👤 Frederick R Cross

Hypothalamic neurons expressing either POMC or AGRP sense nutritional state directly and indirectly and transmit these neuropeptide signals to other brain centres through the melanocortin 3 and 4 receptors. MC4R is primarily concerned with the control of appetite and energy expenditure while MC3R is more closely related to the control of linear growth and the timing of puberty. The role of MC3R in the long-term control of energy balance and body composition is less clear, particularly in humans. We have undertaken studies in humans, domestic dogs and mice with the goal of clarifying the relative impact of MC3R deficiency on energy balance, growth and sexual development. By studying three large consanguineously enriched cohorts, totalling approximately 300K people, we identified nine individuals who are homozygous for functionally null MC3R variants. The body mass index (BMI) of the homozygous MC3R variant carriers was not significantly different from that of age, sex and demographically matched controls, with six of the nine homozygotes having a BMI <30 kg/m Show less

Diverse haematological neoplasms are driven by tyrosine kinase (TK) fusion genes formed by recurrent or non-recurrent genomic rearrangements. The resulting chimeric proteins often present excellent targets for treatment with kinase inhibitors, and the fusion transcripts or genomic junctions can be used as specific targets for molecular monitoring. Whilst the TK genes involved are generally well characterised (e.g. ABL1, PDGFRA, FGFR1), the fusion partners are very diverse, presenting a challenge for detection and characterisation of these structural variants (SV) using current diagnostic methods. We assessed the ability of targeted nanopore sequencing using adaptive sampling to detect fusion genes in myeloid neoplasms. We sequenced genomic DNA from patients (n = 20) with a known or suspected TK gene fusion and identified rearrangements in 18 cases, including all cases with a known TK fusion, typical and atypical BCR::ABL1 rearrangements, an 843Kb deletion causing a FIP1L1::PDGFRA fusion, novel AGAP2::PDGFRB and NFIA::PDGFRB fusions, and a complex CCDC88C::PDGFRB rearrangement with multiple translocation events. The approach was fast (<72 h/sample from DNA to result), flexible with minimal hands-on laboratory time, and provided accurate, patient-specific characterisation of genomic breakpoints. Show less

"Nonclassical" myeloproliferative neoplasms (MPNs) and myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) represent a heterogeneous group of malignancies characterized by a wide range of clinical manifestations. Unlike classical MPNs, there is no standardized management approach for these conditions, particularly concerning the indications for and management of allogeneic hematopoietic cell transplantation. To address this gap, the European Society for Blood and Marrow Transplantation (EBMT) Practice Harmonization and Guidelines (PH&G) Committee and the Chronic Malignancies Working Party (CMWP) have collaborated to develop shared guidelines aimed at optimizing the selection and management of patients with these rare forms of neoplasms. A comprehensive review of the literature from the publication of the revised fourth edition of the (2016) World Health Organization classification onward was conducted. A multidisciplinary group of experts in the field convened to produce this document, which was developed through multiple rounds of draft circulation. Key recommendations include the early identification of potential transplant candidates, particularly in cases of chronic neutrophilic leukemia, chronic eosinophilic leukemia (CEL)/CEL, not otherwise specified (CEL-NOS), myeloid/lymphoid neoplasm with eosinophilia and tyrosine kinase gene fusions with FGFR1, JAK2, ABL1, and FLT3 rearrangements, MDS/MPN with neutrophilia/atypical chronic myeloid leukemia, and MDS/MPN, NOS. For patients with MPN, NOS/MPN unclassifiable, standard recommendations for myelofibrosis should be applied. Similarly, in MDS/MPN with thrombocytosis, transplantation is recommended on the basis of established MDS guidelines. Given the current lack of robust evidence, this document will serve as a valuable resource to guide future research activities, providing a framework for addressing critical unanswered questions and advancing the field. Show less

