Replication Licensing Research Articles

SEC-SAXS/MC Ensemble Structural Studies of the Microtubule Binding Protein Cdt1 Show Monomeric, Folded-Over Conformations.

Cdt1 is a mixed folded protein critical for DNA replication licensing and it also has a "moonlighting" role at the kinetochore via direct binding to microtubules and the Ndc80 complex. However, it is unknown how the structure and conformations of Cdt1 could allow it to participate in these multiple, unique sets of protein complexes. While robust methods exist to study entirely folded or unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. In this work, we employ orthogonal biophysical and computational techniques to provide structural characterization of mitosis-competent human Cdt1. Thermal stability analyses shows that both folded winged helix domains1 are unstable. CD and NMR show that the N-terminal and linker regions are intrinsically disordered. DLS shows that Cdt1 is monomeric and polydisperse, while SEC-MALS confirms that it is monomeric at high concentrations, but without any apparent inter-molecular self-association. SEC-SAXS enabled computational modeling of the protein structures. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble of structures. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with SAXS. The best-fit models have the N-terminal and linker disordered regions extended into the solution and the two folded domains close to each other in apparent "folded over" conformations. We hypothesize the best-fit Cdt1 conformations could be consistent with a function as a scaffold protein that may be sterically blocked without binding partners. Our study also provides a template for combining experimental and computational techniques to study mixed-folded proteins.

The Plasmodium falciparum histone methyltransferase SET10 participates in a chromatin modulation network crucial for intraerythrocytic development.

The lifecycle progression of the malaria parasite Plasmodium falciparum requires precise tuning of gene expression including histone methylation. The histone methyltransferase PfSET10 was previously described as an H3K4 methyltransferase involved in var gene regulation, making it a prominent antimalarial target. In this study, we investigated the role of PfSET10 in the blood stages of P. falciparum in more detail, using tagged PfSET10-knockout (KO) and -knockdown (KD) lines. We demonstrate a nuclear localization of PfSET10 with peak protein levels in schizonts. PfSET10 deficiency reduces intraerythrocytic growth but has no effect on gametocyte commitment and maturation. Screening of the PfSET10-KO line for histone methylation variations reveals that lack of PfSET10 renders the parasites unable to mark H3K18me1, while no reduction in the H3K4 methylation status could be observed. Comparative transcriptomic profiling of PfSET10-KO schizonts shows an upregulation of transcripts particularly encoding proteins linked to red blood cell remodeling and antigenic variation, suggesting a repressive function of the histone methylation mark. TurboID coupled with mass spectrometry further highlights an extensive nuclear PfSET10 interaction network with roles in transcriptional regulation and mRNA processing, DNA replication and repair, and chromatin remodeling. The main interactors of PfSET10 include ApiAP2 transcription factors, epigenetic regulators like PfHDAC1, chromatin modulators like PfMORC and PfISWI, mediators of RNA polymerase II, and DNA replication licensing factors. The combined data pinpoint PfSET10 as a histone methyltransferase essential for H3K18 methylation that regulates nucleic acid metabolic processes in the P. falciparum blood stages as part of a comprehensive chromatin modulation network.IMPORTANCEThe fine-tuned regulation of DNA replication and transcription is particularly crucial for the rapidly multiplying blood stages of malaria parasites and proteins involved in these processes represent important drug targets. This study demonstrates that contrary to previous reports the histone methyltransferase PfSET10 of the malaria parasite Plasmodium falciparum promotes the methylation of histone 3 at lysine K18, a histone mark to date not well understood. Deficiency of PfSET10 due to genetic knockout affects genes involved in intraerythrocytic development. Furthermore, in the nuclei of blood-stage parasites, PfSET10 interacts with various protein complexes crucial for DNA replication, remodeling, and repair, as well as for transcriptional regulation and mRNA processing. In summary, this study highlights PfSET10 as a methyltransferase affecting H3K18 methylation with critical functions in chromatin maintenance during the development of P. falciparum in red blood cells.

First report of Sclerotinia minor causing soft rot of Scabiosa atropurpurea in California.

