CRISPR/Cas9 edited SlGT30 improved both drought resistance and fruit yield through endoreduplication.

There is often a trade-off effect between different agronomic traits due to gene pleiotropy, leading to a negative correlation between yield and resistance. Consequently, using gene-editing techniques to develop superior traits becomes challenging. Genetic resources that defy this constraint are scarce but hold great potential as targets for improvement through the utilisation of CRISPR. Transcription factors are critical in modulating numerous gene expressions across diverse biological processes. Here, we found that the trihelix transcription factor SlGT30 plays a role in drought resistance and tomato fruit development. We edited the SlGT30 gene with CRISPR/Cas9 technology and found that the knockout lines showed decreased stomata density in the leaves and large fruits. Subsequent examination revealed that cell ploidy was impacted in the leaves and fruits of SlGT30 knockout lines. SlGT30 knockout affected cell size through the endoreduplication pathway, manifested in decreased stomata density and reduced water loss. Consequently, this resulted in an enhancement of drought resistance. For the fruit, both cell size and cell number increased in the fruit pericarp of knockout lines, improving the fruit size and weight accordingly. Therefore, SlGT30 represents a promising candidate gene for gene editing in breeding practice.

Different responses of Sinorhizobium sp. upon Pb and Zn exposure: Mineralization versus complexation.

Lead (Pb) and zinc (Zn) have been discharged into environment and may negatively impact ecological security. Rhizobia has gained attention due to their involvement in the restoration of metal polluted soils. However, little is known about the responses of rhizobia under Pb and Zn stress, especially the roles played by extracellular polymeric substances (EPS) in the resistance of these two metals. Here, Sinorhizobium sp. C10 was isolated from soil around a mining area and was exposed to a series of Pb/Zn treatments. The cell morphology and surface mineral crystals, EPS content and fluorescent substances were determined. In addition, the extracellular polysaccharides and proteins were characterized by attenuated total reflection infrared spectroscopy (ATR-IR) and X-ray photoelectron spectroscopy (XPS). The results showed that Zn stress induced the synthesis of EPS by C10 cells. Functional groups of polysaccharides (CO) and proteins (C-O/C-N) were involved in complexation with Zn. In contrast, C10 resisted Pb stress by forming lead phosphate (Pb3(PO4)2) on the cell surface. Galactose (Gal) and tyrosine played key roles in resistance to the Zn toxicity, whereas glucosamine (N-Glc) was converted to glucose in large amounts during extracellular Pb precipitation. Together, this study demonstrated that C10 possessed different strategies to detoxify the two metals, and could provide basis for bioremediation of Pb and Zn polluted sites.

The SlWRKY57-SlVQ21/SlVQ16 module regulates salt stress in tomato.

Salt stress is a major abiotic stress which severely hinders crop production. However, the regulatory network controlling tomato resistance to salt remains unclear. Here, we found that the tomato WRKY transcription factor WRKY57 acted as a negative regulator in salt stress response by directly attenuating the transcription of salt-responsive genes (SlRD29B and SlDREB2) and an ion homeostasis gene (SlSOS1). We further identified two VQ-motif containing proteins SlVQ16 and SlVQ21 as SlWRKY57-interacting proteins. SlVQ16 positively, while SlVQ21 negatively modulated tomato resistance to salt stress. SlVQ16 and SlVQ21 competitively interacted with SlWRKY57 and antagonistically regulated the transcriptional repression activity of SlWRKY57. Additionally, the SlWRKY57-SlVQ21/SlVQ16 module was involved in the pathway of phytohormone jasmonates (JAs) by interacting with JA repressors JA-ZIM domain (JAZ) proteins. These results provide new insights into how the SlWRKY57-SlVQ21/SlVQ16 module finely tunes tomato salt tolerance.

A comparative study of the accumulation and detoxification of copper and zinc in Chlamydomonas reinhardtii: The role of extracellular polymeric substances.

