P = 0.0038) than in normal A1AT phenotype. We found that patients with abnormal A1AT phenotypes had significantly higher vasculitis activity (BVAS) as well as anti-PR3 antibodies concentration. We conclude that A1AT deficiency should be considered in all patients with GPA.The deficiency of alpha-1 protease inhibitor, or alpha-1-antitrypsin (A1AT), predisposes to chronic lung diseases and extrapulmonary pathology. Besides classical manifestations, such as pulmonary emphysema and liver disease, alpha-1-antitrypsin deficiency (A1ATD) is also known to be associated with granulomatosis with polyangiitis (GPA or Wegener's granulomatosis). The aim of our study was to evaluate the frequency of allelic isoforms of A1AT and their clinical significance among GPA patients. Detailed clinical information, including Birmingham Vasculitis Activity Score (BVAS), incidence of lung involvement, anti-proteinase 3 (PR3) antibodies concentrations, and other laboratory data were collected in 38 GPA patients. We also studied serum samples obtained from 46 healthy donors. In all collected samples A1AT phenotyping by isoelectrofocusing (IEF) and turbidimetric A1AT measurement were performed. Abnormal A1AT variants were found in 18.4% (7/38) of cases: 1 ZZ, 4 MZ, 2 MF, and only 1 MZ in control group (2%). The mean A1AT concentration in samples with atypical A1AT phenotypes was significantly lower ( Granulomatosis with polyangiitis  is a disease manifested by necrotizing granulomatous inflammation affecting predominantly small to medium vessels and associated with presence of antineutrophil cytoplasmic antibodies (ANCA) in blood. Upper respiratory tract, eyes, lungs, and kidneys are typical target organs; rarely skin, joints, and nervous system are also involved . This tyA1AT is an acute phase protein belonging to the serine proteases inhibitors family and is capable of inactivating many proteases including proteinase 3 that is recognized as the main autoantigenic target in GPA (PR3) . Alpha-1The most common and normally functioning A1AT allelic form is PiM, so healthy human phenotype is designated as PiMM. There are more than 100 genetic A1AT variants, among which PiZ and PiS are the most common and clinically significant. A1ATD becomes clinically manifested in individuals carrying mutation in both gene Pi alleles, especially in PiZZ variant, whereas in heterozygous state the defect is partly compensated by normal allele that is found in individuals with PiMZ and PiMS phenotypes . HeterozAccording to the statistical data of American Thoracic Society/European Respiratory Society (ATS/ERS), the frequency of Z-alleles in GPA patients in Europe varies from 9 to 17.6% . HoweverThe aim of our study was to evaluate the frequency of pathogenic A1AT alleles among Russian GPA patients and to find out whether A1AT phenotype influences vasculitis activity.Serum samples and clinical data were provided by Saint-Petersburg Clinical Rheumatology Hospital number 25. 38 sera were obtained from individuals, suffering from active anti-PR3-positive GPA. 46 samples of healthy blood donors were collected as a control group. To estimate the clinical significance of different A1AT phenotypic variants, we collected detailed clinical and laboratory data, including Birmingham Vasculitis Activity Score (BVAS), the incidence of lung involvement, and anti-PR3 antibodies concentrations, measured by enzyme-linked immunosorbent assay (ELISA) with a commercial kit .We determined A1AT phenotypes in all collected samples by IEF with immunoblotting with the use of horizontal electrophoresis system . PH gradient was created by adding narrow specter ampholytes pH 4.2–4.9 . The A1AT molecules focused within agarose gel were blotted onto nitrocellulose paper and selectively stained by horseradish peroxidase conjugated goat anti-A1AT antibodies . A1AT phenotypes were assessed by comparing A1AT migration patterns with control PiMM, PiMZ, and PiMS samples.Electrophoretic patterns of main A1AT isoforms migration by IEF are demonstrated in Besides phenotyping, quantitative A1AT measurements were performed with turbidimetric commercial kit .t-test or Mann-Whitney U test, depending on whether the distribution was Gaussian. All categorical variables were compared with exact Fisher test. Differences between the groups were considered to be significant at a P value of <0.05. Statistical analysis was performed using Graph Pad Prism 4.0 software.To estimate statistical significance we compared clinical and laboratory parameters of the groups, using unpaired We observed 38 GPA patients receiving inpatient treatment at specialized rheumatologic clinic. The