👤 Daniel M Levine

The apolipoprotein E epsilon 4 (APOE ε4) gene is associated with an increased risk of developing sporadic Alzheimer's disease (AD). Several studies have focused on declarative memory, where episodic memory deficits are reported in ε4 carriers, while semantic memory has received much less attention. To clarify whether the impact of APOE ε4 on declarative memory is specific to episodic memory, we administered a novel measure of autobiographical memory, the Semantic Autobiographical Interview. Thirty-eight healthy older adults were recruited, 19 ε4 carriers and 19 noncarriers, matched in age, education, and gender. The groups did not significantly differ in any neuropsychological tests except for recognition memory, where ε4 carriers showed reduced performance. On the original Autobiographical Interview (AI), results revealed a reduced number of target details in carriers. Together, these results suggest a reduction of episodic specificity in ε4 carriers. In contrast, carriers had very similar semantic production to noncarriers, whether it was for off-task semantic details in the AI, or on-task general and personal semantic details produced in the Semantic Autobiographical Interview. These results suggest that older adults retain the gist of their personal experience and that the semanticization of their autobiographical narratives is robust and less sensitive to risk for AD than episodic memory. (PsycInfo Database Record (c) 2026 APA, all rights reserved). Show less

The assessment of serious illness communication (SIC) competence has, to date, primarily utilized tools that are profession-specific and not explicitly designed using competency-based or person-centered frameworks. To address these gaps, we developed and validated a new tool, the Assessment of Clinical Encounters - Communication Tool (ACE-CT). We convened a national panel of interprofessional SIC experts to develop and validate the ACE-CT using a three-phase multi-method approach. Phase 1 focused on item development through review of existing validated tools, and a Bayesian process in which panel members assessed item quality and item-domain correlation. Phase 2 involved item refinement and preliminary validation through stimulated recall interviews using a think-aloud technique. Phase 3 consisted of psychometric analyses for which panel members used the tool to assess video-recorded standardized patient encounters from interprofessional clinicians completing a SIC professional development intervention. In Phase 1, 37 relevant items from previously validated tools were identified, of which 11 items were removed due to redundance. Through the Bayesian process, 14 items were removed and 1 item was generated. Through Phase 2, 2 items were generated, 2 items were combined into 1, and remaining items were refined to optimize measurability and understandability. In Phase 3, reliability was demonstrated through evidence of high internal consistency and moderate reproducibility, both over time and across raters. The tool was found to be responsive and have sound construct validity through evidence of congruence, convergence and credibility. Raters found the tool to be intuitive, easy to complete, and that it accurately captured their perception of the quality of communication observed. The ACE-CT provides a reliable and valid approach to assessing SIC competence among interprofessional clinicians. Through its person-centered orientation, the ACE-CT provides an opportunity to objectively assess elements of SIC that patients and families value. Show less

Lipoprotein(a) [Lp(a)] is associated with atherosclerotic cardiovascular disease. An Lp(a) threshold of ≥125 nmol/L is commonly used to identify individuals at higher risk for events, but there is a paucity of data on individuals of Hispanic/Latino descent. The purpose of this study was to provide a comprehensive evaluation of Lp(a) and its association with 10-year cardiovascular disease risk and mortality among Hispanic/Latino adults in the United States. We evaluated the association between Lp(a) and myocardial infarction (MI), ischemic stroke, and all-cause mortality among 16,117 Hispanic Community Health Study/Study of Latinos individuals. Event rates were compared across Lp(a) quintiles. Multivariable Cox proportional hazards models assessed the relationship between events and Lp(a) across increasing quintiles, log-transformed Lp(a), and ≥125 nmol/L vs <125 nmol/L. Sampling weights and survey methods were used to account for the stratified probability sampling of the cohort. Among the Hispanic Community Health Study/Study of Latinos target population (median age 41.1 years, 52.4% women), the median Lp(a) was 19.7 nmol/L (Q1-Q3: 7.3-60.6 nmol/L), with 11.4% having Lp(a) ≥125 nmol/L, and the highest Lp(a) quintile defined as >77 nmol/L. Over a median follow-up of 9.8 years, 883 events (135 MI, 99 stroke, 649 all-cause mortality) occurred. The age-adjusted incidence rate of the composite events (MI, stroke, and all-cause mortality) was 505.2 per 100,000 person-years. After multivariable adjustment, each 1-SD increase in log-transformed Lp(a) was associated with a higher risk of MI (HR: 1.47; 95% CI: 1.14-1.89). Compared with Lp(a) <125 nmol/L, elevated Lp(a) ≥125 nmol/L conferred an increased risk of MI (HR: 2.29; 95% CI: 1.45-3.63), all-cause mortality (HR: 1.43; 95% CI: 1.05-1.93), and composite events (HR: 1.56; 95% CI: 1.22-2.01), but not stroke. Findings were consistent when comparing the highest Lp(a) quintile to the lower 4 quintiles, but the elevated risk was observed only for MI and composite events. Hispanic/Latino individuals with elevated Lp(a) are at an increased risk of MI and all-cause mortality. Although Lp(a) ≥125 nmol/L is a valid risk threshold, Hispanics/Latinos show a continuous relationship between increasing Lp(a) levels and MI risk. Show less

