Faculty Bibliography

Establishment and maintenance of cellular sex identity is essential for reproduction. Sex identity of somatic and germline cells must correspond for sperm or oocytes to be produced, with mismatched identity causing infertility in all organisms from flies to humans. In adult Drosophila testes, Chronologically inappropriate morphogenesis (Chinmo) is required for maintenance of male somatic identity. Loss of chinmo leads to feminization of the male soma, including adoption of female-specific cell morphologies and gene expression. However, the degree to which feminized somatic cells engage female-specific cellular behaviors or influence the associated XY germline is unknown. Using extended live imaging, we find that chinmo-depleted somatic cells acquire cell behaviors characteristic of ovarian follicle cells, including incomplete cytokinesis and rotational migration. Importantly, migration in both contexts require the basement membrane protein Perlecan and adhesion protein E-cadherin. Finally, we find that sex- converted soma non-autonomously induce expression of an early oocyte specification protein in XY germ cells. Taken together, our work reveals a dramatic transformation of somatic cell behavior during sex conversion and provides a powerful model to study soma-derived induction of oocyte identity.

INTRODUCTION/BACKGROUND:Presenilin (PS) gene mutations cause memory impairment in early-onset familial Alzheimer's disease (FAD), but the underlying mechanisms remain unclear. METHODS:activity and CREB phosphorylation in the primary motor cortex. RESULTS:levels are altered in a cortical layer and neuron type-specific manner in PS1 mutant mice as compared to WT control mice. Notably, while running caused a significant increase of CREB phosphorylation in WT mice, it led to a significant decrease of CREB phosphorylation in layer 5 neurons of mutant mice. DISCUSSION/CONCLUSIONS:activity and CREB phosphorylation in deep cortical layers are early events leading to memory impairment in the PS1 mutation-related familial form of AD.

In Escherichia coli, ribose-5-phosphate (R5P) biosynthesis occurs via two distinct pathways: an oxidative branch of the pentose phosphate pathway (PPP) originating from glucose-6-phosphate, and a reversed non-oxidative branch originating from fructose-6-phosphate, which relies on transaldolases TalA and TalB. Remarkably, we found that disrupting the oxidative PPP branch by deleting the zwf gene significantly increased bacterial susceptibility to killing by a variety of antibiotics. Surprisingly, additional mutations in the talA and talB genes further enhanced bacterial sensitivity to oxidative stress and antibiotic-mediated killing though they had little impact on the minimal inhibitory concentrations (MICs). The hypersensitivity observed in the zwf talAB mutant could be fully reversed by the processes that either utilize R5P or limited its accumulation. Specifically, activating the purine biosynthetic regulon or inhibiting nucleoside catabolism via deoB gene inactivation, which blocks the conversion of ribose-1-phosphate to R5P, restored bacterial tolerance. Furthermore, enhancing the biosynthesis of cell wall component ADP-heptose from sedoheptulose-7-phosphate suppressed antibiotic killing of the zwf talAB mutant. Biochemical analysis confirmed a direct link between elevated intracellular R5P levels and increased bacterial susceptibility to antibiotics-induced killing. These findings suggest that targeting the PPP could be a promising strategy for developing new therapeutic approaches aimed at potentiating clinically relevant antibiotics.IMPORTANCERecent studies have revealed the crucial role of bacterial cell's metabolic status in its susceptibility to the lethal action of antibacterial drugs. However, there is still no clear understanding of which key metabolic nodes are optimal targets to improve the effectiveness of bacterial infection treatment. Our study establishes that the disruption of the canonical pentose phosphate pathway induces one-way anabolic synthesis of pentose phosphates (aPPP) in E. coli cells, increasing the killing efficiency of various antibiotics. It is also demonstrated that the activation of ribose-5-phosphate utilization processes restores bacterial tolerance to antibiotics. We consider the synthesis of ribose-5-phosphate to be one of the determining factors of bacterial cell stress resistance. Understanding bacterial metabolic pathways, particularly the aPPP's role in antibiotic sensitivity, offers insights for developing novel adjuvant therapeutic strategies to enhance antibiotic potency.

Pancreatic ductal adenocarcinoma (PDAC) emerges from mutant KRAS-harboring but dormant low-grade pancreatic intraepithelial neoplasia (PanIN). To examine the role of oxidative stress, a putative PDAC risk factor, we established an organoid-based transformation system. Although the prototypic oxidant H₂O₂ induced organoid transformation, its effect was nonmutational and was mediated by the oxidant-responsive transcription factor NRF2, which induced the histone methyltransferase EZH2. Congruently, nonoxidizing NRF2 activators triggered organoid malignant conversion through NRF2 and EZH2, establishing a hitherto unknown epigenetic mechanism underlying PanIN-to-PDAC progression. While NRF2 induced EZH2 gene transcription in mouse and human PDAC, EZH2, a general repressor, coactivated transcription of NRF2-encoding NFE2L2 and interacted with other transcription factors to induce genes that sustain PDAC metabolic demands. The self-amplifying NRF2-EZH2 epigenetic loop also accounted for inflammation-driven PanIN-to-PDAC progression in vivo and was upregulated in established human PDAC, whose malignancy was maintained by NRF2 binding to the EZH2 promoter.

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.