Extracellular polymeric substances (EPS) form an interface between microalgae and the surrounding water environment. Copper (Cu) and zinc (Zn) are essential micronutrients but may negatively affect microbial growth when their concentrations reach toxic thresholds. However, how EPS affect the accumulation and resistance of Cu and Zn in microalgae remains largely unknown. Here, we investigated EPS production upon Cu/Zn exposure and compared the tolerance strategies to the two metals by Chlamydomonas reinhardtii with and without EPS. Microalgal EPS synthesis was induced by Cu/Zn treatments, and the functional groups of polysaccharides and proteins were involved in complexation with metal ions. The extraction of EPS aggravated the toxicity and reduced the removal of metals from solution, but the effect was more pronounced for Cu than for Zn. Copper bound on the cell surface accounted for 54.6 ± 2.0 % of the Cu accumulated by C. reinhardtii, whose EPS components strongly correlated with Cu adsorption. In contrast, 74.3 ± 3.0 % of accumulated Zn was absorbed in cells, and glutathione synthesis was significantly induced. Redundancy and linear correlation analyses showed that the polysaccharide, protein and DNA contents in EPS were significantly correlated with Cu accumulation, absorption and adsorption but not with Zn. Data fitted to a Michaelis-Menten model further showed that the EPS-intact cells had higher binding capacity for Cu2+ but not for Zn2+. These differential impacts of EPS on Cu/Zn sorption and detoxification contribute to a more comprehensive understanding of the roles of microalgal EPS in the biogeochemical cycle of metals.

SlWRKY45 interacts with jasmonate-ZIM domain proteins to negatively regulate defense against the root-knot nematode Meloidogyne incognita in tomato.

Parasitic root-knot nematodes (RKNs) cause a severe reduction in crop yield and seriously threaten agricultural production. The phytohormones jasmonates (JAs) are important signals regulating resistance to multiple biotic and abiotic stresses. However, the molecular mechanism for JAs-regulated defense against RKNs in tomato remains largely unclear. In this study, we found that the transcription factor SlWRKY45 interacted with most JA-ZIM domain family proteins (JAZs), key repressors of the JA signaling. After infection by the RKN Meloidogyne incognita, the slwrky45 mutants exhibited lower gall numbers and egg numbers per gram of roots than wild type, whereas overexpression of SlWRKY45 attenuated resistance to Meloidogyne incognita. Under M. incognita infection, the contents of jasmonic acid (JA) and JA-isoleucine (JA-Ile) in roots were repressed by SlWRKY45-overexpression. Furthermore, SlWRKY45 bound to and inhibited the promoter of the JA biosynthesis gene ALLENE OXIDE CYCLASE (AOC), and repressed its expression. Overall, our findings revealed that the SlJAZ-interaction protein SlWRKY45 attenuated RKN-regulated JA biosynthesis and repressed defense against the RKN M. incognita in tomato.

SlVQ15 interacts with jasmonate-ZIM domain proteins and SlWRKY31 to regulate defense response in tomato.

Botrytis cinerea is one of the most widely distributed and harmful pathogens worldwide. Both the phytohormone jasmonate (JA) and the VQ motif-containing proteins play crucial roles in plant resistance to B. cinerea. However, their crosstalk in resistance to B. cinerea is unclear, especially in tomato (Solanum lycopersicum). In this study, we found that the tomato VQ15 was highly induced upon B. cinerea infection and localized in the nucleus. Silencing SlVQ15 using virus-induced gene silencing reduced resistance to B. cinerea. Overexpression of SlVQ15 enhanced resistance to B. cinerea, while disruption of SlVQ15 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) technology increased susceptibility to B. cinerea. Furthermore, SlVQ15 formed homodimers. Additionally, SlVQ15 interacted with JA-ZIM domain proteins, repressors of the JA signaling pathway, and SlWRKY31. SlJAZ11 interfered with the interaction between SlVQ15 and SlWRKY31 and repressed the SlVQ15-increased transcriptional activation activity of SlWRKY31. SlVQ15 and SlWRKY31 synergistically regulated tomato resistance to B. cinerea, as silencing SlVQ15 enhanced the sensitivity of slwrky31 to B. cinerea. Taken together, our findings showed that the SlJAZ-interacting protein SlVQ15 physically interacts with SlWRKY31 to cooperatively control JA-mediated plant defense against B. cinerea.

Contrasting detoxification mechanisms of Chlamydomonas reinhardtii under Cd and Pb stress.