Parkinson's disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer a unique experimental platform to advance our understanding of PD etiology by enabling the generation of disease-relevant cell types carrying patient mutations along with isogenic control cells. To facilitate this approach, we generated a collection of 65 human stem cell lines genetically engineered to harbor high risk or causal variants in genes associated with PD ( Show less

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m Show less

Over the past decade, our understanding of the molecular drivers of pediatric low-grade glioma (PLGG) has expanded dramatically. These tumors are predominantly driven by RAS/MAPK pathway activating alterations (fusions and point mutations), most frequently in BRAF, FGFR1, and NF1. Furthermore, additional second hits in tumor suppressor genes (TP53, ATRX, CDKN2A) can portend more aggressive behaviour. Accordingly, comprehensive molecular profiling-specifically genetic sequencing, often plus copy number profiling-has become critical for guiding the diagnosis and management of PLGG. In this review, we discuss the most important genetic alterations that inform on classification and prognosis of PLGG, highlighting their diagnostic and therapeutic relevance. Show less

The molecular characterization of male breast cancer (MaBC) has received limited attention in research, mostly because of its low incidence rate, accounting for only 0.5% to 1% of all reported cases of breast cancer each year. Managing MaBC presents significant challenges, with most treatment protocols being adapted from those developed for female breast cancer. Utilizing whole-genome sequencing (WGS) and state-of-the-art analyses, the genomic features of 10 MaBC cases (n = 10) were delineated and correlated with clinical and histopathologic characteristics. Using fluorescence in situ hybridization, an additional cohort of 18 patients was interrogated to supplement WGS findings. The genomic landscape of MaBC uncovered significant genetic alterations that could influence diagnosis and treatment. We found common somatic mutations in key driver genes, such as FAT1, GATA3, SMARCA4, and ARID2. Our study also mapped out structural variants that impact cancer-associated genes, such as ARID1A, ESR1, GATA3, NTRK1, and NF1. Using a WGS-based classifier, homologous recombination deficiency (HRD) was identified in 2 cases, both presenting with deleterious variants in BRCA2. Noteworthy was the observation of FGFR1 amplification in 21% of cases. Altogether, we identified at least 1 potential therapeutic target in 8 of the 10 cases, including high tumor mutational burden, FGFR1 amplification, and HRD. Our study is the first WGS characterization of MaBC, which uncovered potentially relevant variants, including structural events in cancer genes, HRD signatures, and germline pathogenic mutations. Our results demonstrate unique genetic markers and potential treatment targets in MaBC, thereby underlining the necessity of tailoring treatment strategies for this understudied patient population. These WGS-based findings add to the growing knowledge of MaBC genomics and highlight the need to expand research on this type of cancer. Show less

Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs. Significance: Given the broad prevalence, comorbidities, and risk of malignant transformation associated with CH, there is an unmet need to identify therapeutic targets. We develop an ex vivo platform to perform CRISPR/Cas9 screens in primary HSPCs. We identify KDM3B and downstream signaling components as genotype-specific dependencies in CH and myeloid malignancies. See related commentary by Khabusheva and Goodell, p. 1768. Show less

Despite significant advances in developing selective JAK2 inhibitors, JAK2 kinase inhibitor (TKI) therapy is ineffective in suppressing the disease. Reactivation of compensatory MEK-ERK and PI3K survival pathways sustained by inflammatory cytokine signaling causes treatment failure. Concomitant inhibition of MAPK pathway and JAK2 signaling showed improved in vivo efficacy compared to JAK2 inhibition alone but lacked clonal selectivity. We hypothesized that cytokine signaling in JAK2 Show less

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins (FPs) are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of acute myeloid leukemia (AML) driven by the most common AF10 FPs, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a detailed map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent antioncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias. Show less

A key mediator of macroautophagy/autophagy induction is the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) consisting of PIK3C3/VPS34, PIK3R4/VPS15, BECN1, and ATG14. Although several proteins are known to enhance or decrease PtdIns3K-C1 activity, our understanding of the molecular regulation of PtdIns3K-C1 is still incomplete. Previously, we identified a Golgi-associated protein, GLIPR2, in a screen for proteins that interact with amino acids 267-284 of BECN1, a region of BECN1 sufficient to induce autophagy when fused to a cell penetrating leader sequence. In this study, we used CRISPR-Cas9-mediated depletion of GLIPR2 in cells and mice to investigate the role of GLIPR2 in the regulation of autophagy and PtdIns3K-C1 activity. Depletion of GLIPR2 in HeLa cells increased autelophagic flux and generation of phosphatidylinositol 3-phosphate (PtdIns3P). Show less

