Genes to Cells

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

BackgroundHeterochromatic long arm of mouse Y chromosome harbors the multicopy species-specific sequences Ssty, Sly, Asty and Orly that are transcribed in testis and have known functions in male fertility. Of these Ssty and Sly encode proteins - yet all the transcripts are not translated. To investigate the roles of these Y-heterochromatic transcripts further, we analyzed them. MethodsMice with 2/3rd deletion of the Y-chromosome (XYRIIIqdel) and its wild type (XYRIII) were used in this study. Bioinformatic approaches, small RNA northern blots, Electrophoretic Mobility Shift Assays, Luciferase reporter assays, dPCR analysis, RT-qPCR assays and western blotting techniques were used to identify piRNAs that regulate autosomal genes. ResultsWe demonstrate that the multicopy gene families from mouse Y-long arm generate piRNAs predominantly in testis. We observed sequences homologous to these piRNAs in the UTRs of a few autosomal genes, which are differentially expressed in the sperms of XYRIIIqdel mice. Furthermore, the Endogenous Retrovirus Element (ERV) LTR, found in the Orly1 transcript identified piRNAs in the database, showed homology to UTRs and associated genomic regions of a few autosomal genes. Orly1 showed a reduction in genomic copy number by digital PCR in XYRIIIqdel mice. One of the four autosomal genes containing the ERV segment in their UTRs, showed a differential testicular protein expression in the mutant mice. ConclusionsThus, we further elucidate that different classes of repeats from Y-chromosome regulate autosomal gene expression via piRNAs. Besides, this study also identified novel roles for a Y-derived ERV in autosomal gene regulation in testis.

BackgroundDynamic chromatin behavior, which is related to chromatin accessibility, plays a critical role in various genome DNA functions such as RNA transcription and DNA replication/repair. Previous studies using highly synchronized cells showed that average local chromatin motion, captured by single-nucleosome imaging and tracking on a second time scale, remained almost constant throughout G1, S, and G2 phases in living human cells, although possible effects of prolonged drug treatments for cell-cycle synchronization could not be excluded. ResultsTo avoid possible effects of prolonged drug treatment, we combined single-nucleosome imaging with Fucci probes to visualize cell-cycle progression through G1, S, and G2. Using HeLa and HCT116 cells expressing H2B-HaloTag and Fucci probes, we found that local nucleosome motion remained similar on average throughout interphase, except for elevated motion in early G1. Transcription inhibition similarly increased nucleosome motion throughout interphase. Local nucleosome motion also increased following replication stress or DNA damage. ConclusionOur findings suggest that near-constant chromatin motion supports housekeeping functions under similar physical conditions during interphase. Our findings also suggest that cells can transiently change chromatin motion to perform ad hoc tasks in response to signals from inside and outside the cell, such as DNA damage.

BackgroundShort-chain fatty acids (SCFAs) are produced by the gut microbiota as secondary metabolites during fermentation process of dietary fibers. Although SCFAs are beneficial for immuno-related diseases because they regulate the gene expression and functions of myeloid cells, the effects of SCFAs on the development of DCs remain unclear. MethodsWe analyzed the effect of SCFAs on the expression levels of surface proteins and mRNAs, and histone modification in Flt3L-induced bone marrow-derived DCs. ResultsSCFAs, particularly butyrate, regulated the expression of surface molecules on mouse bone marrow-derived dendritic cells (DCs): increases in MHCII, CD86, CD11b, and LPAM-1 (4{beta}7) levels and the ratio of CD11c+/PDCA-1-/B220- conventional DCs (cDCs) to CD11c+/PDCA-1+/B220+ plasmacytoid DCs (pDCs). Experiments using inhibitors of histone deacetylase (HDAC) and Gi proteins, and GPR109A deficient mice indicated that butyrate regulated DCs by suppression of HDACs and not through a stimulatory effect on G protein-coupled receptors. Butyrate and the HDAC inhibitor, trichostatin A (TSA), increased the cDC/pDC ratio, surface LPAM-1 and Itga4 mRNA, while the mRNA level of Itgb7 was not affected by butyrate and was reduced by TSA. ChIP assays showed that butyrate and TSA increased histone acetylation in the Itga4 and Spi1 genes. Furthermore, the butyrate treatment increased the levels of Spi1 mRNA and PU.1 protein and decreased those of Spib/SpiB in DCs. In knockdown (KD) experiments using siRNAs, the gene expression of Itga4 was decreased by KD of Spi1 or Irf8, and cDC/pDC ratio decreased by Spi1 KD. ConclusionsButyrate controls the gene expression and development of DCs through epigenetic regulation and DC-related transcription factors.