Chlamydomonas reinhardtii has been frequently investigated for its resistance to metals; however, few studies have systematically compared the intracellular and extracellular processes involved in the detoxification of Cd and Pb by this microalga. We found that C. reinhardtii was more tolerant to Pb (concentration for 50% of the maximal effect; EC50: 29.48 ± 8.83 mg L-1) than to Cd (EC50: 12.48 ± 1.30 mg L-1) after 96 h of exposure. Extracellular polymeric substances (EPS), intracellular starch granules, lipid droplets, and glutathione were significantly increased under Cd and Pb treatments. Lead-containing particles were formed outside of the cells exposed to 30 mg L-1 of Pb, whereas no minerals were present when Cd was added. Various EPS functional groups, including -COOH, C-O-C (polysaccharides), and amide I and II (proteins), were involved in the interactions with Cd and Pb. The Pb removal rate (60.46-78.27%) by C. reinhardtii was higher than that of Cd (50.61-59.38%), and the microalgal cells with intact EPS bound more metals than those without EPS. Adsorption accounted for 79.62% of the total Cd accumulation in the low-Cd treatment, whereas absorption dominated the Pb accumulation at low Pb concentrations. The distributions of Cd and Pb in and out of the microalgal cells were reversed when the concentrations of the two metals increased. The detoxification strategies of C. reinhardtii for Cd and Pb were completely different, and these findings may assist in the phycoremediation of metal pollution in aquatic environments.

Sorption and transformation of arsenic by extracellular polymeric substances extracted from Synechocystis sp. PCC6803.

Cyanobacteria widely distribute in the aqueous ecosystem and produce abundant extracellular polymeric substances (EPS), yet little is known about how the quantity and composition of cyanobacterial EPS change upon As exposure, and what are functions of these complex biopolymers in the As sorption and transformation processes. Here we extracted the EPS from Synechocystis sp. PCC6803, characterized their properties, quantified their components upon exposure to arsenite (As(III))/arsenate (As(V)) treatments, and investigated As binding and speciation as affected by the levels of EPS and solution pH. The total binding sites, zeta potential and reducing power of EPS were 17.47 mmol g-1, -19.72 mV and 1.71. The amounts of EPS increased by 22-65.3% and 13.8-39% when the cells were treated with 10-500 µM As(III) and As(V) respectively. The As removal was influenced by the EPS doses and solution pH, with 52.8% at pH 8.5 for As(III) and 49.5% at pH 4.5 for As(V) at 300 mg L-1 EPS. In addition, As speciation was transformed with the addition of EPS. As(V) and As(III) respectively accounted for 4.9-20.3% and 6.5-26.7% of the total dissolved As after the EPS were added (100-300 mg L-1) to the As(III) and As(V) solutions. Fourier transform infrared spectroscopy (FTIR) and three-dimensional excitation-emission fluorescence spectra (3D-EEM) revealed that As was bound to functional groups such as C═O, ─NH, and ─OH in the EPS via surface complexation/hydrophobic interactions. Taken together, this study demonstrated that the EPS extracted from Synechocystis were capable to bind and transform As and could be potentially applied to remove or detoxify As in solutions.

A phase 3 study of tezacaftor in combination with ivacaftor in children aged 6 through 11 years with cystic fibrosis.

BACKGROUND: Tezacaftor/ivacaftor is a new treatment option in many regions for patients aged ≥12 years who are homozygous (F/F) or heterozygous for the F508del-CFTR mutation and a residual function (F/RF) mutation. This Phase 3, 2-part, open-label study evaluated the pharmacokinetics (PK), safety, tolerability, and efficacy of tezacaftor/ivacaftor in children aged 6 through 11 years with these mutations. METHODS: Part A informed weight-based tezacaftor/ivacaftor dosages for part B. The primary objective of part B was to evaluate the safety and tolerability of tezacaftor/ivacaftor through 24 weeks; the secondary objective was to evaluate efficacy based on changes from baseline in percentage predicted forced expiratory volume in 1 s (ppFEV1), growth parameters, sweat chloride, and the Cystic Fibrosis Questionnaire-Revised (CFQ-R) respiratory domain score. RESULTS: After PK analysis in part A, 70 children received ≥1 dose of tezacaftor/ivacaftor in part B; 67 children completed treatment. Exposures in children aged 6 through 11 years were within the target range for those observed in patients aged ≥12 years. The safety profile of tezacaftor/ivacaftor was generally similar to prior studies in patients aged ≥12 years. One child discontinued treatment for a serious adverse event of constipation. Tezacaftor/ivacaftor treatment improved sweat chloride levels and CFQ-R respiratory domain scores, mean ppFEV1 remained stable in the normal range, and growth parameters remained stable over 24 weeks. CONCLUSIONS: Tezacaftor/ivacaftor was generally safe and well tolerated, and improved CFTR function in children aged 6 through 11 years with CF with F/F and F/RF genotypes, supporting tezacaftor/ivacaftor use in this age group. NCT02953314.

