Unraveling the complex evolutionary history of lepidopteran chromosomes through ancestral chromosome reconstruction and novel chromosome nomenclature.

BACKGROUND: Lepidoptera is one of the most species-rich animal groups, with substantial karyotype variations among species due to chromosomal rearrangements. Knowledge of the evolutionary patterns of lepidopteran chromosomes still needs to be improved. RESULTS: Here, we used chromosome-level genome assemblies of 185 lepidopteran insects to reconstruct an ancestral reference genome and proposed a new chromosome nomenclature. Thus, we renamed over 5000 extant chromosomes with this system, revealing the historical events of chromosomal rearrangements and their features. Additionally, our findings indicate that, compared with autosomes, the Z chromosome in Lepidoptera underwent a fast loss of conserved genes, rapid acquisition of lineage-specific genes, and a low rate of gene duplication. Moreover, we presented evidence that all available 67 W chromosomes originated from a common ancestor chromosome, with four neo-W chromosomes identified, including one generated by fusion with an autosome and three derived through horizontal gene transfer. We also detected nearly 4000 inter-chromosomal gene movement events. Notably, Geminin is transferred from the autosome to the Z chromosome. When located on the autosome, Geminin shows female-biased expression, but on the Z chromosome, it exhibits male-biased expression. This contributes to the sexual dimorphism of body size in silkworms. CONCLUSIONS: Our study sheds light on the complex evolutionary history of lepidopteran chromosomes based on ancestral chromosome reconstruction and novel chromosome nomenclature.

Alterations in the spatiotemporal expression pattern of geminin during human epidermal morphogenesis.

INTRODUCTION: Geminin, a (25 kDa) protein, was originally identified as a key regulator of DNA replication licensing in the cell cycle and of cell fate during embryonic nervous system formation. Although geminin is involved in mechanisms underlying the regulation of transcription and patterning in embryonic development, its expression and possible significance in human epidermal morphogenesis remains unknown. METHODS: Forty-one skin biopsy specimens obtained from human fetuses (10th to 23rd week of estimated gestational age) were processed for immunohistochemistry using a primary rabbit polyclonal antibody against geminin. RESULTS: Distinct and statistically significant qualitative and quantitative alterations in the spatiotemporal expression pattern of geminin were observed in the developing human epidermis. CONCLUSIONS: The highly ordered expression of geminin in different layers of fetal human epidermis reported here for the first time suggests that this protein may play a significant role in epidermal morphogenesis. However, the mechanisms underlying the alterations of the geminin expression pattern during fetal development at the molecular level remain to be elucidated. Further studies are now warranted to address whether the expression pattern of geminin in the developing human epidermis is disturbed in fetuses with genodermatoses and whether these disturbances might be important for prenatal diagnosis of genodermatoses.

Cas9-Geminin and Cdt1-fused anti-CRISPR protein synergistically increase editing accuracy.

Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly. In contrast, we found that regulation of SpyCas9 activity with an anti-CRISPR protein (AcrIIA4) fused to Chromatin licensing and DNA replication factor 1 (Cdt1) significantly increases HDR efficiency and reduces off-target effects. Here, another anti-CRISPR protein, AcrIIA5, was applied, and the combined use of Cas9-Gem and Anti-CRISPR+Cdt1 showed synergistic enhancement of HDR efficiency. The method may be applicable to various anti-CRISPR/CRISPR-Cas combinations.

RAD51/geminin/γH2AX immunohistochemical expression predicts platinum-based chemotherapy response in ovarian high-grade serous carcinoma.