In May of 2019, Scabiosa atropurpurea samples with brown discoloration, soft rot of the crown and lower stem, with presence of white mycelium and black sclerotia (Supp. Fig. 1A, B) were collected from a 0.10 ha open field diversified cut flower production in San Luis Obispo County, CA. Approximately 30 to 40% of the scabiosa crop planted in a quarter of the field, exhibited symptoms. Symptomatic crowns and lower stems from five plants were surface disinfested by rinsing in 0.1% Tween 20, soaking in 70% ethanol for 30 s, 1% sodium hypochlorite for 2 min and sterile water. Disinfested tissue was placed in 1/10 potato dextrose agar (PDA) and incubated at 20°C (12 h photoperiod). Resulting colonies (n = 5) formed abundant white mycelia, with black sclerotia formed on the outer edge of the plates after two weeks (Suppl. Fig. 1C). Sclerotia (n = 50) had an average size of 1.6 (± 0.19) mm in diameter. Morphological identification resulted in Sclerotinia sp. (Hao et al., 2003). The pathogen was further identified by DNA extraction of two hyphal tipped isolates, followed by amplification and sequencing of the rDNA internal transcribed spacer (ITS) region, ITS1/ITS4 (White et al. 1990), calmodulin (CaM), CAL-228F/CAL-737R (Carbone and Kohn, 1999), and DNA replication licensing factor Mcm7 Mcm7-709for/Mcm7-1348rev (Schmitt et al., 2009). NCBI BLAST searches with consensus sequences for each maker revealed 99 to 100% identity with S. minor ex-types for all loci (Supp. Table 1). A maximum parsimony multilocus phylogenetic analysis clustered Californian isolates with reference strains of S. minor (Supp. Fig. 1F). Sequences were deposited in GenBank (Supp. Table 1). Pathogenicity tests were conducted with isolate CS435, which was transferred onto PDA plates and incubated at 20°C for one week. Inoculum consisted of CS435 infested PDA plugs (1 cm3). In the greenhouse, the experiment was set as a complete randomized design and observed for six weeks. Fourteen-week-old scabiosa 'Merlot Red' grown in 3.78 L pots (n = 6), were inoculated by wounding plants at 0.5 cm above the crowns with a 1 mm probe. Inoculum was placed directly on top of the wound and was secured with parafilm. Negative control plants (n = 6) were wounded as above and inoculated with PDA plugs. In experiment 1(19.4 (± 3) °C, RH 46.9), 83% of plants exhibited yellowing of the lower leaves and wilting at one week post inoculation (wpi). Symptoms progressed over time until wilting, major leaf and stem necrosis, was observed in all inoculated plants (Supp. Fig 1E, D). Plant mortality incidence at five wpi was 83%. Pathogen signs including white mycelia and black sclerotia were also observed. In experiment 2 (20.0 (± 10) °C, RH 39.6), 66% of the total plants were symptomatic at five wpi: 33% exhibited yellowing of the lower leaves and wilting, and 33% of plants died. Disease did not develop in non-inoculated plants in either experiment. S. minor was successfully reisolated from surface disinfested tissue of at least 50% (Exp. 1) and 100% (Exp. 2) of inoculated plants, yielding identical sequences to those of the inoculated isolate. S. minor is a soil-borne pathogen that infects tomato, lettuce, brassica, and sunflower crops in California. This study stablishes S. minor as a new soil-borne pathogen of Scabiosa in California. Soil-borne pathogens are a recurrent issue in the cut flower industry, characterizing new pathogens in these crops can inform crop advisors and disease diagnostician to improve disease management in the ornamental industry.

Replication licensing regulated by a short linear motif within an intrinsically disordered region of origin recognition complex

In eukaryotes, the origin recognition complex (ORC) faciliates the assembly of pre-replicative complex (pre-RC) at origin DNA for replication licensing. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit is crucial for this process. Removing a segment (residues 176-200) from Orc2-IDR or mutating a key isoleucine (194) significantly inhibits replication initiation across the genome. These Orc2-IDR mutants are capable of assembling the ORC-Cdc6-Cdt1-Mcm2-7 intermediate, which exhibits impaired ATP hydrolysis and fails to be convered into the subsequent Mcm2-7-ORC complex and pre-RC. These defects can be partially rescued by the Orc2-IDR peptide. Moreover, the phosphorylation of this Orc2-IDR region by S cyclin-dependent kinase blocks its binding to Mcm2-7 complex, causing a defective pre-RC assembly. Our findings provide important insights into the multifaceted roles of ORC in supporting origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase.

Dynamics of amylopectin granule accumulation during the course of the chronic Toxoplasma infection is linked to intra-cyst bradyzoite replication.