group was heterogeneous by gender (16 men and 22 women) and by age (18–77 years old). All samples were anti-PR-3 ANCA-positive.N = 13) and interstitial fibrosis (N = 7). All patients had a different degree of vasculitis activity, which was evaluated with BVAS clinical scale. In GPA patients, we found 18.4% (7/38) pathologic A1AT variants. Following A1AT phenotypes were identified: 4 MZ, 1 ZZ, and 2 MF. Among 46 samples of healthy donor's sera 1 heterozygous PiMZ phenotype was found.Pulmonary involvement in vasculitis was found in 64.5% (20/31) of cases and was presented by cavitating infiltrates  and pathological (N = 7) A1AT phenotypes were 1840 mg/L ± 127.2 and 970.0 mg/L ± 167.6, respectively . A1AT concentrations in both groups are presented in To find out clinical significance of pathogenic A1AT allelic forms, we analyzed GPA patients depending on A1AT phenotype. Mean A1AT concentrations in groups with normal  in GPA patients with abnormal A1AT phenotypes .Concentrations of anti-PR3 antibodies were also significantly higher  in patients with abnormal A1AT phenotypes were also statistically higher than in patients with normal phenotypes: 35.70 mm/h ± 3.278 and 55.67 mm/h ± 2.031, respectively, N = 7) and only in 65% (20/31) patients with normal A1AT, although this difference was not statistically significant.We noted that pulmonary involvement was found in all GPA patients with abnormal A1AT phenotype (To analyze the incidence of abnormal phenotypic variants of A1AT among Russian GPA patients, we studied 38 serum samples from individuals suffering from this disease. We determined A1AT phenotype and measured its concentration in all serum samples. We found abnormal A1AT phenotypes in 18.4% of cases, over the number shown in other publications: 9–17.6% [We found higher BVAS activity, greater concentrations of anti-PR3 antibodies, and higher ESR in GPA patients carrying atypical alleles of A1AT. These data suggest that GPA is more severe in these individuals and more effective treatment in such cases may be considered.Our findings can be explained by the fact that A1AT modulates PR3 activity and therefore it is presumably an important protective factor in systemic vasculitis. Tissue inflammation induces PR3 expression by neutrophils; this is attended by oxidative burst and partial neutrophil degranulation. PR3 also stimulates IL-8 production by endotheliocytes and monocytes and promotes increase of eicosanoids level and release of leucotriene B4, launching aggressive necrotizing inflammatory cascade in GPA –18. It wThe interaction of A1AT and PR3 may have a considerable influence on severity of inflammatory process in GPA and can be impaired in case of abnormal A1AT phenotype.The case of successful A1AT replacement treatment of individual suffering from GPA and A1ATD has been reported. When this patient with A1ATD and GPA, resistant to standard therapy, was given replacement therapy with A1AT, she developed stable remission of GPA with regression of skin lesions and improvement of pulmonary parameters .The laboratory testing for pathological A1AT alleles is not usually ordered for patients with systemic vasculitides in routine clinical practice. Considering relatively high frequency of pathological A1AT phenotype carriage among GPA patients and its clinical significance we may conclude that the relevance of A1ATD is underestimated. During medical examination of every GPA patient, the possibility of A1AT deficiency should be considered.


Inflammation is believed to be a contributing factor to many chronic diseases. The influence of vitamin D deficiency on inflammation is being explored but studies have not demonstrated a causative effect.Low serum 25(OH)D is also found in healthy persons exposed to adequate sunlight. Despite increased vitamin D supplementation inflammatory diseases are increasing. The current method of determining vitamin D status may be at fault. The level of 25(OH)D does not always reflect the level of 1,25(OH)2D. Assessment of both metabolites often reveals elevated 1,25(OH)2D, indicating abnormal vitamin D endocrine function.This article reviews vitamin D's influence on the immune system, examines the myths regarding vitamin D photosynthesis, discusses ways to accurately assess vitamin D status, describes the risks of supplementation, explains the effect of persistent infection on vitamin D metabolism and presents a novel immunotherapy which provides evidence of an infection connection to inflammation.Some authorities now believe that low 25(OH)D is a consequence of chronic inflammation rather than the cause. Research points to a bacterial etiology