The fifth edition of the World Health Organization (WHO) classification and the International Consensus Classification (ICC) both include a category "myeloid/lymphoid neoplasms (MLN) with eosinophilia (eo) and tyrosine kinase (TK) gene fusions" (WHO, MLN-TK; ICC, M/LN-eo-TK). This rare group comprises phenotypically and prognostically heterogeneous disorders, which present a significant diagnostic challenge. The rapid and reliable identification of patients with MLN-TK may be delayed due to genetic complexity and significant phenotypic differences, including the chronic phase and primary/secondary blast phase (BP) of myeloid, lymphoid, or mixed phenotype in the bone marrow (BP-BM) and/or at extramedullary sites (extramedullary disease [EMD]). As a result, the entire armamentarium of conventional molecular genetic and cytogenetic techniques complemented by modern sequencing technologies, such as RNA sequencing or whole-genome sequencing, are often required to identify an underlying TK fusion. TK inhibitors (TKIs) with variable efficacy are available for all fusion genes, but a long-term favorable clinical course under TKI monotherapy is currently only observed in MLN-PDGFRA/PDGFRB fusion genes on imatinib. Because primary/secondary BP-BM/EMD occurs more frequently in MLN-FGFR1/JAK2/FLT3/ETV6::ABL1, a sequential combination of selective TKIs with or without prior intensive chemotherapy, rarely local radiotherapy, and/or subsequent allogeneic hematopoietic cell transplantation should be considered. Show less

In a registry-based analysis of 135 patients with "myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions" (MLN-TK; FIP1L1::PDGFRA, n = 78; PDGFRB, diverse fusions, n = 26; FGFR1, diverse, n = 9; JAK2, diverse, n = 11; ETV6::ABL1, n = 11), we sought to evaluate the disease-defining characteristics. In 81/135 (60%) evaluable patients, hypereosinophilia (>1.5 × 10 Show less

The relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called "helper" cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies. Show less

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. This requires the Nedd4 family HECT E3 ubiquitin ligase Wwp2 and a tumor-suppressing transmembrane protein adaptor Tmem127. Here, through a proteomic screen of dendritic cells, we found that SteD targets the plasma membrane protein CD97 for degradation by a similar mechanism. SteD enhanced ubiquitination of CD97 on K555 and mutation of this residue eliminated the effect of SteD on CD97 surface levels. We showed that CD97 localises to and stabilises the immunological synapse between dendritic cells and T cells. Removal of CD97 by SteD inhibited dendritic cell-T cell interactions and reduced T cell activation, independently of its effect on MHCII. Therefore, SteD suppresses T cell immunity by two distinct processes. Show less

The inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) cause significant morbidity and are increasing in prevalence among all populations, including African Americans. More than 200 susceptibility loci have been identified in populations of predominantly European ancestry, but few loci have been associated with IBD in other ethnicities. We performed 2 high-density, genome-wide scans comprising 2345 cases of African Americans with IBD (1646 with CD, 583 with UC, and 116 inflammatory bowel disease unclassified) and 5002 individuals without IBD (controls, identified from the Health Retirement Study and Kaiser Permanente database). Single-nucleotide polymorphisms (SNPs) associated at P < 5.0 × 10 We detected SNPs at HLA-DRB1, and African-specific SNPs at ZNF649 and LSAMP, with associations of genome-wide significance for UC. We detected SNPs at USP25 with associations of genome-wide significance for IBD. No associations of genome-wide significance were detected for CD. In addition, 9 genes previously associated with IBD contained SNPs with significant evidence for replication (P < 1.6 × 10 We performed a genome-wide association study of African Americans with IBD and identified loci associated with UC in only this population; we also replicated IBD, CD, and UC loci identified in European populations. The detection of variants associated with IBD risk in only people of African descent demonstrates the importance of studying the genetics of IBD and other complex diseases in populations beyond those of European ancestry. Show less