OBJECTIVE: The RAD51 assay is a recently developed functional assay for homologous recombination deficiency (HRD) that reflects real-time HRD status. We aimed to identify the applicability and predictive value of RAD51 immunohistochemical expression in pre- and post-neoadjuvant chemotherapy (NAC) samples of ovarian high-grade serous carcinoma (HGSC). METHODS: We evaluated the immunohistochemical expression of RAD51/geminin/γH2AX in ovarian HGSC before and after NAC. RESULTS: In pre-NAC tumors (n=51), 74.5% (39/51) showed at least 25% of γH2AX-positive tumor cells, suggesting endogenous DNA damage. The RAD51-high group (41.0%, 16/39) showed significantly worse progression-free survival (PFS) compared to the RAD51-low group (51.3%, 20/39) (p=0.032). In post-NAC tumors (n=50), the RAD51-high group (36.0%, 18/50) showed worse PFS (p=0.013) and tended to present worse overall survival (p=0.067) compared to the RAD51-low group (64.0%, 32/50). RAD51-high cases were more likely to progress than RAD51-low cases at both 6 months and 12 months (p=0.046 and p=0.019, respectively). Of 34 patients with matched pre- and post-NAC RAD51 results, 44% (15/34) of pre-NAC RAD51 results were changed in the post-NAC tissue, and the RAD51 high-to-high group showed the worst PFS, while the low-to-low group showed the best PFS (p=0.031). CONCLUSION: High RAD51 expression was significantly associated with worse PFS in HGSC, and post-NAC RAD51 status showed higher association than pre-NAC RAD51 status. Moreover, RAD51 status can be evaluated in a significant proportion of treatment-naïve HGSC samples. As RAD51 status dynamically changes, sequential follow-up of RAD51 status might reflect the biological behavior of HGSCs.

Introduction of a Geminin mScarlet Reporter into H2B-mTurq2 hiPSCs for Live-cell Imaging of Proliferation and Cell Cycling.

We previously generated a doxycycline-inducible H2B-mTurq2 reporter in hiPSCs to track cells and study cell division and apoptosis. To improve visualization of cycling cells, we introduced a ubiquitously transcribed mScarletI-Geminin (GMMN) (1-110) into the previously untargeted second AAVS1 allele. Fusion to the N-terminal part of GMNN provided tightly controlled mScarletI expression during the cell cycle. mScarletI fluorescence increased gradually from the S-phase through the M-phase of the cell cycle and was lost at the metaphase-anaphase transition. The resulting hiPSC reporter line generated, which we named ProLiving, is a valuable tool to study cell division and cell cycle characteristics in living hiPSC-derived cells.

Multi-candidate immunohistochemical markers to assess radiation response and prognosis in prostate cancer: results from the CHHiP trial of radiotherapy fractionation.

BACKGROUND: Protein markers of cellular proliferation, hypoxia, apoptosis, cell cycle checkpoints, growth factor signalling and inflammation in localised prostate tumours have previously shown prognostic ability. A translational substudy within the CHHiP trial of radiotherapy fractionation evaluated whether these could improve prediction of prognosis and assist treatment stratification following either conventional or hypofractionated radiotherapy. METHODS: Using case:control methodology, patients with biochemical or clinical failure after radiotherapy (BCR) were matched to patients without recurrence according to established prognostic factors (Gleason score, presenting PSA, tumour-stage) and fractionation schedule. Immunohistochemical (IHC) staining of diagnostic biopsy sections was performed and scored for HIF1α, Bcl-2, Ki67, Geminin, p16, p53, p-chk1 and PTEN. Univariable and multivariable conditional logistic regression models, adjusted for matching strata and age, estimated the prognostic value of each IHC biomarker, including interaction terms to determine BCR prediction according to fractionation. FINDINGS: IHC results were available for up to 336 tumours. PTEN, Geminin, mean Ki67 and max Ki67 were prognostic after adjusting for multiple comparisons and were fitted in a multivariable model (n = 212, 106 matched pairs). Here, PTEN and Geminin showed significant prediction of prognosis. No marker predicted BCR according to fractionation. INTERPRETATION: Geminin or Ki67, and PTEN, predicted response to radiotherapy independently of established prognostic factors. These results provide essential independent external validation of previous findings and confirm a role for these markers in treatment stratification. FUNDING: Cancer Research UK (BIDD) grant (A12518), Cancer Research UK (C8262/A7253), Department of Health, Prostate Cancer UK, Movember Foundation, NIHR Biomedical Research Centre at Royal Marsden/ICR.