Chromosome segregation fidelity during meiosis is critical for genome integrity, with aneuploidy causing infertility, miscarriages, and congenital anomalies. In the oocytes of many species, spindle assembly occurs in the absence of centrosomes that normally function as microtubule-organizing centers at the poles. Such acentrosomal spindles are believed to pose significant challenges for accurate chromosome segregation compared to centrosomal organized spindles. Previous work in Drosophila has shown that the chromosomal passenger complex (CPC) is required for acentrosomal spindle assembly. We found that heterochromatin protein-1 (HP1) plays a critical role in regulating CPC localization and spindle assembly. Furthermore, HP1 moves to the microtubules, where it has roles in building a functional spindle and interacts with the CPC to regulate chromosome biorientation. These results indicate that spindle assembly is mediated by multiple interactions between the CPC, HP1, and the chromosomes, and provide insights into the mechanisms that restricts spindle assembly to the chromosomes in Drosophila oocytes.

Heterochromatin is a membraneless compartment within the cell nucleus. In recent years, a controversy arose on whether heterochromatin organization is driven by liquid-liquid phase separation or not. While many heterochromatin proteins were shown to undergo liquid-liquid phase separation in vitro, other studies reported that this does not happen in cells. Here, we tested the ability of heterochromatin proteins to generate heterochromatin barrier compartments in cells. We found that, while several proteins (H1.0, H1.4, HP1alpha, HP1beta, Mbd1, Mbd2 and MeCP2) form barrier compartments in mouse and/or human cells this differs between cell types. In addition, not all compartments in the same cell form barriers. We established and experimentally validated a model that predicted the ability to form barrier compartments is dependent on the protein accumulation in heterochromatin followed by the competition between compartments for the nucleoplasm pool of the protein and resulted in larger size for the barrier compartments. These findings resolve the existing controversy and rationalize how in cells heterochromatin compartments form and compete to establish dynamic barriers to the entry and exit of its components. HighlightsHeterochromatin barrier formation differs between proteins, cell lines and heterochromatin compartments within the cell. Barrier formation depends on heterochromatin anchors, including ligands and other scaffolds. Barrier compartments are defined by their larger size and higher protein enrichment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/729812v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a63021org.highwire.dtl.DTLVardef@a20362org.highwire.dtl.DTLVardef@8c2390org.highwire.dtl.DTLVardef@72dde3_HPS_FORMAT_FIGEXP M_FIG C_FIG

In this study, we investigate alliance formation and complexity in male Indo-Pacific bottlenose dolphins around Mikura Island using five years of data collected through underwater observations. Focusing on 18 mature males, we examined affiliative behaviors (proximity and rubbing), consortships, and associations. To determine male relationships, we evaluated a simple model (small units) and a complex model (large units). In both models, units were identified by association, and models were evaluated by the extent to which affiliative behaviors and consortship were concentrated within units. All types of behaviors were more concentrated within units determined by the complex model than the simple model, and thus the former was further investigated. The unit sizes determined by the complex model were three, seven, and eight, and variation in association frequency was observed within units. Within units, two to four males engaged in a single consortship irrespective of association frequency. Given that units are mediated by affiliative and cooperative relationships, it is reasonable to interpret units as alliances. Considering the variation in both alliance size and within-alliance relationships, as well as the fact that only a few males cooperate in a single consortship, we suggest that a multi-level structure is plausible in the Mikura community.