The challenge of developing effective pharmacodynamic biomarkers for preclinical and clinical testing of FGFR signaling inhibition is significant. Assays that rely on the measurement of phospho-protein epitopes can be limited by the availability of effective antibody detection reagents. Transcript profiling enables accurate quantification of many biomarkers and provides a broader representation of pathway modulation. To identify dynamic transcript biomarkers of FGFR signaling inhibition by AZD4547, a potent inhibitor of FGF receptors 1, 2, and 3, a gene expression profiling study was performed in FGFR2-amplified, drug-sensitive tumor cell lines. Consistent with known signaling pathways activated by FGFR, we identified transcript biomarkers downstream of the RAS-MAPK and PI3K/AKT pathways. Using different tumor cell lines in vitro and xenografts in vivo, we confirmed that some of these transcript biomarkers (DUSP6, ETV5, YPEL2) were modulated downstream of oncogenic FGFR1, 2, 3, whereas others showed selective modulation only by FGFR2 signaling (EGR1). These transcripts showed consistent time-dependent modulation, corresponding to the plasma exposure of AZD4547 and inhibition of phosphorylation of the downstream signaling molecules FRS2 or ERK. Combination of FGFR and AKT inhibition in an FGFR2-mutated endometrial cancer xenograft model enhanced modulation of transcript biomarkers from the PI3K/AKT pathway and tumor growth inhibition. These biomarkers were detected on the clinically validated nanoString platform. Taken together, these data identified novel dynamic transcript biomarkers of FGFR inhibition that were validated in a number of in vivo models, and which are more robustly modulated by FGFR inhibition than some conventional downstream signaling protein biomarkers. Mol Cancer Ther; 15(11); 2802-13. ©2016 AACR. Show less

The objective of the study was to investigate how inflammatory cytokines, IL-1β, and TNF-α control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKKβ significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38α/β2/δ, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38γ played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1β as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1β and TNF-α activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function. Show less

In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle. Show less

Imaging single cells with fluorescent markers over multiple cell cycles is a powerful tool for unraveling the mechanism and dynamics of the cell cycle. Over the past ten years, microfluidic techniques in cell biology have emerged that allow for good control of growth environment. Yet the control and quantification of transient gene expression in unperturbed dividing cells has received less attention. Here, we describe a microfluidic flow cell to grow Saccharomyces Cerevisiae for more than 8 generations (approximately 12 hrs) starting with single cells, with controlled flow of the growth medium. This setup provides two important features: first, cells are tightly confined and grow in a remarkably planar array. The pedigree can thus be determined and single-cell fluorescence measured with 3 minutes resolution for all cells, as a founder cell grows to a micro-colony of more than 200 cells. Second, we can trigger and calibrate rapid and transient gene expression using reversible administration of inducers that control the GAL1 or MET3 promoters. We then show that periodic 10-20 minutes gene induction pulses can drive many cell division cycles with complete coherence across the cell cluster, with either a G1/S trigger (cln1 cln2 cln3 MET3-CLN2) or a mitotic trigger (cdc20 GALL-CDC20). In addition to evident cell cycle applications, this device can be used to directly measure the amount and duration of any fluorescently scorable signal-transduction or gene-induction response over a long time period. The system allows direct correlation of cell history (e.g., hysteresis or epigenetics) or cell cycle position with the measured response. Show less

Familial hypertrophic cardiomyopathy (HCM) is a leading cause of sudden cardiac death among young and apparently healthy people. Autosomal dominant mutations within genes encoding sarcomeric proteins have been identified. An autosomal recessive form of HCM has been discovered in a group of Amish children that is associated with poor prognosis and death within the first year of life. Affected patients experienced progressive cardiac failure despite maximal medical treatment. Postmortem histology showed myofibre disarray and myocyte loss consistent with refractory clinical deterioration in affected infants. To conduct a genome-wide screen for linkage and try to identify an autozygous region which cosegregates with the infant cardiac phenotype An autozygous region of chromosome 11 which cosegregates with the infant cardiac phenotype was identified. This region contained the MYBPC3 gene, which has previously been associated with autosomal dominant adult-onset HCM. Sequence analysis of the MYBPC3 gene identified a splice site mutation in intron 30 which was homozygous in all affected infants. All surviving patients with the homozygous MYBPC3 gene mutations (3330+2T>G) underwent an orthotopic heart transplantation. Homozygous mutations in the MYBPC3 gene have been identified as the cause of severe infantile HCM among the Amish population. Show less