High expression of GMNN predicts malignant progression and poor prognosis in ACC.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare endocrine neoplasm, which is characterized by poor prognosis and high recurrence rate. Novel and reliable prognostic and metastatic biomarkers are lacking for ACC patients. This study aims at screening potential prognostic biomarkers and therapeutic targets of ACC through bioinformatic methods and immunohistochemical (IHC) analysis. METHODS: In the present study, by using the Gene Expression Omnibus (GEO) database we identified differentially expressed genes (DEGs) in ACC and validated these DEGs in The Cancer Genome Atlas (TCGA) ACC cohort. A DEGs-based signature was additionally constructed and we assessed its prognosis and prescient worth for ACC by survival analysis and nomogram. Immunohistochemistry (IHC) was used to verify the relationship between hub gene-GMNN expressions and clinicopathologic outcomes in ACC patients. RESULTS: A total of 24 DEGs correlated with the prognosis of ACC were screened from the TCGA and GEO databases. Five DEGs were subsequently selected in a signature which was closely related to the survival rates of ACC patients and GMNN was identified as the core gene in this signature. Univariate and multivariate Cox regression showed that the GMNN was an independent prognostic factor for ACC patients (P < 0.05). Meanwhile, GMNN was closely related to the OS and PFI of ACC patients treated with mitotane (P < 0.001). IHC confirmed that GMNN protein was overexpressed in ACC tissues compared with normal adrenal tissues and significantly correlated with stage (P = 0.011), metastasis (P = 0.028) and Ki-67 index (P = 0.014). CONCLUSIONS: GMNN is a novel tumor marker for predicting the malignant progression, metastasis and prognosis of ACC, and may be a potential therapeutic target for ACC.

p53/p21 pathway activation contributes to the ependymal fate decision downstream of GemC1.

Multiciliated ependymal cells and adult neural stem cells are components of the adult neurogenic niche, essential for brain homeostasis. These cells share a common glial cell lineage regulated by the Geminin family members Geminin and GemC1/Mcidas. Ependymal precursors require GemC1/Mcidas expression to massively amplify centrioles and become multiciliated cells. Here, we show that GemC1-dependent differentiation is initiated in actively cycling radial glial cells, in which a DNA damage response, including DNA replication-associated damage and dysfunctional telomeres, is induced, without affecting cell survival. Genotoxic stress is not sufficient by itself to induce ependymal cell differentiation, although the absence of p53 or p21 in progenitors hinders differentiation by maintaining cell division. Activation of the p53-p21 pathway downstream of GemC1 leads to cell-cycle slowdown/arrest, which permits timely onset of ependymal cell differentiation in progenitor cells.

Different expression of DNMT1, PCNA, MCM2, CDT1, EZH2, GMNN and EP300 genes in lymphomagenesis of low vs. high grade lymphoma.

Tumour cells develop by accumulating changes in the genome that result in changes of main cellular processes. Aberrations of basic processes such as replication and chromatin reassembly are particularly important for genomic (in)stability. The aim of this study was to analyse the expression of genes whose products are crucial for the regulation of replication and chromatin reassembly during lymphomagenesis (DNMT1, PCNA, MCM2, CDT1, EZH2, GMNN, EP300). Non-tumour B cells were used as a control, and follicular lymphoma (FL) and the two most common groups of diffuse large B cell lymphoma (DLBCL) samples were used as a model for tumour progression. The results showed that there are significant changes in the expression of the analysed genes in lymphomagenesis, but also that these changes do not display linearity when assessed in relation to the degree of tumour aggression. Additionally, an integrated bioinformatics analysis of the difference in the expression of selected genes between tumour and non-tumour samples, and between tumour samples (FL vs. DLBCL) in five GEO datasets, did not show a consistent pattern of difference among the datasets.