The contribution of amylopectin granules (AG), comprised of a branched chain storage homopolymer of glucose, to the maintenance and progression of the chronic Toxoplasma gondii infection has remained undefined. Here we describe the role of AG in the physiology of encysted bradyzoites by using a custom developed imaging-based application AmyloQuant that permitted quantification of relative levels of AG within in vivo derived tissue cysts during the initiation and maturation of the chronic infection. Our findings establish that AG are dynamic entities, exhibiting considerable heterogeneity among tissue cysts at all post infection time points examined. Quantification of relative AG levels within tissue cysts exposes a previously unrecognized temporal cycle defined by distinct phases of AG accumulation and utilization over the first 6 weeks of the chronic phase. This AG cycle is temporally coordinated with overall bradyzoite mitochondrial activity implicating amylopectin in the maintenance and progression of the chronic infection. In addition, the staging of AG accumulation and it rapid utilization within encysted bradyzoites was associated with a burst of coordinated replication. As such our findings suggest that AG levels within individual bradyzoites, and across bradyzoites within tissue cysts may represent a key component in the licensing of bradyzoite replication, intimately linking stored metabolic potential to the course of the chronic infection. This extends the impact of AG beyond the previously assigned role that focused exclusively on parasite transmission. These findings force a fundamental reassessment of the chronic Toxoplasma infection, highlighting the critical need to address the temporal evolution of this crucial stage in the parasite life cycle.

Assembly, Activation, and Helicase Actions of MCM2-7: Transition from Inactive MCM2-7 Double Hexamers to Active Replication Forks.

In this review, we summarize the processes of the assembly of multi-protein replisomes at the origins of replication. Replication licensing, the loading of inactive minichromosome maintenance double hexamers (dhMCM2-7) during the G1 phase, is followed by origin firing triggered by two serine-threonine kinases, Cdc7 (DDK) and CDK, leading to the assembly and activation of Cdc45/MCM2-7/GINS (CMG) helicases at the entry into the S phase and the formation of replisomes for bidirectional DNA synthesis. Biochemical and structural analyses of the recruitment of initiation or firing factors to the dhMCM2-7 for the formation of an active helicase and those of origin melting and DNA unwinding support the steric exclusion unwinding model of the CMG helicase.

CDT1, transcriptionally regulated by E2F2, promotes lung adenocarcinoma progression

CDT1, a gene that shows excessive expression in various malignancies, functions as a pivotal regulator of replication licensing. In this study, we observed a positive correlation in expression between CDT1 and E2F2 among patients with lung adenocarcinoma (LUAD). Our findings substantiated that E2F2 directly interacted with the promoter region of CDT1, as confirmed by ChIP-qPCR assays, and depletion of E2F2 resulted in a downregulation of CDT1 expression in LUAD cell lines by gene interference technology. Furthermore, we identified an upregulation of CDT1 mRNA level in Chinese LUAD samples. Notably, in the loss-of-function assays, depletion of CDT1 in LUAD cell lines inhibited cell proliferation, migration, and invasion. Concurrently, it promoted cell apoptosis and induced G0/G1 phase arrest using MTT, flow cytometry, and Transwell assays, reinforcing its role as an oncogene.Furthermore, enhanced tumor ablation was determined in a CDT1-downregulated LUAD tumor-bearing nude mouse model. Collectively, our results strongly suggest that E2F2 positively regulates CDT1 expression and actively participates in the progression of lung adenocarcinoma, thereby providing valuable insights into identifying novel therapeutic targets for LUAD treatment.

Endonucleosis mediates internalization of cytoplasm into the nucleus

Setd8 regulates transcription elongation, mitotic DNA condensation, DNA damage response and replication licensing. Here we show that, in mitogen-stimulated liver-specific Setd8-KO mice, most of the hepatocytes are eliminated by necrosis but a significant number of them survive via entering a stage exhibiting several senescence-related features. Setd8-deficient hepatocytes had enlarged nuclei, chromosomal hyperploidy and nuclear engulfments progressing to the formation of intranuclear vesicles surrounded by nuclear lamina. These vesicles contain glycogen, cytoplasmic proteins and even entire organelles. We term this process “endonucleosis”. Intranuclear vesicles are absent in hepatocytes of Setd8/Atg5 knockout mice, suggesting that the process requires the function of the canonical autophagy machinery. Endonucleosis and hyperploidization are temporary, early events in the surviving Setd8-deficient cells. Larger vesicles break down into microvesicles over time and are eventually eliminated. The results reveal sequential events in cells with extensive DNA damage, which function as part of survival mechanisms to prevent necrotic death.