Molecular noise in gene expression can generate substantial variability in protein concentration. However, its effect on the precision of a natural eukaryotic circuit such as the control of cell cycle remains unclear. We use single-cell imaging of fluorescently labelled budding yeast to measure times from division to budding (G1) and from budding to the next division. The variability in G1 decreases with the square root of the ploidy through a 1N/2N/4N ploidy series, consistent with simple stochastic models for molecular noise. Also, increasing the gene dosage of G1 cyclins decreases the variability in G1. A new single-cell reporter for cell protein content allows us to determine the contribution to temporal G1 variability of deterministic size control (that is, smaller cells extending G1). Cell size control contributes significantly to G1 variability in daughter cells but not in mother cells. However, even in daughters, size-independent noise is the largest quantitative contributor to G1 variability. Exit of the transcriptional repressor Whi5 from the nucleus partitions G1 into two temporally uncorrelated and functionally distinct steps. The first step, which depends on the G1 cyclin gene CLN3, corresponds to noisy size control that extends G1 in small daughters, but is of negligible duration in mothers. The second step, whose variability decreases with increasing CLN2 gene dosage, is similar in mothers and daughters. This analysis decomposes the regulatory dynamics of the Start transition into two independent modules, a size sensing module and a timing module, each of which is predominantly controlled by a different G1 cyclin. Show less

In the yeast Saccharomyces cerevisiae it has long been thought that cells must reach a critical cell size, called the "setpoint," in order to allow the Start cell cycle transition. Recent evidence suggests that this setpoint is lowered when ribosome biogenesis is slowed. Here we present evidence that yeast can sense ribosome biogenesis independently of mature ribosome levels and protein synthetic capacity. Our results suggest that ribosome biogenesis directly promotes passage through Start through Whi5, the yeast functional equivalent to the human tumor suppressor Rb. When ribosome biogenesis is inhibited, a Whi5-dependent mechanism inhibits passage through Start before significant decreases in both the number of ribosomes and in overall translation capacity of the cell become evident. This delay at Start in response to decreases in ribosome biogenesis occurs independently of Cln3, the major known Whi5 antagonist. Thus ribosome biogenesis may be sensed at multiple steps in Start regulation. Ribosome biogenesis may thus both delay Start by increasing the cell size setpoint and independently may promote Start by inactivating Whi5. Show less

Cell cycle "Start" in budding yeast involves induction of a large battery of G1/S-regulated genes, coordinated with bud morphogenesis. It is unknown how intra-Start coherence of these events and inter-Start timing regularity are achieved. We developed quantitative time-lapse fluorescence microscopy on a multicell-cycle timescale, for following expression of unstable GFP under control of the G1 cyclin CLN2 promoter. Swi4, a major activator of the G1/S regulon, was required for a robustly coherent Start, as swi4 cells exhibited highly variable loss of cooccurrence of regular levels of CLN2pr-GFP expression with budding. In contrast, other known Start regulators Mbp1 and Cln3 are not needed for coherence but ensure regular timing of Start onset. The interval of nuclear retention of Whi5, a Swi4 repressor, largely accounts for wild-type mother-daughter asymmetry and for variable Start timing in cln3 mbp1 cells. Thus, multiple pathways may independently suppress qualitatively different kinds of noise at Start. Show less