Pharmacokinetic and Drug-Drug Interaction Profiles of the Combination of Tezacaftor/Ivacaftor.

Drug-drug interaction (DDI) studies are described for tezacaftor/ivacaftor, a new cystic fibrosis transmembrane conductance regulator modulator therapy for the treatment of cystic fibrosis. Three phase I DDI studies were conducted in healthy subjects to characterize the DDI profile of tezacaftor/ivacaftor with cytochrome P450 (CYP)3A substrates, CYP3A inhibitors, and a permeability glycoprotein (P-gp) substrate. The effects of steady-state tezacaftor/ivacaftor on the pharmacokinetics (PKs) of digoxin (a P-gp substrate), midazolam, and ethinyl estradiol/norethindrone (CYP3A substrates) were evaluated. Effects of strong (itraconazole) and moderate (ciprofloxacin) CYP3A inhibitors on tezacaftor/ivacaftor PKs were also determined. Tezacaftor/ivacaftor increased digoxin area under the curve (AUC) by 30% but did not affect midazolam, ethinyl estradiol, or norethindrone exposures. Itraconazole increased the AUC of tezacaftor 4-fold and ivacaftor 15.6-fold. Ciprofloxacin had no significant effect on tezacaftor or ivacaftor exposure. Coadministration of tezacaftor/ivacaftor may increase exposure of sensitive P-gp substrates. Tezacaftor/ivacaftor is unlikely to impact exposure of drugs metabolized by CYP3A, including hormonal contraceptives. Strong CYP3A inhibitors significantly increase the exposures of tezacaftor and ivacaftor.

Safety, pharmacokinetics, and pharmacodynamics of lumacaftor and ivacaftor combination therapy in children aged 2-5 years with cystic fibrosis homozygous for F508del-CFTR: an open-label phase 3 study.

BACKGROUND: The efficacy, safety, and tolerability of lumacaftor and ivacaftor are established in patients aged 6 years and older with cystic fibrosis, homozygous for the F508del-CFTR mutation. We assessed the safety, pharmacokinetics, pharmacodynamics, and efficacy of lumacaftor and ivacaftor in children aged 2-5 years. METHODS: In this multicentre, phase 3, open-label, two-part study, we enrolled children aged 2-5 years, weighing at least 8 kg at enrolment, with a confirmed diagnosis of cystic fibrosis who were homozygous for the F508del-CFTR mutation. Children received lumacaftor 100 mg and ivacaftor 125 mg (bodyweight <14 kg) or lumacaftor 150 mg and ivacaftor 188 mg (bodyweight ≥14 kg) orally every 12 h for 15 days in part A (to assess pharmacokinetics and safety) and for 24 weeks in part B (to assess safety, pharmacokinetics, pharmacodynamics, and efficacy). Children could participate in part A, part B, or both. Children were enrolled into part A at five sites in the USA and into part B at 20 sites in North America (USA, 17 sites; Canada, three sites). The primary endpoints of the study were the pharmacokinetics (part A) and safety (part B) of lumacaftor and ivacaftor; all analyses were done in children who received at least one dose of lumacaftor and ivacaftor. Secondary endpoints in part A were safety and pharmacokinetics of the metabolites of lumacaftor and ivacaftor, and in part B included pharmacokinetics in children who received at least one dose of lumacaftor and ivacaftor and absolute changes from baseline in sweat chloride concentration, growth parameters, and markers of pancreatic function. This study is registered with ClinicalTrials.gov, number NCT02797132. FINDINGS: The study was done from May 13, 2016, to Sept 8, 2017. 12 children enrolled in part A, 11 of whom completed the 15-day treatment period and enrolled in part B. 60 children enrolled in part B, 56 of whom completed the 24-week treatment period. Safety and pharmacokinetics were consistent with the well characterised safety profile of lumacaftor and ivacaftor. In part B, most children (59 [98%] of 60 children) had one or more treatment-emergent adverse events; most events were mild to moderate in severity. The most common adverse events were cough (38 [63%] of 60), vomiting (17 [28%]), pyrexia (17 [28%]), and rhinorrhoea (15 [25%]). Serious adverse events occurred in four children: infective pulmonary exacerbation of cystic fibrosis (n=2), gastroenteritis viral (n=1), and constipation (n=1). Three (5%) of 60 children discontinued treatment because of elevated serum aminotransferase concentrations. Mean sweat chloride concentrations decreased by 31·7 mmol/L, biomarkers of pancreatic function improved (fecal elastase-1 concentrations increased and serum immunoreactive trypsinogen concentrations decreased), and growth parameters increased at week 24. INTERPRETATION: Lumacaftor and ivacaftor were generally safe and well tolerated in children aged 2-5 years with cystic fibrosis for 24 weeks. Efficacy findings also suggest that early intervention with lumacaftor and ivacaftor has the potential to modify the course of disease. FUNDING: Vertex Pharmaceuticals Incorporated.