pathogenesis for an inflammatory disease process which results in high 1,25(OH)2D and low 25(OH)D. Immunotherapy, directed at eradicating persistent intracellular pathogens, corrects dysregulated vitamin D metabolism and resolves inflammatory symptoms. Inflammation is involved in many chronic diseases and concern has been raised about the influence of vitamin D deficiency on inflammatory processes. When studies found an association between inflammatory diseases and low serum 25-hydroxyvitamin D (25(OH)D), further research found evidence of low vitamin D in a large segment of the general population. This led some authorities to declare a world-wide epidemic of vitamin D deficiency and to recommend vitamin D supplementation. Experts are debating the definition of vitamin D deficiency and the appropriate vitamin D doses, while further research is being done to determine if vitamin D supplementation has the intended effect.According to some current definitions of vitamin D deficiency, even healthy persons, exposed to adequate sunlight, are unable to acquire enough vitamin D without supplementation. Often reiterated causes of vitamin D deficiency can be disputed in the light of more current research. In the absence of definitive studies, authorities are questioning the wisdom of supplementing the general population with vitamin D. The definition of Vitamin D deficiency needs re-evaluation in view of the fact that low 25(OH)D is found in both healthy and sick individuals. Concerns about vitamin D deficiency merit a closer look at the current method of determining vitamin D status because the level of 25(OH)D does not always reflect the level of 1,25-dihydroxyvitamin-D 2D). Analysis of this active metabolite may reveal elevated 1,25(OH)2D) in the presence of low 25(OH)D and lead to a diagnosis of abnormal vitamin D endocrine system function.An infectious pathogenesis posits that intracellular bacteria disrupt the vitamin D regulated immune system, resulting in persistent infection and chronic inflammation. In the clinical setting, a novel immunotherapy is demonstrating the ability to resolve vitamin D metabolism dysfunction, restore immune function, and thus, eliminate infection and reduce inflammation. This review ponders the question, “Is low 25(OH)D a cause of, or a consequence of inflammation?” The answer is found in the evidence that adds persistent intracellular infection to the equation.3 is photosynthesized in the skin or when vitamin D2 or D3 is ingested. Vitamin D is transported to the liver where it is hydroxylated by an enzyme  to produce 25(OH)D [The sequential metabolic processes that convert biologically inactive, parental vitamin D into active metabolites begin when vitamin D 25(OH)D  Fig. 1)3 is photThe influence of 1,25(OH)2D on the immune system is one of its most important roles. 1,25(OH)2D regulates the immune system via the VDR which is present in most immune cell types, particularly in antigen-presenting cells (APCs) such as monocytes, macrophages and dendritic cells . 1,25(OHConcerns about vitamin D deficiency arose when studies showed patients with autoimmune diseases have lower levels of serum 25(OH)D and study subjects given vitamin D had lower rates of autoimmune diseases and fewer markers of inflammation , 13. HowDeficient: 0–40 ng/mlSufficient: 40–80 ng/mlHigh normal: 80–100 ng/mlThe Vitamin D Council definition :DeficienDeficiency ≤ 20 ng/mlInsufficiency = 20–29 ng/mlThe Endocrine Society definition :DeficienRisk/deficiency ≤12 ng/mlRisk/insufficiency = 12–20 ng/mlSufficient = 20 ng/mlThe Institute of Medicine definition :Risk/defIn 2006, the Merck Manual listed 25–40 ng/ml as the normal 25(OH)D range . RecentlIs low 25(OH)D among the general population an accurate assessment of vitamin D deficiency? Many reasons are cited for the current ‘epidemic’ of vitamin D ‘deficiency’ but closer examination reveals these beliefs are based on outdated or limited studies and can be challenged with more recent research.3 which is photosynthesized [3 formation, simulating the process with a fixed dose of UVB radiation and concluded that while racial pigmentation has a photo-protective effect, it does not prevent the generation of normal levels of active vitamin D metabolites. Persons with dark skin also compensate for low 25(OH)D by rapidly converting it to the active 1,25(OH)2D metabolite, thus allowing them to maintain adequate vitamin D status [Melanin pigmentation is only one factor that determines the amount of vitamin Dthesized , 21. Bogthesized  measuredthesized  investigD status . Skin piClothing is a barrier to vitamin D photosynthesis but this is an issue only