Regions of the budding yeast G1 cyclin Cln3 were characterized using mutational analysis and viability assays to identify functionally relevant and novel mutant alleles of CLN3. Cyclin proteins are conserved, and Cln3 contains a region with homology to the cyclin box, which is thought to mediate physical interactions with the cyclin-dependent kinase. CLN3 was found to have characteristics similar to the conserved cyclin fold found in higher eukaryotic cyclin boxes, which consist of five alpha-helices. Peptide linker sequences inserted within helices 1, 2, 3 and 5 resulted in a loss of Cln3 function, showing cyclin fold structure similar to that previously observed for the G1 cyclin Cln2. A clustered-charge-to-alanine scan mutagenesis revealed two regions of Cln3 important for Cln3-dependent viability. The first region encompasses the conserved cyclin box. The second region is identified with alanine substitutions located well past the cyclin box, just prior to the C-terminal region of Cln3 important for protein stability. Cln3 with mutational changes in each of these regions are expressed at steady-state levels higher than wild-type Cln3, and show some defect in binding to Cdc28. The conserved hydrophobic patch domain (HPD) of cyclins is present within the first helix of the cyclin box. Alanine substitutions introduced into the HPD of Cln3 and Cln2 show functional defects while maintaining physical interaction with Cdc28 as measured by co-immunoprecipitation assay. Show less

We derived novel, testable predictions from a mathematical model of the budding yeast cell cycle. A key qualitative prediction of bistability was confirmed in a strain simultaneously lacking cdc14 and G1 cyclins. The model correctly predicted quantitative dependence of cell size on gene dosage of the G1 cyclin CLN3, but it incorrectly predicted strong genetic interactions between G1 cyclins and the anaphase-promoting complex specificity factor Cdh1. To provide constraints on model generation, we determined accurate concentrations for the abundance of all nine cyclins as well as the inhibitor Sic1 and the catalytic subunit Cdc28. For many of these we determined abundance throughout the cell cycle by centrifugal elutriation, in the presence or absence of Cdh1. In addition, perturbations to the Clb-kinase oscillator were introduced, and the effects on cyclin and Sic1 levels were compared between model and experiment. Reasonable agreement was obtained in many of these experiments, but significant experimental discrepancies from the model predictions were also observed. Thus, the model is a strong but incomplete attempt at a realistic representation of cell cycle control. Constraints of the sort developed here will be important in development of a truly predictive model. Show less

Different G(1) cyclins confer functional specificity to the cyclin-dependent kinase (Cdk) Cdc28p in budding yeast. The Cln3p G(1) cyclin is localized primarily to the nucleus, while Cln2p is localized primarily to the cytoplasm. Both binding to Cdc28p and Cdc28p-dependent phosphorylation in the C-terminal region of Cln2p are independently required for efficient nuclear depletion of Cln2p, suggesting that this process may be physiologically regulated. The accumulation of hypophosphorylated Cln2 in the nucleus is an energy-dependent process, but may not involve the RAN GTPase. Phosphorylation of Cln2p is inefficient in small newborn cells obtained by elutriation, and this lowered phosphorylation correlates with reduced Cln2p nuclear depletion in newborn cells. Thus, Cln2p may have a brief period of nuclear residence early in the cell cycle. In contrast, the nuclear localization pattern of Cln3p is not influenced by Cdk activity. Cln3p localization requires a bipartite nuclear localization signal (NLS) located at the C terminus of the protein. This sequence is required for nuclear localization of Cln3p and is sufficient to confer nuclear localization to green fluorescent protein in a RAN-dependent manner. Mislocalized Cln3p, lacking the NLS, is much less active in genetic assays specific for Cln3p, but more active in assays normally specific for Cln2p, consistent with the idea that Cln3p localization explains a significant part of Clnp functional specificity. Show less