Varying outcomes of triple-negative breast cancer in different age groups-prognostic value of clinical features and proliferation.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive disease lacking specific biomarkers to guide treatment decisions. We evaluated the combined prognostic impact of clinical features and novel biomarkers of cell cycle-progression in age-dependent subgroups of TNBC patients. METHODS: One hundred forty seven TNBC patients with complete clinical data and up to 18 year follow-up were collected from Turku University Hospital, Finland. Eight biomarkers for cell division were immunohistochemically detected to evaluate their clinical applicability in relation to patient and tumor characteristics. RESULTS: Age at diagnosis was the decisive factor predicting disease-specific mortality in TNBC (p = 0.002). The established prognostic features, nodal status and Ki-67, predicted survival only when combined with age. The outcome and prognostic features differed significantly between age groups, middle-aged patients showing the most favorable outcome. Among young patients, only lack of basal differentiation predicted disease outcome, indicating 4.5-fold mortality risk (p = 0.03). Among patients aged > 57, the established prognostic features predicted disease outcome with up to 3.0-fold mortality risk for tumor size ≥ 2 cm (p = 0.001). Concerning cell proliferation, Ki-67 alone was a significant prognosticator among patients aged > 57 years (p = 0.009). Among the studied cell cycle-specific biomarkers, only geminin predicted disease outcome, indicating up to 6.2-fold increased risk of mortality for tumor size < 2 cm (p = 0.03). CONCLUSION: Traditional clinical features do not provide optimal prognostic characterization for all TNBC patients. Young age should be considered as an additional adverse prognostic feature in therapeutic considerations. Increased proliferation, as evaluated using Ki-67 or geminin immunohistochemistry, showed potential in detecting survival differences in subgroups of TNBC.

Distinctive expression of DNA replication factors in squamous cell carcinomas of the lip, face and oral cavity.

OBJECTIVE: Uncontrolled proliferation and aberrations in cell-cycle progression are fundamental issues in cancer. In this study we aimed to determine and compare deoxyribonucleic acid (DNA) replication licensing factors at the mRNA and protein levels among squamous cell carcinomas (SCCs) of the lip, facial-skin and oral cavity. MATERIALS AND METHODS: A total of 103 lip, oral and face SCCs were immunohistochemically stained with MCM2 (mini-chromosome maintenance 2), geminin, and ki67, and their labeling-indices were calculated. Also, 57 SCCs from the same regions along with their adjacent normal tissues underwent quantitative reverse transcription-polymerase chain reaction analysis. RESULTS: All three proteins were overexpressed in the studied SCCs, but only geminin (P = 0.004) showed significant difference among the three regions, with higher levels in oral SCCs compared to lip (P = 0.005) and skin (P = 0.024) tumors. Geminin expression did not differ between skin- and lip-SCCs (P = 0.822). MCM2/ki67 ratio was higher in oral- compared to skin-neoplasms (P = 0.039), but no difference was found in geminin/ki67 among the SCC-subsites. There were significant differences in MCM2 and geminin mRNA between carcinomatous- and normal-tissues in all tumors, but not among the three locations. CONCLUSION: MCM2 and geminin are involved in the tumorigenesis of lip, face and oral SCC at both mRNA- and protein-levels. Geminin may have a role in the site-specific biologic behavior of SCC. Skin SCCs had the highest proportion of licensed non-proliferating cells, while actively proliferating cells were more prominent in oral tumors. Regarding DNA replication, lip SCCs seem to be closer to skin tumors compared to their oral counterparts.

Geminin is essential for DNA re-replication in the silk gland cells of silkworms.

Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.

Cell kinetic markers in cutaneous squamous and basal cell carcinoma of the head and neck.