Recent preclinical and clinical data suggests enhanced metastatic fitness of hybrid epithelial/mesenchymal (E/M) phenotypes, but mechanistic details regarding their survival strategies during metastasis remain unclear. Here, we investigate immune-evasive strategies of hybrid E/M states. We construct and simulate the dynamics of a minimalistic regulatory network encompassing the known associations among regulators of EMT (epithelial-mesenchymal transition) and PD-L1, an established immune-suppressor. Our simulations for the network consisting of SLUG, ZEB1, miR-200, CDH1 and PD-L1, integrated with single-cell and bulk RNA-seq data analysis, elucidate that hybrid E/M cells can have high levels of PD-L1, similar to those seen in cells with a full EMT phenotype, thus obviating the need for cancer cells to undergo a full EMT to be immune-evasive. Specifically, in breast cancer, we show the co-existence of hybrid E/M phenotypes, enhanced resistance to anti-estrogen therapy and increased PD-L1 levels. Our results underscore how the emergent dynamics of interconnected regulatory networks can coordinate different axes of cellular fitness during metastasis. Show less

Genomic analyses of chronic lymphocytic leukemia (CLL) identified somatic mutations and associations of clonal diversity with adverse outcomes. Clonal evolution likely has therapeutic implications but its dynamic is less well studied. We studied clonal composition and prognostic value of seven recurrently mutated driver genes using targeted next-generation sequencing in 643 CLL patients and found higher frequencies of mutations in TP53 (35 vs. 12%, p < 0.001) and SF3B1 (20 vs. 11%, p < 0.05) and increased number of (sub)clonal (p < 0.0001) mutations in treated patients. We next performed an in-depth evaluation of clonal evolution on untreated CLL patients (50 "progressors" and 17 matched "non-progressors") using a 404 gene-sequencing panel and identified novel mutated genes such as AXIN1, SDHA, SUZ12, and FOXO3. Progressors carried more mutations at initial presentation (2.5 vs. 1, p < 0.0001). Mutations in specific genes were associated with increased (SF3B1, ATM, and FBXW7) or decreased progression risk (AXIN1 and MYD88). Mutations affecting specific signaling pathways, such as Notch and MAP kinase pathway were enriched in progressive relative to non-progressive patients. These data extend earlier findings that specific genomic alterations and diversity of subclones are associated with disease progression and persistence of disease in CLL and identify novel recurrently mutated genes and associated outcomes. Show less

Carbohydrate response element binding protein (ChREBP) is a key transcriptional regulator of de novo lipogenesis (DNL) in response to carbohydrates and in hepatic steatosis. Mechanisms underlying nutrient modulation of ChREBP are under active investigation. Here we identify host cell factor 1 (HCF-1) as a previously unknown ChREBP-interacting protein that is enriched in liver biopsies of nonalcoholic steatohepatitis (NASH) patients. Biochemical and genetic studies show that HCF-1 is O-GlcNAcylated in response to glucose as a prerequisite for its binding to ChREBP and subsequent recruitment of OGT, ChREBP O-GlcNAcylation, and activation. The HCF-1:ChREBP complex resides at lipogenic gene promoters, where HCF-1 regulates H3K4 trimethylation to prime recruitment of the Jumonji C domain-containing histone demethylase PHF2 for epigenetic activation of these promoters. Overall, these findings define HCF-1's interaction with ChREBP as a previously unappreciated mechanism whereby glucose signals are both relayed to ChREBP and transmitted for epigenetic regulation of lipogenic genes. Show less

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10-6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations. Show less

Tubular lipid binding proteins (TULIPs) have become a focus of interest in the cell biology of lipid signalling, lipid traffic and membrane contact sites. Each tubular domain has an internal pocket with a hydrophobic lining that can bind a hydrophobic molecule such as a lipid. This allows TULIP proteins to carry lipids through the aqueous phase. TULIP domains were first found in a large family of extracellular proteins related to the bacterial permeability-inducing protein (BPI) and cholesterol ester transfer protein (CETP). Since then, the same fold and lipid transfer capacity have been found in SMP domains (so-called for their occurrence in synaptotagmin, mitochondrial and lipid binding proteins), which localise to intracellular membrane contact sites. Here the methods for identifying known TULIPs are described, and used to find previously unreported TULIPs, one in the silk polymer and another in prokaryotes illustrated by the E. coli protein YceB. The bacterial TULIP alters views on the likely evolution of the domain, suggesting its presence in the last universal common ancestor. The major function of TULIPs is to handle lipids, but we still do not know how they work in detail, or how many more remain to be discovered. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann. Show less