Function of CENPJ/CPAP is essential for centriole duplication and cilia biogenesis. Recently, we showed that CPAP is also an integral Endosomal Sorting Complexes Required for Transport (ESCRT)-0-like protein that recruits ESCRT-I protein TSG101 to early endosome (EE) and positively regulates multi-vesicular body (MVB) formation. Sequential recruitment of the ESCRT protein complexes and AAA+ ATPase VPS4B to EE facilitates MVB biogenesis. VPS4B is critical for ESCRT-III disassembly/recycling and contributes to membrane fission in several cellular processes. Here, we report that CPAP is critical for the protein stability and EE localization of VPS4B, and this function is independent from its role as an ESCRT-0. Other VPS4B-dependent cellular processes such as exosome release, cytokinesis, and retroviral budding are also compromised under CPAP deficiency. Interaction with CPAP prevents the proteasome degradation of VPS4B. The stability and EE localization of VPS4B can be attributed to two different C-terminal domains in CPAP. Overall, these observations provide evidence that CPAP is critical for VPS4B function and suggest that distinct pools of CPAP may be involved in its ESCRT-0 and VPS4B stabilization roles.

An expectation of a hypothesis that proposes cell-to-cell signalling pathways are redundant due to the redundancy of pathway terminal transcription factors (TFs) was tested by screening 35 signalling ligands (SLs) for rescue of a decapentaplegic (dpp) hypomorphic wing growth phenotype. The screen identified three examples of partial rescue: Hedgehog (HH), Semphorin 1a (SEMA1A) and Wnt ortholog 2 (WNT2). HH overexpression with dppGAL4 may increase the expression of DPP activity from the hypomorphic dpp alleles. However, SEMA1A and WNT2 did not phenocopy ectopic expression of HH or DPP and neither SEMA1A nor WNT2 were required for wing growth suggesting substitution of DPP for partial restoration of wing growth. The WNT2 rescue was dependent on the Frizzled 4 (FZ4) WNT receptor excluding the possibility that WNT2 weakly binds the DPP receptor. Although examples of phenotypic nonspecificity of SL function were identified, this is an expectation, and not direct proof, of the hypothesis of TF redundancy. Screen Report SummaryAn expectation of a hypothesis proposing that cell-to-cell signalling pathways are redundant due to the redundancy of the pathway terminal transcription factors was tested by screening for replacement of one signalling ligand (DPP; SLa) with another SLb for wing growth. Three non-DPP SLs were identified in the screen of 35SLs: HH, SEMA1A and WNT2. Genetic analysis of Sema1a and Wnt2 suggests functional complementation of dpp for wing growth suggesting that SEMA1A and WNT2 partially replace DPP for wing growth. Therefore, an expectation of the hypothesis is met.

Turbot (Scophthalmus maximus) is an important aquaculture species, but the genomic organization and expressed diversity of its antibody repertoire remain incompletely characterized. In this study, we annotated the immunoglobulin heavy chain (IGH) locus using the haplotype resolved fScoMax1.1 genome assembly, and we used this as a reference to profile the expressed turbot IgM, IgD and IgT repertoires in skin and spleen. The primary IGH locus was located on chromosome 19, spanned approximately 72 kb, and contained 25 IGHV genes, including 24 functional genes and one pseudogene, together with three IGHD, seven IGHJ and three IGHC genes corresponding to IgT, IgM and IgD. Comparison with the alternate fScoMax1.1 haplotype and a second turbot genome assembly showed conserved IGHD, IGHJ and IGHC content, whereas IGHV gene number differed among assemblies. High throughput 5RACE repertoire sequencing revealed isotype and tissue associated differences in expressed IGH diversity. IgM represented the dominant productive repertoire in both skin and spleen and showed the highest clonotypic diversity, particularly in spleen. IgD displayed an intermediate profile, whereas IgT was more enriched in skin and exhibited the strongest clonal restriction. IGHV subgroup usage was dominated by IGHV3 in IgM and IgD, whereas IgT showed a distinct profile characterized by preferential use of IGHV4, especially in skin. Gene level analysis further showed broad IGHV-IGHJ pairing in IgM and IgD, with preferential use IGHJ3 segment, while IgT sequences paired exclusively with IGHJT. Clonotype sharing between skin and spleen was isotype dependent, being strongest for IgT, intermediate for IgM, and negligible for IgD, suggesting that clonal expansion did not necessarily predict inter tissue trafficking. Together, these results provide a curated genomic and expressed repertoire framework for turbot IGH genes and reveal isotype specific organization of antibody diversity, with IgT displaying a particular repertoire pattern.