INTRODUCTION: Proliferation markers play a significant role in the biologic behavior of tumors. Geminin is a known inhibitor of the cell cycle and DNA replication and has not been previously reported in cutaneous basal and squamous cell carcinomas of the head and neck. OBJECTIVES: We aimed to investigate proliferation markers ki67, MCM2, and geminin in head and neck cutaneous basal and squamous cell carcinomas. METHODS: Forty cases of each tumor were immuostained with ki67, MCM2, and geminin followed by assessment of labeling indices (LIs). MCM2/ki67- and geminin/ki67-ratios were also determined; t-test was used for statistical analysis (p<0.05). RESULTS: There was no significant difference in ki67 (p=0.06) and MCM2 (p=0.46) between cutaneous basal and squamous cell carcinomas; however, geminin LI was significantly higher in squamous cell carcinomas compared to cutaneous basal cell carcinomas (p<0.001). Only geminin/ki67 showed a significant difference between the two tumors with the ratio showing significantly higher numbers in squamous cell carcinomas (p=0.015). CONCLUSIONS: Geminin could be regarded as an effective factor in the pathogenesis of head and neck cutaneous cutaneous basal cell carcinomas and squamous cell carcinomas and may be one of the responsible elements in the difference between the biologic behavior of these tumors.

The molecular underpinning of geminin-overexpressing triple-negative breast cancer cells homing specifically to lungs.

Triple-negative breast cancer (TNBCs) display lung metastasis tropism. However, the mechanisms underlying this organ-specific pattern remains to be elucidated. We sought to evaluate the utility of blocking extravasation to prevent lung metastasis. To identify potential geminin overexpression-controlled genetic drivers that promote TNBC tumor homing to lungs, we used the differential/suppression subtractive chain (D/SSC) technique. A geminin overexpression-induced lung metastasis gene signature consists of 24 genes was discovered. We validated overexpression of five of these genes (LGR5, HAS2, CDH11, NCAM2, and DSC2) in worsening lung metastasis-free survival in TNBC patients. Our data demonstrate that LGR5-induced ß-catenin signaling and stemness in TNBC cells are geminin-overexpression dependent. They also demonstrate for the first-time expression of RSPO2 in mouse lung tissue only and exacerbation of its secretion in the circulation of mice that develop geminin overexpressing/LGR5+-TNBC lung metastasis. We identified a novel extravasation receptor complex, consists of CDH11, CD44v6, c-Met, and AXL on geminin overexpressing/LGR5+-TNBC lung metastatic precursors, inhibition of any of its receptors prevented geminin overexpressing/LGR5+-TNBC lung metastasis. Overall, we propose that geminin overexpression in normal mammary epithelial (HME) cells promotes the generation of TNBC metastatic precursors that home specifically to lungs by upregulating LGR5 expression and promoting stemness, intravasation, and extravasation in these precursors. Circulating levels of RSPO2 and OPN can be diagnostic biomarkers to improve risk stratification of metastatic TNBC to lungs, as well as identifying patients who may benefit from therapy targeting geminin alone or in combination with any member of the newly discovered extravasation receptor complex to minimize TNBC lung metastasis.

Gene expression signature as a surrogate marker of microvascular invasion on routine hepatocellular carcinoma biopsies.

BACKGROUND & AIMS: Microvascular invasion (MVI), a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma (HCC), is only detectable by microscopic examination of the surgical specimen. We aimed to define a transcriptomic signature associated with MVI in HCC than can be applied to formalin-fixed paraffin-embedded (FFPE) biopsies for use in clinical practice. METHODS: To identify a gene expression signature related to MVI by using NanoString technology, we selected a set of 200 genes according to the literature and RNA-sequencing data obtained from a cohort of 150 frozen HCC samples previously published. We used 178 FFPE-archived HCC samples, including 109 surgical samples for the training set and 69 paired pre-operative biopsies for the validation set. In 14 cases of the training set, a paired biopsy was available and was also analyzed. RESULTS: We identified a 6-gene signature (ROS1, UGT2B7, FAS, ANGPTL7, GMNN, MKI67) strongly associated with MVI in the training set of FFPE surgical HCC samples, with 82% accuracy (sensitivity 82%, specificity 81%, AUC 0.82). The NanoString gene expression was highly correlated in 14 paired surgical/biopsy HCC samples (mean R: 0.97). In the validation set of 69 FFPE HCC biopsies, the 6-gene NanoString signature predicted MVI with 74% accuracy (sensitivity 73%, specificity 76%, AUC 0.74). Moreover, on multivariate analysis, the MVI signature was associated with overall survival in both sets (hazard ratio 2.29; 95% CI 1.03-5.07; p = 0.041). CONCLUSION: We defined a 6-gene signature that can accurately predict MVI in FFPE HCC biopsy samples, which is also associated with overall survival, although its survival impact must be confirmed by extensive study with further clinical data. LAY SUMMARY: Microvascular invasion, a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma, is only detectable by microscopic examination of a surgical specimen. In this study, we defined a relevant surrogate signature of microvascular invasion in hepatocellular carcinoma that may be applied in clinical practice with routine tumor biopsy and integrated into the therapeutic strategy.