for people who cover themselves from head to toe . It takeAlthough pollution can block some ultraviolet radiation, even in urban areas of high pollution 50 % of UV rays reach the ground . A signi3 [3 production in a 70-year-old compared to a 20-year-old, the skin has such a high capacity to make vitamin D3 that elders exposed to sunlight will produce an adequate amount of vitamin D3 to satisfy their vitamin D requirement [As the skin ages, there is a decline in the cutaneous levels of 7-dehydrocholesterol, resulting in a marked reduction of the skin’s capacity to produce vitamin D3 . Howeveruirement , 34.3. However, other researchers who conducted more recent studies refute this hypothesis. Kimlin et al. [A 1988 study by Webb et al.  is oftenn et al.  report, n et al.  states, n et al.  report t3, is a result of an inflammatory process.Acceptance of these vitamin D myths regarding photosynthesis prevents consideration of the alternate hypothesis—low serum 25(OH)D, despite adequate photosynthesis of vitamin DMany studies of healthy subjects have found levels of 25(OH)D that, by some vitamin D definitions, are declared deficient (hypovitaminosis-D) , 39. VitSince low 25(OH)D is found in both healthy persons and those with autoimmune or chronic inflammatory diseases, assessing vitamin D status with the measurement of an additional clinical marker may be helpful. It is asserted that low levels of 25(OH)D accurately reflect vitamin D status; however, measurement of 1,25(OH)2D often demonstrates a positive correlation of elevated 1,25(OH)2D to inflammatory diseases D indicates a current or potential disease state, then increasing 25(OH)D by supplementing with vitamin D should provide some symptom relief and/or protection. So far, there is scant evidence for this hypothesis , 57. Acc3 photosynthesized from sunlight [Decreases in vitamin D levels are a marker of deteriorating health. Ageing and inflammatory processes involved in disease occurrence and clinical course reduce vitamin D concentrations, which would explain why vitamin D deficiency is reported in a wide range of disorders. We postulate that inflammation is the common factor between most non-skeletal health disorders and low 25(OH)D concentrations. Inflammatory processes involved in disease occurrence and clinical course would reduce 25(OH)D, which would explain why low vitamin D status is reported in a wide range of disorders. However, increases in 25(OH)D have no effect on inflammatory processes or on disorders at the origin of these processes.Consequently, more vitamin D experts are beginning to reconsider vitamin D supplementation among the general population . Recommesunlight . Howeversunlight . A studysunlight  cast dousunlight  concludesunlight  advised A 2014 meta-analysis by Bolland et al.  on the eIf evidence indicates that most people get adequate vitamin D from sunlight exposure but healthy persons are found to be ‘deficient’ by recent standards, what is the explanation for this phenomenon? Vitamin D proponents use a disease deficiency model to explain low levels of 25(OH)D. Their hypothesis states low 25(OH)D causes chronic diseases; however, a pathogenesis has not been elucidated . Low serThe existence of bacteria which are capable of invading human cells has been known for over a century and are described by many authors , 71. TheDomingue  commenteCWD bacteria are considered communicable but not contagious; protective immunity depends on an effective cell-mediated immune response . It is nPathogens gain many advantages by parasitizing immune cells and altering nuclear receptor activity. Tissue invasion provides a privileged niche with access to host protein and iron, sequestration from immune response, and a means for persistence . In the It is theorized that bacteria have developed some of these strategies in order to invade host cells and remain undetected within cellular cytoplasm. Many bacterial pathogens form antibiotic-tolerant persister cells which can replicate within macrophages. In this form they can cause subclinical infection and have been associated with chronic diseases , 97, 98.17 cells and contribute to the development of chronic inflammatory conditions [Macrophage microbicidal mechanisms are responsible for the control and elimination of pathogens. 