The G(1) cyclins of budding yeast drive cell cycle initiation by different mechanisms, but the molecular basis of their specificity is unknown. Here we test the hypothesis that the functional specificity of G(1) cyclins is due to differential subcellular localization. As shown by indirect immunofluorescence and biochemical fractionation, Cln3p localization appears to be primarily nuclear, with the most obvious accumulation of Cln3p to the nuclei of large budded cells. In contrast, Cln2p localizes to the cytoplasm. We were able to shift localization patterns of truncated Cln3p by the addition of nuclear localization and nuclear export signals, and we found that nuclear localization drives a Cln3p-like functional profile, while cytoplasmic localization leads to a partial shift to a Cln2p-like functional profile. Therefore, forcing Cln3p into a Cln2p-like cytoplasmic localization pattern partially alters the functional specificity of Cln3p toward that of Cln2p. These results suggest that there are CLN-dependent cytoplasmic and nuclear events important for cell cycle initiation. This is the first indication of a cytoplasmic function for a cyclin-dependent kinase. The data presented here support the idea that cyclin function is regulated at the level of subcellular localization and that subcellular localization contributes to the functional specificity of Cln2p and Cln3p. Show less

The completion of DNA synthesis in yeast is monitored by a checkpoint that requires MEC1 and RAD53. Here we show that deletion of the Saccharomyces cerevisiae G1 cyclins CLN1 and CLN2 suppressed the essential requirement for MEC1 function. Wild-type levels of CLN1 and CLN2, or overexpression of CLN1, CLN2, or CLB5, but not CLN3, killed mec1 strains. We identified RNR1, which encodes a subunit of ribonucleotide reductase, as a high-copy suppressor of the lethality of mec1 GAL1-CLN1. Northern analysis demonstrated that RNR1 expression is reduced by CLN1 or CLN2 overexpression. Because limiting RNR1 expression would be expected to decrease dNTP pools, CLN1 and CLN2 may cause lethality in mec1 strains by causing initiation of DNA replication with inadequate dNTPs. In contrast to mec1 mutants, MEC1 strains with low dNTPs would be able to delay S phase and thereby remain viable. We propose that the essential function for MEC1 may be the same as its checkpoint function during hydroxyurea treatment, namely, to slow S phase when nucleotides are limiting. In a cln1 cln2 background, a prolonged period of expression of genes turned on at the G1-S border, such as RNR1, has been observed. Thus deletion of CLN1 and CLN2 could function similarly to overexpression of RNR1 in suppressing mec1 lethality. Show less

The Saccharomyces cerevisiae cell cycle is arrested in G1 phase by the mating factor pathway. Genetic evidence has suggested that the G1 cyclins Cln1, Cln2, and Cln3 are targets of this pathway whose inhibition results in G1 arrest. Inhibition of Cln1- and Cln2-associated kinase activity by the mating factor pathway acting through Far1 has been described. Here we report that Cln3-associated kinase activity is inhibited by mating factor treatment, with dose response and timing consistent with involvement in cell cycle arrest. No regulation of Cln3-associated kinase was observed in a fus3 kss1 strain deficient in mating factor pathway mitogen-activated protein (MAP) kinases. Inhibition occurs mainly at the level of specific activity of Cln3-Cdc28 complexes. Inhibition of the C-terminally truncated Cln3-1-associated kinase is not observed; such truncations were previously identified genetically as causing resistance to mating factor-induced cell cycle arrest. Regulation of Cln3-associated kinase specific activity by mating factor treatment requires Far1. Overexpression of Far1 restores inhibition of C-terminally truncated Cln3-1-associated kinase activity. G2/M-arrested cells are unable to regulate Cln3-associated kinase, possibly because of cell cycle regulation of Far1 abundance. Inhibition of Cln3-associated kinase activity by the mating factor pathway may allow this pathway to block the earliest step in normal cell cycle initiation, since Cln3 functions as the most upstream G1-acting cyclin, activating transcription of the G1 cyclins CLN1 and CLN2 as well as of the S-phase cyclins CLB5 and CLB6. Show less