Phosphorylation of Orc6 During Mitosis Regulates DNA Replication and Ribosome Biogenesis.

The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication.

The Crk4-Cyc4 complex regulates G2/M transition in Toxoplasma gondii

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4–TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4–TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4–Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.

CUL4A Ubiquitin Ligase Is an Independent Predictor of Overall Survival in Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with dismal prognosis. Genomic instability due to defects in cell-cycle regulation/mitosis or deficient DNA-damage repair is a major driver of PDAC progression with clinical relevance. Deregulation of licensing of DNA replication leads to DNA damage and genomic instability, predisposing cells to malignant transformation. While overexpression of DNA replication-licensing factors has been reported in several human cancer types, their role in PDAC remains largely unknown. We aimed here to examine the expression and prognostic significance of the DNA replication-licensing factors chromatin licensing and DNA replication factor 1 (CDT1), cell-division cycle 6 (CDC6), minichromosome maintenance complex component 7 (MCM7) and also of the ubiquitin ligase regulator of CDT1, cullin 4A (CUL4A), in PDAC. Expression levels of CUL4, CDT1, CDC6 and MCM7 were evaluated by immunohistochemistry in 76 formalin-fixed paraffin-embedded specimens of PDAC patients in relation to DNA-damage response marker H2AX, clinicopathological parameters and survival. We also conducted bioinformatics analysis of data from online available databases to corroborate our findings. CUL4A and DNA replication-licensing factors were overexpressed in patients with PDAC and expression of CDT1 positively correlated with H2AX. Expression of CUL4A and CDT1 positively correlated with lymph node metastasis. Importantly, elevated CUL4A expression was associated with reduced overall survival and was an independent indicator of poor prognosis on multivariate analysis. Our findings implicate CUL4A, CDT1, CDC6 and MCM7 in PDAC progression and identify CUL4A as an independent prognostic factor for this disease.

Qualitative rather than quantitative phosphoregulation shapes the end of meiosis I in budding yeast

Exit from mitosis is brought about by dramatic changes in the phosphoproteome landscape. A drop in Cyclin-dependent kinase (Cdk) activity, the master regulatory kinase, and activation of counteracting phosphatases such as Cdc14 in budding yeast, results in ordered substrate dephosphorylation, allowing entry into a new cell cycle and replication licensing. In meiosis however, two cell divisions have to be executed without intermediate DNA replication, implying that global phosphorylation and dephosphorylation have to be adapted to the challenges of meiosis. Using a global time-resolved phosphoproteomics approach in budding yeast, we compared the phosphoproteome landscape between mitotic exit and the transition from meiosis I to meiosis II. We found that unlike exit from mitosis, Cdk phosphomotifs remain mostly stably phosphorylated at the end of meiosis I, whereas a majority of Cdk-unrelated motifs are reset by dephosphorylation. However, inducing an artificial drop of Cdk at metaphase of meiosis I leads to ordered substrate dephosphorylation, comparable to mitosis, indicating that phosphoregulation of substrates at the end of meiosis I is thus mainly qualitatively rather than quantitatively ordered.

Methodology to Create Auxin-Inducible Degron Tagging System to Control Expression of a Target Protein in Mammalian Cell Lines.

The auxin-inducible degron (AID) system is a versatile tool in cell biology and genetics, enabling conditional protein regulation through auxin-induced degradation. Integrating CRISPR/Cas9 with AID expedites tagging and depletion of a required protein in human and mouse cells. The mechanism of AID involves interactions between receptors like TIR1 and the AID tag fused to the target protein. The presence of auxin triggers protein ubiquitination, leading to proteasome-mediated degradation. We have used AID to explore the mitotic functions of the replication licensing protein CDT1. Swift CDT1 degradation via AID upon auxin addition achieves precise mitotic inhibition, revealing defects in mitotic spindle structure and chromosome misalignment. Using live imaging, we found that mitosis-specific degradation of CDT1 delayed progression and chromosome mis-segregation. AID-mediated CDT1 inhibition surpasses siRNA-based methods, offering a robust approach to probe CDT1's mitotic roles. The advantages of AID include targeted degradation and temporal control, facilitating rapid induction and reversal of degradation-contrasting siRNA's delayed RNA degradation and protein turnover. In summary, the AID technique enhances precision, control, and efficiency in studying protein function and regulation across diverse cellular contexts. In this article, we provide a step-by-step methodology for generating an efficient AID-tagging system, keeping in mind the important considerations that need to be adopted to use it for investigating or characterizing protein function in a temporally controlled manner. Key features • The auxin-inducible degron (AID) system serves as a versatile tool, enabling conditional protein regulation through auxin-induced degradation in cell biology and genetics. • Integration of CRISPR/Cas9 knock-in technology with AID expedites the tagging and depletion of essential proteins in mammalian cells. • AID's application extends to exploring the mitotic functions of the replication licensing protein CDT1, achieving precise mitotic inhibition and revealing spindle defects and chromosome misalignment. • The AID system and its diverse applications advance the understanding of protein function and cellular processes, contributing to the study of protein regulation and function.