Clinical drug-drug interaction assessment of ivacaftor as a potential inhibitor of cytochrome P450 and P-glycoprotein.

Ivacaftor is approved in the USA for the treatment of cystic fibrosis (CF) in patients with a G551D-CFTR mutation or one of eight other CFTR mutations. A series of in vitro experiments conducted early in the development of ivacaftor indicated ivacaftor and metabolites may have the potential to inhibit cytochrome P450 (CYP) 2C8, CYP2C9, CYP3A, and CYP2D6, as well as P-glycoprotein (P-gp). Based on these results, a series of clinical drug-drug interaction (DDI) studies were conducted to evaluate the effect of ivacaftor on sensitive substrates of CYP2C8 (rosiglitazone), CYP3A (midazolam), CYP2D6 (desipramine), and P-gp (digoxin). In addition, a DDI study was conducted to evaluate the effect of ivacaftor on a combined oral contraceptive, as this is considered an important comedication in CF patients. The results indicate ivacaftor is a weak inhibitor of CYP3A and P-gp, but has no effect on CYP2C8 or CYP2D6. Ivacaftor caused non-clinically significant increases in ethinyl estradiol and norethisterone exposure. Based on these results, caution and appropriate monitoring are recommended when concomitant substrates of CYP2C9, CYP3A and/or P-gp are used during treatment with ivacaftor, particularly drugs with a narrow therapeutic index, such as warfarin.

Elevated sphingosine-1-phosphate promotes sickling and sickle cell disease progression.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.

PMA induces SnoN proteolysis and CD61 expression through an autocrine mechanism.

Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-ß family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APC(Cdh1) ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation.

Proteome-wide profiling of activated transcription factors with a concatenated tandem array of transcription factor response elements.

Transcription factors (TFs) are families of proteins that bind to specific DNA sequences, or TF response elements (TFREs), and function as regulators of many cellular processes. Because of the low abundance of TFs, direct quantitative measurement of TFs on a proteome scale remains a challenge. In this study, we report the development of an affinity reagent that permits identification of endogenous TFs at the proteome scale. The affinity reagent is composed of a synthetic DNA containing a concatenated tandem array of the consensus TFREs (catTFRE) for the majority of TF families. By using catTFRE to enrich TFs from cells, we were able to identify as many as 400 TFs from a single cell line and a total of 878 TFs from 11 cell types, covering more than 50% of the gene products that code for the DNA-binding TFs in the genome. We further demonstrated that catTFRE pull-downs could quantitatively measure proteome-wide changes in DNA binding activity of TFs in response to exogenous stimulation by using a label-free MS-based quantification approach. Applying catTFRE on the evaluation of drug effects, we described a panoramic view of TF activations and provided candidates for the elucidation of molecular mechanisms of drug actions. We anticipate that the catTFRE affinity strategy will find widespread applications in biomedical research.

Pharmacokinetic/pharmacodynamic modeling of the antiretroviral activity of the CCR5 antagonist Vicriviroc in treatment experienced HIV-infected subjects (ACTG protocol 5211).