The G1 cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits. Show less

We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function. Show less

The three budding yeast CLN genes appear to be functionally redundant for cell cycle Start: any single CLN gene is sufficient to promote Start, while the cln1 cln2 cln3 triple mutant is Start defective and inviable. Both quantitative and apparently qualitative differences between CLN genes have been reported, but available data do not in general allow distinction between qualitative functional differences as opposed to simply quantitative differences in expression or function. To determine if there are intrinsic qualitative differences between Cln proteins, we compared CLN2, CLN3, and crippled (but still partially active) CLN2 genes in a range of assays that differentiate genetically between CLN2 and CLN3. The results suggest that different potencies of Cln2, Cln3, and Cln2 mutants in functional assays cannot be accounted for by a simple quantitative model for their action, since Cln3 is at least as active as Cln2 and much more active than the Cln2 mutants in driving Swi4/Swi6 cell cycle box (SCB)-regulated transcription and cell cycle initiation in cln1 cln2 cln3 bck2 strains, but Cln3 has little or no activity in other assays in which Cln2 and the Cln2 mutants function. Differences in Cln protein abundance are unlikely to account for these results. Cln3-associated kinase is therefore likely to have an intrinsic in vivo substrate specificity distinct from that of Cln2-associated kinase, despite their functional redundancy. Consistent with the idea that Cln3 may be the primary transcriptional activator of CLN1, CLN2, and other genes, the activation of CLN2 transcription was found to be sensitive to the gene dosage of CLN3 but not to the gene dosage of CLN2. Show less

In budding yeast, one of three G1 cyclins is required for progression though START, when cells commit to a further round of cell division. We have identified mutations in ALG1 (ERC14), a gene required for N-glycosylation, which are inviable in a cln1 cln2 background but are rescued by over-expression of CLNs. CLN1 and CLN2 are much more efficient than CLN3 in rescuing the erc14-1 allele. The erc14-1 allele results in a significant N-glycosylation defect, and no rescue of this defect by CLN1 over-expression was detected. These data suggest that CLN over-expression could be allowing cells to live with lower levels of N-glycosylation, possibly by overcoming a checkpoint sensitive to N-glycosylation capacity. A plasmid suppressor of alg1, PSA1, encodes a 361 amino-acid protein with homology to NDP-hexose pyrophosphorylases, the enzymes that catalyze the formation of activated sugar nucleotides. PSA1 is an essential gene, and PSA1 transcription is nearly co-ordinately regulated with CLN2 transcription, peaking near START. Co-ordinate regulation of glycosylation, sugar nucleotide metabolism, and cell-cycle progression through G1 may be a feature that ensures adequate cell-wall precursors are present before bud emergence. Show less

The yeast Saccharomyces cerevisiae has three G1 cyclin (CLN) genes with overlapping functions. To analyze the functions of the various CLN genes, we examined mutations that result in lethality in conjunction with loss of cln1 and cln2. We have isolated alleles of RAD27/ERC11/YKL510, the yeast homolog of the gene encoding flap endonuclease 1, FEN-1.cln1 cln2 rad27/erc11 cells arrest in S phase; this cell cycle arrest is suppressed by the expression of CLN1 or CLN2 but not by that of CLN3 or the hyperactive CLN3-2. rad27/erc11 mutants are also defective in DNA damage repair, as determined by their increased sensitivity to a DNA-damaging agent, increased mitotic recombination rates, and increased spontaneous mutation rates. Unlike the block in cell cycle progression, these phenotypes are not suppressed by CLN1 or CLN2. CLN1 and CLN2 may activate an RAD27/ERC11-independent pathway specific for DNA synthesis that CLN3 is incapable of activating. Alternatively, CLN1 and CLN2 may be capable of overriding a checkpoint response which otherwise causes cln1 cln2 rad27/erc11 cells to arrest. These results imply that CLN1 and CLN2 have a role in the regulation of DNA replication. Consistent with this, GAL-CLN1 expression in checkpoint-deficient, mec1-1 mutant cells results in both cell death and increased chromosome loss among survivors, suggesting that CLN1 overexpression either activates defective DNA replication or leads to insensitivity to DNA damage. Show less