Prognostic value of HPV 16/18 genotyping and geminin mRNA quantification in low-grade cervical squamous intraepithelial lesion.

Low-grade cervical squamous intraepithelial lesion is a precancerous neoplasia that has appreciable probability to evolve into malignancy. To explore the prognostic value of HPV 16/18 genotyping and geminin mRNA quantification in predicting the progressiveness of LSIL. We recruited 212 participants who were negative for intraepithelial lesion or malignancy (NILM 76), low-grade squamous intraepithelial lesion (LSIL 85), high-grade squamous intraepithelial lesion (HSIL 36) and cervical intraepithelial neoplasia grade cervical cancer grade 3, (CIN3 15) patients. Tissues were obtained during excisional treatment. HPV 16/18 genotyping and geminin mRNA qRT-PCR were performed. HPV 16/18 positivity rate and geminin mRNA level were integrated with the clinical parameters into a multivariate logistic model. Area under curve was yielded based on receiver operation curve derived from this multivariate logistic model. Follow-up visits were performed to LSIL patients with progression. HSIL patients have higher HPV 16/18 positivity rate and geminin mRNA levels than LSIL. Among HSIL, CIN3 have higher HPV 16/18 positivity rate and geminin mRNA levels. Multivariate logistic analysis showed that HPV 16/18 positivity and geminin mRNA expression status are independent factors for differentiating HSIL and LSIL. The baseline HPV 16/18 positivity rate and geminin mRNA levels of 18 LSIL patients who developed HSIL are significantly higher than non-progressive LSIL patients. The values examined at follow-up timepoints were also higher than baseline. These results suggest that geminin is implicated in the progression of LSIL and combining HPV 16/18 genotyping and geminin mRNA qRT-PCR could potentially differentiating the progressive LSIL and improve the efficacy of clinical intervention.

Proteome-wide mapping of short-lived proteins in human cells.

Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.

SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition.

Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.

Live Imaging and Analysis of Cilia and Cell Cycle Dynamics with the Arl13bCerulean-Fucci2a Biosensor and Fucci Tools.

The cell and cilia cycles are inextricably linked through the dual functions of the centrioles at both the basal body of cilia and at mitotic centrosomes. How cilia assembly and disassembly, either through slow resorption or rapid deciliation, are coordinated with cell cycle progression remains unclear in many cell types and developmental paradigms. Moreover, little is known about how additional cilia parameters including changes in ciliary length or frequency of distal tip shedding change with cell cycle stage. In order to explore these questions, we have developed the Arl13bCerulean-Fucci2a tricistronic cilia and cell cycle biosensor (Ford et al., Dev Cell 47:509-523.e7, 2018). This reporter allowed us to document the heterogeneity in ciliary behaviors during the cell cycle at a population level. Without the need for external stimuli, it revealed that in several cell types and in the developing embryo cilia persist beyond the G1/S checkpoint. Here, we describe the generation of stable cell lines expressing Arl13bCerulean-Fucci2a and open-source software to aid morphometric profiling of the primary cilium with cell cycle phases, including changes in cilium length. This resource will allow the investigation of multiple morphometric questions relating to cilia and cell cycle biology.

Single-cell transcriptome analysis defines heterogeneity of the murine pancreatic ductal tree.

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA-sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.