DDI2 and DDI3 (DDI2/3) are duplicated genes in Saccharomyces cerevisiae that exhibit strong induction by a transcription factor Fzf1 in response to chemical treatments like cyanamide (CY) and methyl methanesulfonate (MMS). Although, like DDI2/3, SSU1, YHB1 and YNR064C also contain an Fzf1-binding consensus sequence CS2 and are coordinately regulated by Fzf1, these genes are only modestly induced by CY and MMS. To identify additional cis-acting elements in the DDI2/3 promoter, we made DDI2/3 promoter deletions in a reporter system and identified upstream repressing sequences (URS) spanning 480 nucleotides. To test a hypothesis that the chromatin structure constitutes the URS, we utilized a yeast strain capable of histone H3/H4 depletion by shifting carbon sources. Following histone depletion, DDI2/3 were strongly induced in an Fzf1 dependent manner, while YHB1 was repressed. Interestingly, under histone depletion conditions, CY or MMS treatment further increased expression of all Fzf1-regulated genes to comparable levels in an Fzf1 dependent manner. A genome-wide MNase-seq analysis showed that CY treatment reduced the nucleosome occupancy at the mapped DDI2/3 URS region in wild-type cells, but not in in fzf1{Delta} cells. These findings collectively indicate that Fzf1 plays dual roles in regulating the DDI2/3 response to CY. Firstly, it binds CS2 and serves as a transcription activator. Secondly, it is required for the chromatin remodeling at URS. This two-tier regulation at the DDI2/3 promoter helps to explain why DDI2/3 achieve much higher fold induction by CY and MMS than other Fzf1-regulated genes, suggesting Fzf1 to be a candidate pioneer transcription factor.

Dynamic FGF/ERK signaling plays key roles in development, regeneration, and disease. We recently developed a zebrafish-optimized optogenetic tool, bOpto-FGF, that enables reversible activation of FGF/ERK signaling in response to blue light ([~]455 nm) by fusing a zebrafish receptor tyrosine kinase domain to the blue light-dimerizing LOV domain. Previously, this tool was introduced into zebrafish embryos by mRNA injection. Here, we develop a novel transgenic zebrafish ubiquitously expressing bOpto-FGF, Tg(ubi:bOpto-FGF), to streamline experimental workflows. We demonstrate robust blue light-mediated activation of FGF/ERK signaling in gastrulation-stage Tg(ubi:bOpto-FGF) homozygous and heterozygous embryos. Light-mediated signaling activation is more spatially uniform in transgenics compared to embryos injected with bOpto-FGF mRNA. Tg(ubi:bOpto-FGF) heterozygotes are light-responsive from late blastula stages through at least 24 hours post-fertilization. Finally, ectopic signaling in response to continuous light exposure starting at late blastula stage is activated within 3 minutes and maintained for at least 75 minutes. This transgenic line provides a powerful and convenient new strategy for experimental manipulation of FGF/ERK signaling dynamics in the vertebrate zebrafish model.

Objective To develop and validate a risk model for predicting postoperative bleeding in patients with thyroid cancer. Methods A total of 2800 consecutive patients diagnosed with thyroid cancer in the Department of Thyroid and Breast Surgery of the Affiliated Hospital of Xuzhou Medical University between January 2020 and December 2023 were retrospectively analyzed. Patients were categorized into two groups based on postoperative bleeding occurrence: bleeding and non-bleeding groups. Univariate and multivariate logistic regression analyses were utilized to screen independent risk factors. Meanwhile, risk prediction models were developed and nomogram . Subgroup analysis was performed to identify independent risk factors. The predictive effects of the models were assessed using the Hosmer-Lemeshow test and receiver operating characteristic (ROC) curves. Results Of the 2800 recruited patients, 50 had postoperative bleeding, with an incidence rate of 1.7%. Multivariate logistic regression analysis showed that age, hypertension, total thyroidectomy, tumor size [&ge;]4 cm, and operation time [&ge;]90 min were the risk factors for postoperative bleeding in thyroid cancer patients (P<0.05). A risk prediction model was established based on the above factors, and the area under the ROC curve was 0.881, with a sensitivity of 94.0%, a specificity of 67.3%, and an accuracy of 74.0%. Decision curve analysis revealed that the model had good predictive ability. Conclusions The constructed risk prediction model has good predictive power and can provide a reference for healthcare professionals to predict the risk of bleeding in patients after thyroid cancer surgery.