Transcriptional induction by the mating pheromone alpha-factor was monitored at different stages of the yeast cell cycle. G2/M-phase and pre-Start cells showed strong FUS1 mRNA induction, whereas in post-Start cells the signaling was reduced significantly. This reduction in signaling activity in post-Start cells was correlated with the presence of CLN1 or CLN2 transcripts and was not observed in synchronized cells lacking functional CLN1 and CLN2 genes. Activation of the Cln-Cdc28p kinase by overexpression of CLN2 from the GAL1 promoter strongly reduced FUS1 mRNA induction. CLN1 overexpression had a similar effect when the FAR1 gene, encoding a negative regulator of CLN1/2 function, was deleted. This reduction of pheromone signaling was specific for CLN1 and CLN2, as it was not observed when CLN3 was overexpressed. Inactivation of the Cln-Cdc28p kinase complex by thermal inactivation of temperature-sensitive Cdc28p prevented repression of FUS1 signaling. CLN2 overexpression suppressed the constitutive signaling and division-arrest phenotypes of cells with a disrupted gpa1 gene, indicating that the site of action for repression is downstream of the alpha-subunit (Gpa1p) of the heterotrimeric G protein. The repression at Start of pheromone signaling by Cln1-Cdc28p or Cln2-Cdc28p kinase complexes may contribute to the acquisition of pheromone resistance as cells execute Start. Show less

Cell cycle START in Saccharomyces cerevisiae requires at least one of the three CLN genes (CLN1, CLN2, or CLN3). A total of 12 mutations bypassing this requirement were found to be dominant mutations in a single gene that we named BYC1 (for bypass of CLN requirement). We also isolated a plasmid that had cln bypass activity at a low copy number; the gene responsible was distinct from BYC1 and was identical to the recently described BCK2 gene. Strains carrying bck2::ARG4 disruption alleles were fully viable, but bck2::ARG4 completely suppressed the cln bypass activity of BYC1. swi4 and swi6 deletion alleles also efficiently suppressed BYC1 cln bypass activity; Swi4 and Swi6 are components of a transcription factor previously implicated in control of CLN1 and CLN2 expression. bck2::ARG4 was synthetically lethal with cln3 deletion, suggesting that CLN1 and CLN2 cannot function in the simultaneous absence of BCK2 and CLN3; this observation correlates with low expression of CLN1 and CLN2 in bck2 strains deprived of CLN3 function. Thus, factors implicated in CLN1 and CLN2 expression and/or function are also required for BYC1 function in the absence of all three CLN genes; this may suggest the involvement of other targets of Swi4, Swi6, and Bck2 in START. Show less

The CLN1, CLN2 and CLN3 gene family of G1-acting cyclin homologs of Saccharomyces cerevisiae is functionally redundant: any one of the three Cln proteins is sufficient for activation of Cdc28p protein kinase activity for cell cycle START. The START event leads to multiple processes (including DNA replication and bud emergence); how Cln/Cdc28 activity activates these processes remains unclear. CLN3 is substantially different in structure and regulation from CLN1 and CLN2, so its functional redundancy with CLN1 and CLN2 is also poorly understood. We have isolated mutations that alter this redundancy, making CLN3 insufficient for cell viability in the absence of CLN1 and CLN2 expression. Mutations causing phenotypes specific for the cell division cycle were analyzed in detail. Mutations in one gene result in complete failure of bud formation, leading to depolarized cell growth. This gene was identified as BUD2, previously described as a non-essential gene required for proper bud site selection but not required for budding and viability. Bud2p is probably the GTPase-activating protein for Rsr1p/Bud1p [Park, H., Chant, I. and Herskowitz, I. (1993) Nature, 365, 269-274]; we find that Rsr1p is required for the bud2 lethal phenotype. Mutations in two other genes (ERC10 and ERC19) result in a different morphogenetic defect: failure of cytokinesis resulting in the formation of long multinucleate tubes. These results suggest direct regulation of diverse aspects of bud morphogenesis by Cln/Cdc28p activity. Show less