The Origin Recognition Complex: From Origin Selection to Replication Licensing in Yeast and Humans.

Understanding human DNA replication through the study of yeast has been an extremely fruitful journey. The minichromosome maintenance (MCM) 2-7 genes that encode the catalytic core of the eukaryotic replisome were initially identified through forward yeast genetics. The origin recognition complexes (ORC) that load the MCM hexamers at replication origins were purified from yeast extracts. We have reached an age where high-resolution cryoEM structures of yeast and human replication complexes can be compared side-by-side. Their similarities and differences are converging as alternative strategies that may deviate in detail but are shared by both species.

Identification of key genes and molecular pathways associated with claw regeneration in mud crab (Scylla paramamosain)

The mud crab (Scylla paramamosain) possesses extensive regenerative abilities, enabling it to replace missing body parts, including claws, legs, and even eyes. Studying the genetic and molecular mechanisms underlying regenerative ability in diverse animal phyla has the potential to provide new insights into regenerative medicine in humans. In the present study, we performed mRNA sequencing to reveal the genetic mechanisms underlying the claw regeneration in mud crab. Several differentially expressed genes (DEGs) were expressed in biological pathways associated with cuticle synthase, collagen synthase, tissue regeneration, blastema formation, wound healing, cell cycle, cell division, and cell migration. The top GO enrichment terms were microtubule-based process, collagen trimer, cell cycle process, and extracellular matrix structural constituent. The most enriched KEGG pathways were ECM-receptor interaction and focal adhesion. The genes encoding key functional proteins, such as collagen alpha, cuticle protein, early cuticle protein, arthrodial cuticle protein, dentin sialophosphoprotein (DSPP), epidermal growth factor receptor (EGFR), kinesin family member C1 (KIFC1), and DNA replication licensing factor mcm2-like (MCM2) were the most significant and important DEGs suspected to participate in claw regeneration. The findings of this research offer a comprehensive and insightful understanding of the genetic and molecular mechanisms underlying claw regeneration in S. paramamosain. By elucidating the specific genes and molecular pathways implicated in this process, our study contributes significantly to the broader field of regenerative biology and offers potential avenues for further exploration in crustacean limb regeneration.

RAD51 Is Required for the Occupancy of Replication Licensing Factors on the Myeloma Genome

Genomic instability and extended replicative potential arise early during oncogenic transformation and contribute to the development and progression of cancer. Although chromosomal instability and replication are distinct pathways, they share certain proteins and activities. DNA replication starts by sequential loading of replication licensing factors at replication origins. We have observed that RAD51 - a keystone protein in stabilizing the genome through homologous recombination (HR) and a dependency in multiple myeloma (MM) - interacts with the MCM complex. In fact, RAD51 knockdown inhibits DNA replication in MM cells. Because RAD51 organizes the initial steps of homologous recombination, we investigated whether RAD51 plays a similar setup role in replication - namely, that RAD51 is required for the occupancy or proper assembly of replication licensing factors (ORC1-ORC6, CDC6, CDT1, MCM2-7) on the MM genome. To investigate this, we either inhibited (by knockdown or inhibitor) or overexpressed RAD51 in MM cells and monitored the occupancy of the replication complex on genomic DNA using ChIP with an antibody against MCM4. Subsequently, the proteins in the complex were released from DNA and identified by Western blotting. Although RAD51-KD (or inhibition) did not reduce the expression or stability of licensing factors, the occupancy of all these factors (MCM4, MCM2, MCM5, MCM6, MCM7, CDC6) on the MM genome was inhibited. Consistently, RAD51-overexpression led to an increase in the genomic occupancy of most of the licensing factors (ORC1, ORC2, MCM4, MCM2, MCM5, MCM6, MCM7, CDC6) tested. This was validated using antibodies against other members of the complex i.e., ORC1 and CDC6. Overall, we demonstrate that RAD51 in MM cells interacts with components of the replicative protein complexes (ORC1-ORC6, CDC6, CDT1, MCM2-7) that license and ensure DNA replication once per cell cycle. Our data also demonstrate that RAD51 is required for the occupancy of most of these factors on genomic DNA in MM cells, which may explain its ability to impact DNA replication in MM cells. We are currently trying to understand if RAD51 brings one or more of these factors to the origin of replication and if some of its activities (such as ATPase, DNA binding) are involved in loading these factors onto DNA. Based on its impact both on replication and HR, RAD51 is an attractive target in myeloma. Our inhibitor studies provide further rationale to study therapeutic strategy to target RAD51 in MM.