The plasmodium of Myxogastoria (a group of amoeboid protists) species often crawls around the forest floor to feed while searching for places to form fruiting bodies for reproduction (sporulation). Certain environmental factors that trigger sporulation have been reported; however, other unknown factors are also expected. In this study, we reported field observations of Physarum rigidum and Fuligo septica. Inspired by the field observation, we examined the effects of multiple factors on sporulation in laboratory experiments using Physarum polycephalum. We found that:(1) there was a critical body size below which sporulation did not occur under our experimental conditions and (2) the plasmodium selected its sporulation sites from the available landscape of the experimental arena: dry and low sites for the majority and dry and high sites for the minority. Further analysis revealed that they preferred the edge area at the high site. We discuss the possible ecological importance of the threshold and location preference

In the terminal segment of the seminiferous tubules, SOX17 expression in the rete testis (RT) epithelium plays a crucial role in the formation of the Sertoli valve (SV), as revealed by phenotypic analyses of RT-specific Sox17 conditional knockout (cKO) mouse testes. In these RT-specific Sox17 cKO testes, SV disruption leads to the backflow of RT fluid into the seminiferous tubules, resulting in defective spermiogenesis and male infertility. Although valve deformation in the Sox17 cKO testes is likely caused indirectly by impaired downstream actions of Sox17 in the RT, the mechanisms by which SOX17 in RT influences SV formation in the seminiferous tubules remain unclear. To address this, we generated a novel AMH-Sox17 transgenic (Tg) mouse line carrying a human AMH promoter-driven Sox17 cDNA cassette. We analyzed the phenotypes of the Sertoli valve and spermatogenesis in AMH-Sox17 Tg mice, as well as in RT-specific Sox17 cKO; AMH-Sox17 Tg double mutant mice. Ectopic SOX17 (SOX17+) expression in Sertoli cells resulted in excessive Sertoli valve structures with acetylated tubulin bundles in the terminal segment of the AMH-Sox17 Tg testes, along with enhanced WNT4/RSPO1 signaling, suggesting the enhanced valve formation of ectopic SOX17+ Sertoli cells by themselves. Moreover, the AMH-Sox17 Tg could partially rescue the SV deformation and infertility in RT-specific Sox17 cKO mice, leading to proper SV formation, normal spermiogenesis and a partial recovery of male fertility in AMH-Sox17 Tg; RT-specific Sox17 cKO double mutant mice. These findings genetically demonstrate that ectopic SOX17+ Sertoli cells can compensate for SOX17 paracrine signaling in the RT, underscoring a key shared downstream pathway between RT and SV. Summary statementThe paracrine actions downstream of ectopic SOX17 expression in the Sertoli cells not only promote the valve formation, but also partially rescue the defective spermiogenesis of the rete testis-specific Sox17-null mice.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that undergoes lytic replication in epithelial and other permissive cell types and can establish latency in peripheral neurons. ICP22 is a multifunctional HSV-1 immediate-early protein that localizes to the nucleus of infected cells; however, its interactions with host cellular factors remain incompletely understood. Here, ICP22 was demonstrated to interact with the human antisense function 1 protein (ASF1), including both ASF1a and ASF1b, in transfected cells and HSV-1-infected cells, respectively. ICP22 also colocalized with ASF1 in the nucleus. ICP22 amino acids 213 to 340 are important for the interaction of ICP22 with ASF1, whereas amino acids 37 to 153 of ASF1a and ASF1b are critical for their interactions with ICP22. Furthermore, ICP22 expression was associated with reduced ASF1-H3.1 co-immunoprecipitation under the tested conditions. ASF1 knockdown also reduced HSV-1-BAC-Luc luciferase output, indicating that ASF1 contributes to efficient infection-associated reporter activity in this study. Collectively, these results indicate that the interaction of HSV-1 ICP22 with ASF1 might help regulate the transcription of viral or cellular genes during HSV-1 infection. Keywords: HSV-1, ICP22, ASF1, histone H3.

