drive RS. Further bioinformatic analysis demonstrated that ZC3H4 preferentially binds to genomic regions prone to TRCs and R loops, where it suppresses ncRNA bursts, functioning as part of the Restrictor complex. Our findings identify ZC3H4 as a crucial factor in maintaining genome integrity, strategically positioned at the critical intersection of DNA and RNA synthesis.

DNA double-strand breaks (DSBs) are harmful lesions and major sources of genomic instability. Studies have suggested that DSBs induce local transcriptional silencing that consequently promotes genomic stability. Several factors have been proposed to actively participate in this process, including Ataxia-telangiectasia mutated (ATM) and Polycomb repressive complex 1 (PRC1). Here, we found that disrupting PRC1 clustering disrupts DSB-induced gene silencing. Interactome analysis of PHC2, a PRC1 subunit that promotes the PRC1 clustering, found several nucleoporins found in the nuclear pore complex (NPC). Similar to PHC2, depleting the nucleoporins also disrupted the DSB-induced gene silencing. We found that some of these nucleoporins, such as NUP107 and NUP43, which are members of the Y-complex of NPC, localize to DSB sites. The presence of nucleoporins and PHC2 at DSB regions was interdependent, suggesting that they act cooperatively in the DSB-induced gene silencing. We further found two structural components within NUP107 to be necessary for the transcriptional repression at DSBs: ATM/ Ataxia telangiectasia and Rad3-related-mediated phosphorylation at the Serine37 residue within the N-terminal disordered tail and the NUP133-binding surface at the C-terminus. These results provide a functional interplay among nucleoporins, ATM, and the Polycomb proteins in the DSB metabolism and underscore their emerging roles in genome stability maintenance.

OBJECTIVE:Identify how surgical resection of pancreatic ductal adenocarcinoma (PDAC) affects systemic minimal residual disease (MRD). METHODS:Pancreatic tumors were generated by orthotopic implantation of tumor cells into the pancreas of immunocompetent mice. Tumor resection was carried out via distal pancreatectomy and splenectomy. Liver metastases and microenvironment immune changes were analyzed in resected vs. non-resected mice. RESULTS:Resection was accompanied by proliferative expansion of liver metastases and an increase in hepatic metastatic burden. Postoperative immune changes predominantly manifested as a time-dependent increase in eosinophils and decrease in neutrophils. The postoperative hepatic eosinophilia was protective of further metastatic progression. The parenchymal findings were detectable in the circulation, and the trends observed in the mouse model modeled those seen in PDAC patients postoperatively. CONCLUSION/CONCLUSIONS:Collectively, we describe a preclinical resection model that offers a means to investigate MRD. Using this model, we delineated effects of surgical resection on metastatic outgrowth and uncovered a protective link between the postoperative hepatic eosinophilia and further metastatic progression.

Around 40% of the US population and 1 in 6 individuals worldwide have obesity, with the incidence surging globally^1,2. Various dietary interventions, including carbohydrate, fat and, more recently, amino acid restriction, have been explored to combat this epidemic^3-6. Here we investigated the impact of removing individual amino acids on the weight profiles of mice. We show that conditional cysteine restriction resulted in the most substantial weight loss when compared to essential amino acid restriction, amounting to 30% within 1 week, which was readily reversed. We found that cysteine deficiency activated the integrated stress response and oxidative stress response, which amplify each other, leading to the induction of GDF15 and FGF21, partly explaining the phenotype^7-9. Notably, we observed lower levels of tissue coenzyme A (CoA), which has been considered to be extremely stable¹⁰, resulting in reduced mitochondrial functionality and metabolic rewiring. This results in energetically inefficient anaerobic glycolysis and defective tricarboxylic acid cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen-rich compounds and amino acids. In summary, our investigation reveals that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism and stress signalling compared with other amino acid restrictions. These findings suggest strategies for addressing a range of metabolic diseases and the growing obesity crisis.

DNA double strand break repair (DSBR) represents a fundamental process required to maintain genome stability and prevent the onset of disease. Whilst cell cycle phase and the chromatin context largely dictate which repair pathway is utilised to restore damaged DNA, it has been recently shown that nuclear actin filaments play a major role in clustering DNA breaks to facilitate DSBR by homologous recombination (HR). However, the mechanism with which nuclear actin and the different actin nucleating factors regulate HR is unclear. Interestingly, patients with biallelic mutations in the actin nucleating factor DIAPH1 exhibit a striking overlap of clinical features with the HR deficiency disorders, Nijmegen Breakage Syndrome (NBS) and Warsaw Breakage Syndrome (WABS). This suggests that DIAPH1 may play a role in regulating HR and that some of the clinical deficits associated with DIAPH1 mutations may be caused by an underlying DSBR defect. In keeping with this clinical similarity, we demonstrate that cells from DIAL (DIAPH1 Loss-of-function) Syndrome patients display an HR repair defect comparable to loss of NBS1. Moreover, we show that this DSBR defect is also observed in a subset of patients with Baraitser-Winter Cerebrofrontofacial (BWCFF) syndrome associated with mutations in ACTG1 (γ-actin) but not ACTB (β-actin). Lastly, we demonstrate that DIAPH1 and γ-actin promote HR-dependent repair by facilitating the relocalisation of the MRE11/RAD50/NBS1 complex to sites of DNA breaks to initiate end-resection. Taken together, these data provide a mechanistic explanation for the overlapping clinical symptoms exhibited by patients with DIAL syndrome, BWCFF syndrome and NBS.