CDC6, a key replication licensing factor, is overexpressed and confers poor prognosis in diffuse large B-cell lymphoma

BackgroundCell division cycle 6 (CDC6) is a key licensing factor in the assembly of pre-replicative complexes at origins of replication. The role of CDC6 in the pathogenesis of in diffuse larger B-cell lymphoma (DLBCL) remains unknown. We aim to investigate the effects of CDC6 on the proliferation, apoptosis and cell cycle regulation in DLBCL cells, delineate its underlying mechanism, and to correlate CDC6 expression with clinical characteristics and prognosis of patients with DLBCL.MethodsInitial bioinformatic analysis was performed to screen the potential role of CDC6 in DLBCL. Lentiviral constructs harboring CDC6 or shCDC6 was transfected to overexpress or knockdown CDC6 in SUDHL4 and OCI-LY7 cells. The cell proliferation was evaluated by CCK-8 assay, cell apoptosis was detected by Annexin-V APC/7-AAD double staining, and cell cycle was measured by flow cytometry. Real time quantitative PCR and western blot was used to characterize CDC6 expression and its downstream signaling pathways. The clinical data of DLBCL patients were retrospectively reviewed, the CDC6 expression in DLBCL or lymph node reactive hyperplasia tissues was evaluated by immunohistochemistry.ResultsIn silico data suggest that CDC6 overexpression is associated with inferior prognosis of DLBCL. We found that CDC6 overexpression increased SUDHL4 or OCI-LY7 cell proliferation, while knockdown of CDC6 inhibited cell proliferation in a time-dependent manner. Upon overexpression, CDC6 reduced cells in G1 phase and did not affect cell apoptosis; CDC6 knockdown led to significant cell cycle arrest in G1 phase and increase in cell apoptosis. Western blot showed that CDC6 inhibited the expression of INK4, E-Cadherin and ATR, accompanied by increased Bcl-2 and deceased Bax expression. The CDC6 protein was overexpressed DLBCL compared with lymph node reactive hyperplasia, and CDC6 overexpression was associated with non-GCB subtype, and conferred poor PFS and OS in patients with DLBCL.ConclusionCDC6 promotes cell proliferation and survival of DLBCL cells through regulation of G1/S cell cycle checkpoint and apoptosis. CDC6 is overexpressed and serves as a novel prognostic marker in DLBCL.

Glutamine Regulates Gene Expression Profiles to Increase the Proliferation of Porcine Intestinal Epithelial Cells and the Expansion of Intestinal Stem Cells.

The intestinal epithelium is known for its rapid self-renewal, and glutamine is crucial in providing carbon and nitrogen for biosynthesis. However, understanding how glutamine affects gene expression in the intestinal epithelium is limited, and identifying the essential genes and signals involved in regulating intestinal epithelial cell growth is particularly challenging. In this study, glutamine supplementation exhibited a robust acceleration of intestinal epithelial cell proliferation and stem cell expansion. RNA sequencing indicated diverse transcriptome changes between the control and glutamine supplementation groups, identifying 925 up-regulated and 1152 down-regulated genes. The up-regulated DEGs were enriched in the KEGG pathway of cell cycle and GO terms of DNA replication initiation, regulation of phosphatidylinositol 3-kinase activity, DNA replication, minichromosome maintenance protein (MCM) complex, and ATP binding, whereas the down-regulated DEGs were enriched in the KEGG pathway of p53 signaling pathway, TNF signaling pathway, and JAK-STAT signaling pathway and GO terms of inflammatory response and intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress. Furthermore, GSEA analysis revealed a significant up-regulation of the cell cycle, DNA replication initiation, ATP-dependent RNA helicase activity, and down-regulation of the TNF signaling pathway. The protein-protein association network of the intersecting genes highlighted the significance of DNA replication licensing factors (MCM3, MCM6, and MCM10) in promoting intestinal epithelial growth in response to glutamine. Based on these findings, we propose that glutamine may upregulate DNA replication licensing factors, leading to increased PI3K/Akt signaling and the suppression of TNF, JAK-STAT, and p53 pathways. Consequently, this mechanism results in the proliferation of porcine intestinal epithelial cells and the expansion of intestinal stem cells.