OBJECTIVE: This substudy of AIDS Clinical Trials Group (ACTG) Protocol 5211 explored the relationship between antiretroviral effect and plasma concentrations of vicriviroc, an investigational CCR5 antagonist for HIV infection. METHODS: Eighty-six treatment-experienced subjects failing their current antiretroviral regimens were randomized to add vicriviroc 5, 10, or 15 mg once daily or placebo for 2 weeks. Beyond week 2, subjects were changed to optimized background antiretroviral treatment while continuing vicriviroc or placebo. Plasma samples collected at weeks 2 and 8 were assayed for vicriviroc concentrations and combined with vicriviroc concentration data from 110 seronegatives enrolled in 5 phase 1 studies. An inhibitory Emax model was used to assess pharmacokinetic (PK)/pharmacodynamic relationships and recursive partitioning was applied to determine the breakpoint in vicriviroc PK parameters associated with virologic suppression. RESULTS: A 2-compartment model was fitted to the drug concentration data. At week 2, a higher vicriviroc Cmin was associated with a greater mean drop in HIV RNA (viral load) and a higher percentage of subjects experiencing a >1 log10 copies/mL drop in viral load. In subjects with Cmin > 54 ng/mL, the mean viral load decrease was 1.35 log10 copies/mL vs. 0.76 log10 with Cmin < 54 ng/mL (P = 0.003, Student t test). At this Cmin breakpoint, 70% of subjects with the higher Cmin had a >1 log drop in HIV RNA, compared with 44% with a lower Cmin (P = 0.048, Fisher exact test). Similar results were seen with an area under the curve breakpoint of 1460 ng h/mL. At weeks 16 and 24, all vicriviroc-treated subjects experienced better viral load responses than placebo recipients, but there was no apparent relationship between PK and change in viral load among these vicriviroc-treated subjects. CONCLUSIONS: There was a positive correlation between vicriviroc Cmin, area under the curve, and viral load changes at week 2 in treatment-experienced HIV-infected subjects receiving no other new active antiretroviral drugs. This correlation did not persist beyond week 16, probably because treatment response at that point also depended on having other active drugs in the regimen.

DNA replication licensing control and rereplication prevention.

Eukaryotic DNA replication is tightly restricted to only once per cell cycle in order to maintain genome stability. Cells use multiple mechanisms to control the assembly of the prereplication complex (pre-RC), a process known as replication licensing. This review focuses on the regulation of replication licensing by posttranslational modifications of the licensing factors, including phosphorylation, ubiquitylation and acetylation. These modifications are critical in establishing the pre-RC complexes as well as preventing rereplication in each cell cycle. The relationship between rereplication and diseases, including cancer and virus infection, is discussed as well.

Calorie restriction effects on silencing and recombination at the yeast rDNA.

Aging research has developed rapidly over the past decade, identifying individual genes and molecular mechanisms of the aging process through the use of model organisms and high throughput technologies. Calorie restriction (CR) is the most widely researched environmental manipulation that extends lifespan. Activation of the NAD(+)-dependent protein deacetylase Sir2 (Silent Information Regulator 2) has been proposed to mediate the beneficial effects of CR in the budding yeast Saccharomyces cerevisiae, as well as other organisms. Here, we show that in contrast to previous reports, Sir2 is not stimulated by CR to strengthen silencing of multiple reporter genes in the rDNA of S. cerevisiae. CR does modestly reduce the frequency of rDNA recombination, although in a SIR2-independent manner. CR-mediated repression of rDNA recombination also does not correlate with the silencing of Pol II-transcribed noncoding RNAs derived from the rDNA intergenic spacer, suggesting that additional silencing-independent pathways function in lifespan regulation.

An increase in mitochondrial DNA promotes nuclear DNA replication in yeast.

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD+-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.

Linker histone H1 represses recombination at the ribosomal DNA locus in the budding yeast Saccharomyces cerevisiae.

Several epigenetic phenomena occur at ribosomal DNA loci in eukaryotic cells, including the silencing of Pol I and Pol II transcribed genes, silencing of replication origins and repression of recombination. In Saccharomyces cerevisiae, studies focusing on the silencing of Pol II transcription and genetic recombination at the ribosomal DNA locus (rDNA) have provided insight into the mechanisms through which chromatin and chromatin-associated factors regulate gene expression and chromosome stability. The core histones, H2A, H2B, H3 and H4, the fundamental building blocks of chromatin, have been shown to regulate silent chromatin at the rDNA; however, the function of the linker histone H1 has not been well characterized. Here, we show that S. cerevisiae histone H1 represses recombination at the rDNA without affecting Pol II gene silencing. The most highly studied repressor of recombination at the rDNA is the Silent information regulator protein Sir2. We find that cells lacking histone H1 do not exhibit a premature-ageing phenotype nor do they accumulate the rDNA recombination intermediates and products that are found in cells lacking Sir2. These results suggest that histone H1 represses recombination at the rDNA by a mechanism that is independent of the recombination pathways regulated by Sir2.