Ewing sarcoma is a pediatric cancer that develops in skeletal elements. The majority of Ewing sarcoma patients carry the aberrant EWSR1-FLI1 fusion gene. Despite trisomy 8 being an additional common aberration associated with a poor prognosis for patients, its induction mechanism remains unknown. When the EWSR1-FLI1 gene is formed, the cell loses one wildtype EWSR1 allele. To elucidate the induction mechanism of trisomy 8, we generated a cell line that allows for the conditional induction of EWSR1-FLI1 expression and EWSR1 knockdown (derived from a single EWSR1 allele. Specifically, the conditional cell line was generated by integrating the Tet-on EWSR1-FLI1 construct into the AAVS locus and adding a miniAID tag at the 5 end of the EWSR1 locus using auxin-degron system. A combination of the EWSR1-FLI1 expression and degradation of one allele-derived EWSR1 induced a high incidence of trisomy 8 within eight days, enhancing colony formation. Mechanistically, trisomy 8 is induced by the haploinsufficiency of EWSR1, and the remaining EWSR1 proteins are likely inhibited by interaction with EWSR1-FLI1. Our data showed that the knockout of EWSR1 alone was sufficient to increase the incidence of trisomy 8. Expression of wild-type EWSR1 in EWSR1 knockout cells rescued the high incidence of trisomy 8. In contrast, the EWSR1:R565A mutant, which lacks the ability to interact with Aurora B kinase, failed to rescue this phenotype. We propose that the combination of EWSR1-FLI1 expression and loss of EWSR1 contributes to the induction of trisomy 8 through the compromised EWSR1-Aurora B pathway. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/726567v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@999891org.highwire.dtl.DTLVardef@1ef6748org.highwire.dtl.DTLVardef@65e475org.highwire.dtl.DTLVardef@179da40_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

Eukaryotic cells control their size by coordinating growth and division. Fission yeast divide at a reproducible cell size due to regulated activation of the cyclin-dependent kinase Cdk1. The nuclear concentration of mitotic cyclin Cdc13 increases in a time-dependent manner to promote Cdk1 activation as cells grow. Here, we show that interphase Cdc13 is stable against degradation and nuclear export, but is diluted by cell growth. Low glucose reduced cell growth rate but not time-dependent accumulation of Cdc13. Uncoupling the rates of cell growth and Cdc13 accumulation resulted in higher concentrations of nuclear Cdc13 despite reduced cell size. This change coincided with reduced activating phosphorylation of Cdk1-T167 and occurred dynamically during abrupt changes in glucose concentration. Mathematical modeling and experiments showed that cells maintain size homeostasis under these conditions. In contrast to low glucose, poor nitrogen reduced both cell growth rate and Cdc13 accumulation rate. Therefore, Cdc13 accumulation is independent of cell growth rate but can be altered by nutrient-specific mechanisms.

The IFN-I (type I interferon) signaling pathway is the first line of defence against foreign pathogens. Stringent control of signalling pathways is necessary to maintain host immune responses and homeostasis. However, the underlying mechanism for its tight regulation is yet completely understood. In this study, we demonstrated that the TRIM family protein tripartite motif-containing 52 (TRIM52) is a novel negative regulator of IFN-{beta} production. Ectopically expressed TRIM52 markedly inhibited the activation of the IFN-{beta} promoter by ectopic expression of cGAS/STING, RIG-IN, or TRIF, MAVS, STING, and TBK1 but not by IRF3/5D, indicating that TRIM52 targets TBK1. TRIM52 also significantly inhibited the IFN-{beta}, ISG54, and ISG56 production, the dimerization of IRF3 and the nuclear localization of IRF3-YFP induced by ectopic expression of TBK1. Co-immunoprecipitation experiment revealed that TRIM52 specifically interacted with TBK1. Furthermore, the TBK1 protein, but not its mRNA, decreased considerably with increasing expression of TRIM52, and TRIM52 did not decrease the expression of the cGAS, STING, or IRF3 proteins. In addition, proteasome inhibitor MG-132 blocked the reduced TBK1 induced by TRIM52, indicating that TRIM52 caused TBK1 degradation via the proteasome pathway. Co-IP and ubiquitination assays demonstrated that TRIM52 promotion of K48-linked ubiquitination of TBK1, which depends on its E3 ubiquitin ligase. Collectively, our findings identify a previously unrecognized role of TRIM52 in regulating the IFN-I signalling pathway through targeting TBK1 for polyubiquitination and degradation.