NFIB facilitates replication licensing by acting as a genome organizer

The chromatin-based rule governing the selection and activation of replication origins in metazoans remains to be investigated. Here we report that NFIB, a member of Nuclear Factor I (NFI) family that was initially purified in host cells to promote adenoviral DNA replication but has since mainly been investigated in transcription regulation, is physically associated with the pre-replication complex (pre-RC) in mammalian cells. Genomic analyses reveal that NFIB facilitates the assembly of the pre-RC by increasing chromatin accessibility. Nucleosome binding and single-molecule magnetic tweezers shows that NFIB binds to and opens up nucleosomes. Transmission electron microscopy indicates that NFIB promotes nucleosome eviction on parental chromatin. NFIB deficiency leads to alterations of chromosome contacts/compartments in both G1 and S phase and affects the firing of a subset of origins at early-replication domains. Significantly, cancer-associated NFIB overexpression provokes gene duplication and genomic alterations recapitulating the genetic aberrance in clinical breast cancer and empowering cancer cells to dynamically evolve growth advantage and drug resistance. Together, these results point a role for NFIB in facilitating replication licensing by acting as a genome organizer, shedding new lights on the biological function of NFIB and on the replication origin selection in eukaryotes.

A GATA2-CDC6 axis modulates androgen receptor blockade-induced senescence in prostate cancer

BackgroundProstate cancer is a major cause of cancer morbidity and mortality in men worldwide. Androgen deprivation therapy (ADT) has proven effective in early-stage androgen-sensitive disease, but prostate cancer gradually develops into an androgen-resistant metastatic state in the vast majority of patients. According to our oncogene-induced model for cancer development, senescence is a major tumor progression barrier. However, whether senescence is implicated in the progression of early-stage androgen-sensitive to highly aggressive castration-resistant prostate cancer (CRPC) remains poorly addressed.MethodsAndrogen-dependent (LNCaP) and –independent (C4-2B and PC-3) cells were treated or not with enzalutamide, an Androgen Receptor (AR) inhibitor. RNA sequencing and pathway analyses were carried out in LNCaP cells to identify potential senescence regulators upon treatment. Assessment of the invasive potential of cells and senescence status following enzalutamide treatment and/or RNAi-mediated silencing of selected targets was performed in all cell lines, complemented by bioinformatics analyses on a wide range of in vitro and in vivo datasets. Key observations were validated in LNCaP and C4-2B mouse xenografts. Senescence induction was assessed by state-of-the-art GL13 staining by immunocytochemistry and confocal microscopy.ResultsWe demonstrate that enzalutamide treatment induces senescence in androgen-sensitive cells via reduction of the replication licensing factor CDC6. Mechanistically, we show that CDC6 downregulation is mediated through endogenous activation of the GATA2 transcription factor functioning as a CDC6 repressor. Intriguingly, GATA2 levels decrease in enzalutamide-resistant cells, leading to CDC6 stabilization accompanied by activation of Epithelial-To-Mesenchymal Transition (EMT) markers and absence of senescence. We show that CDC6 loss is sufficient to reverse oncogenic features and induce senescence regardless of treatment responsiveness, thereby identifying CDC6 as a critical determinant of prostate cancer progression.ConclusionsWe identify a key GATA2-CDC6 signaling axis which is reciprocally regulated in enzalutamide-sensitive and -resistant prostate cancer environments. Upon acquired resistance, GATA2 repression leads to CDC6 stabilization, with detrimental effects in disease progression through exacerbation of EMT and abrogation of senescence. However, bypassing the GATA2-CDC6 axis by direct inhibition of CDC6 reverses oncogenic features and establishes senescence, thereby offering a therapeutic window even after acquiring resistance to therapy.