Increased fructose consumption is a contributor to the burgeoning epidemic of non-alcoholic fatty liver disease (NAFLD). Recent evidence indicates that the metabolic hormone FGF21 is regulated by fructose consumption in humans and rodents and may play a functional role in this nutritional context. Here, we sought to define the mechanism by which fructose ingestion regulates FGF21 and determine whether FGF21 contributes to an adaptive metabolic response to fructose consumption. We tested the role of the transcription factor carbohydrate responsive-element binding protein (ChREBP) in fructose-mediated regulation of FGF21 using ChREBP knockout mice. Using FGF21 knockout mice, we investigated whether FGF21 has a metabolic function in the context of fructose consumption. Additionally, we tested whether a ChREBP-FGF21 interaction is likely conserved in human subjects. Hepatic expression of In summary, ChREBP and FGF21 constitute a signaling axis likely conserved in humans that mediates an essential adaptive response to fructose ingestion that may participate in the pathogenesis of NAFLD and liver fibrosis. Show less

The strong male predominance in Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) remains inadequately explained, but sex hormones might be involved. We hypothesized that single nucleotide polymorphisms (SNPs) in the androgen pathway influence risk of developing BE and EAC. This genetic-epidemiological analysis included 14 studies from Australia, Europe and North America. Polymorphisms in 16 genes coding for the androgen pathway were analyzed using a gene-based approach: versatile gene-based test association study. This method evaluates associations between a trait and all SNPs within a specific gene rather than each SNP marker individually as in a conventional GWAS. The data were stratified for sex, body-mass index, waist-to-hip ratio, tobacco smoking and gastroesophageal reflux status. Included were data from 1,508 EAC patients, 2,383 BE patients and 2,170 control participants. SNPs within the gene CYP17A1 were associated with risk of BE in the sexes combined (p = 0.002) and in males (p = 0.003), but not in females separately (p = 0.3). This association was found in tobacco smokers (p = 0.003) and in BE patients without reflux (p = 0.004), but not in nonsmokers (p = 0.2) or those with reflux (p = 0.036). SNPs within JMJD1C were associated with risk of EAC in females (p = 0.001). However, none of these associations replicated in a subsequent sample. Fourteen other genes studied did not reach statistically significant levels of association with BE, EAC or the combination of BE and EAC, after correcting for the number of genes included in the analysis. In conclusion, genetic variants in the androgen-related genes CYP17A1 and JMJD1C might be associated with risk of BE and EAC, respectively, but replication data with larger sample sizes are needed. Show less

Osteochondroma is the most common benign bone tumor. However, there are infrequent studies karyotyping solitary osteochondromas. Retrospective review of the University of California, Los Angeles pathology database was performed for karyotype analyses (N = 522 specimens). Two previously undescribed karyotypes were identified. First, was a karyotype showing paracentric inversion of chromosome 7. Second, was a karyotype showing monosomy 3, 6 and 13. Abnormal karyotypes may be more frequently encountered in osteochondroma than previously understood. However, the clinical significance of these abnormalities are yet unknown. Show less

DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P<0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21-23% of primary ovarian cancers but in 81-84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P<0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival. Show less

Mitogen-activated protein kinase (MAPK) pathway signaling plays an important role in the majority of non-small-cell lung cancers (NSCLCs). In a prior microarray analysis of epidermal growth factor receptor (EGFR) inhibition in NSCLC cell lines, we noted that several dual specificity phosphatases (DUSPs) were among the most highly and immediately regulated genes. DUSPs act as natural terminators of MAPK signal transduction and therefore, we hypothesized a tumor suppressive role via feedback mechanisms. In the current study, we focus on the assessment of DUSP6, a cytoplasmic DUSP with high specificity for extracellular signal-regulated kinase (ERK). We demonstrate that DUSP6 expression tracks in tandem with ERK inhibition and that regulation of DUSP6 is mediated at the promoter level by ETS1, a well-known nuclear target of activated ERK. Small interfering RNA knockdown in DUSP6-high H441 lung cancer cells significantly increased ERK activation and cellular proliferation, whereas plasmid-driven overexpression in DUSP6-low H1975 lung cancer cells significantly reduced ERK activation and cellular proliferation and promoted apoptosis. Also, DUSP6 overexpression synergized with EGFR inhibitor treatment in EGFR-mutant HCC827 cells. Our results indicate that DUSP6 expression is regulated by ERK signaling and that DUSP6 exerts antitumor effects via negative feedback regulation, pointing to an important feedback loop in NSCLC. Further studies assessing the tumor suppressive role of DUSP6 and strategies aimed at modulation of its activity are warranted. 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

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