In healthy cells, cyclin D1 is expressed during the G1 phase of the cell cycle, where it activates CDK4 and CDK6. Its dysregulation is a well-established oncogenic driver in numerous human cancers. The cancer-related function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G-Two and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unrecognized substrate of cyclin D1-CDK4/6 in tumor cells overexpressing cyclin D1 during G1 and subsequent phases. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 across all cell cycle phases. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1's role in cell cycle control and oncogenesis beyond RB phosphorylation.

Reactome (reactome.org) is a manually curated, peer-reviewed, open-source, open-access pathway knowledgebase of essential human cellular functions. Reactome includes viral life cycles that capture a broad range of virus-induced human pathology. Here, we describe a workflow using collaborative curation strategies, orthoinference procedures, and literature triage to rapidly create reliable molecular models of emergent viruses. The resulting pathway data set rigorously details viral infection pathways, interactions with normal human biological processes, and potential therapeutic compounds.

The tumour-suppressor protein BRCA2 has a central role in homology-directed DNA repair by enhancing the formation of RAD51 filaments on resected single-stranded DNA generated at double-stranded DNA breaks and stimulating RAD51 activity^1,2. Individuals with BRCA2 mutations are predisposed to cancer; however, BRCA2-deficient tumours are often responsive to targeted therapy with PARP inhibitors (PARPi)^3-6. The mechanism by which BRCA2 deficiency renders cells sensitive to PARPi but with minimal toxicity in cells heterozygous for BRCA2 mutations remains unclear. Here we identify a previously unknown role of BRCA2 that is directly linked to the effect of PARP1 inhibition. Using biochemical and single-molecule approaches, we demonstrate that PARPi-mediated PARP1 retention on a resected DNA substrate interferes with RAD51 filament stability and impairs RAD51-mediated DNA strand exchange. Full-length BRCA2 protects RAD51 filaments and counteracts the instability conferred by PARPi-mediated retention by preventing the binding of PARP1 to DNA. Extending these findings to a cellular context, we use quantitative single-molecule localization microscopy to show that BRCA2 prevents PARPi-induced PARP1 retention at homologous-recombination repair sites. By contrast, BRCA2-deficient cells exhibit increased PARP1 retention at these lesions in response to PARPi. These results provide mechanistic insights into the role of BRCA2 in maintaining RAD51 stability and protecting homologous-recombination repair sites by mitigating PARPi-mediated PARP1 retention.

Topoisomerase I, a nickase that allows swiveling of the DNA substrate, is highly enriched in the ribosomal DNA (rDNA) from yeast to humans, but its function at this locus remains poorly understood. S. cerevisiae mutants lacking topoisomerase I (top1) exhibit pronounced rDNA instability and accumulate bubbles of single-stranded DNA (ssDNA) on 35S ribosomal RNA genes, suggesting a role in relieving transcription-associated topological stress. However, Top1-cleavage complexes are most highly enriched in the rDNA-encoded replication-fork barrier, a genetically encoded source of rDNA instability, and only weakly in the 35S promoter, leading to the proposal that top1-associated rDNA instability may be linked to the fork barrier. Here, we show that the rDNA instability phenotypes of top1 mutants, including increased formation of extrachromosomal rDNA circles, elevated genetic marker loss, and instability of critically short rDNA arrays, are independent of the replication fork barrier. In addition, we link Top1 binding at the 35S promoter to the formation of a DNA species with a long ssDNA tail, which originates in the 35S promoter region and is undetectable in top1 mutants. This DNA species is abundant in wild-type cells, occurs independently of S phase, and may be the resolution product of the ssDNA bubbles seen in top1 mutants. Whether formation of this DNA species is important for rDNA stability remains unclear, but our findings link Top1 to highly active DNA metabolism in the 35S promoter.

Human embryonic carcinoma (hEC) cells are derived from teratocarcinomas, exhibit robust proliferation, have a high differentiation potential, are the malignant counterparts of human embryonic stem cells (hESCs), and are considered hESC-like. The chromosomal passenger complex (CPC), made up of the microtuble binding protein Borealin, the kinase Aurora-B, the CPC-stabilizing inner centromere protein (INCENP), and the inhibitor of apoptosis family member Survivin, regulates cell division and is active exclusively during mitosis in somatic cells. The anaphase-promoting complex/cyclosome and its cofactor Cdh1 (APC/C^Cdh1) is a ubiquitylating complex that catalyzes the degradation of Aurora-B and Borealin in somatic cells but has low activity during interphase in hESCs. Here, we found that Borealin and Aurora-B exhibited sustained stability throughout the cell cycle of hEC cells due to low APC/C^Cdh1 activity. In contrast with somatic cells, CPC activity persisted across the cell cycle of hEC cells because of diminished APC/C^Cdh1 activity. Disrupting the CPC complex by depleting its constituents triggered spontaneous differentiation in hEC cells. As hEC cells differentiated, APC/C^Cdh1 activation curtailed CPC activity. Inactivating the CPC by pharmacologically inhibiting Aurora-B induced hEC cell differentiation by activating the epithelial-to-mesenchymal transition (EMT) program. Hence, APC/C^Cdh1-mediated termination of CPC activity triggered hEC cell differentiation. Collectively, these findings demonstrate a role for the CPC in governing hESC cell fate.