1,25(OH)2D production and action in macrophages activates toll-like receptors to increase expression of the AMP cathelicidin which kills infectious invaders , 102. Whnditions , 105. Annditions ; autoimmnditions , 108. ThIn an essay on the renin–angiotensin system (RAS) and immune response, Smith  postulatMycobacterium tuberculosis down-regulates VDR activity .Mycobacterium leprae inhibits VDR activity through down-regulation of CYP27B1 in monocytes .Aspergillus fumigatus secretes a toxin capable of down-regulating the VDR in macrophages .Epstein–Barr virus lowers VDR activity .HIV completely shuts down VDR activity .In VDR knockout mice, a circumstance that closely mimics extreme VDR dysregulation, 1,25(OH)2D levels increase by a factor of ten .Studies also point to immune system depression and elevated 1,25(OH)2D in chronic diseases :3 levels [Sarcoidosis patients are deficient in cathelicidin despite healthy vitamin D3 levels .1,25(OH)2D is high (>60 pg/ml) in 42 % of Crohn’s patients and the source of the active vitamin D may be the inflamed intestine .1,25(OH)2D is elevated in the synovial fluid of patients with RA (rheumatoid arthritis) .Crohn’s disease decreases expression of cathelicidin .High levels of 1,25(OH)2D may result when down-regulation of the VDR by bacterial ligands prevents the receptor from expressing enzymes necessary to keep 1,25(OH)2D in a normal range . ElevateNumerous examples can be found in which pathogens express antigens that cross-react with host antigens or induce local inflammatory responses that can lead to autoimmune responses through a very complex set of circumstances . The preVitamin D appears to have a positive effect on autoimmune disease due to immune system suppression , 135 andIn a healthy individual, the complex interplay between innate and adaptive immunity cooperates to mount an appropriate response to infection through regulation of the vitamin D endocrine system . The imm3, 25(OH)D and 1,25(OH)2D following sun exposure.Because extra-renal production of 1,25(OH)2D is primarily dependent on the availability of 25(OH)D , supplemInflammatory cytokines activate CYP27B1, an enzyme that causes more 25(OH)D to be converted to 1,25(OH)2D .The microbial-repressed VDR cannot transcribe CYP24A1 (formerly 24-hydroxylase), an enzyme that breaks down excess 1,25(OH)2D .3 to 25(OH)D so 25(OH)D is down-regulated [Excess 1,25(OH)2D binds the PXR (pregnane X receptor), to inhibit conversion of vitamin Degulated .1,25(OH)2D inhibits the hepatic synthesis of 25(OH)D .We hypothesize that when nucleated cells are parasitized by intracellular bacteria, extra-renal production of 1,25(OH)2D increases, the kidneys lose control of 1,25(OH)2D production, and pro-hormone 25(OH)D decreases due to rapid conversion to 1,25(OH)2D. The following mechanisms are thought to be responsible D may be a consequence of the inflammatory process. More studies are concluding that suboptimal circulating levels of vitamin D appear to be caused by the disease process. Waldronn et al.  found se3, vitamin D2 and 1,25(OH)2D, etc.). This measurement may not, however, provide enough information to assess vitamin D endocrine function. The clinical utility of measuring 1,25(OH)D is not fully understood, but it is clear that associations are being made between this active metabolite of vitamin D and disease states [1,25(OH)2D has a short half-life (hours) and fluctuates rapidly.However, a high result may be discovered even at trough level.1,25(OH)2D levels are regulated by PTH, calcium, phosphate.This is not true if extra-renal production is prevalent .1,25(OH)2D does not decrease until 25(OH)D is very low.A low 25(OH)D may be a sign that 1,25(OH)2D is abnormally high .1,25(OH)2D is only over-produced in hypercalcemic disease states such as sarcoidosis.Studies show this is not true .1,25(OH)2D may be elevated as a result of up-regulation of the CYP27B1 enzyme.This begs the question, Why is this enzyme elevated ?Assessing dysregulated vitamin D metabolism has the potential to guide clinical practice , 153. Vie states . 1,25(OHMeasuring both 25(OH)D and 1,25(OH)2D  as clinical markers in chronic disease is more likely to provide a true picture of vitamin D status, than measuring 25(OH)D alone , 156 (TaThe ability to mount an appropriate immune system response to intracellular infection is highly dependent on a competent VDR . When it®) was estimated to have a Ki value in the low nanomolar range, similar to the Ki values of the natural VDR ligands [transcribes CYP24A1 and CYP3A4 2D production) .represses CYP27B1 (the enzyme that hydroxylates 25(OH)D to 1,25(OH)2D) so less 1,25(OH)2D is produced .Angiotensin receptor blockers (ARBs) have been shown, via in silico molecular modeling, to modulate VDR activation . The mos ligands . Olmesar ligands . OlmesarImprovement of glycemic control and insulin resistance was only observed in the olmesartan group and these effects of olmesartan might be mediated by an anti-inflammatory action .Olmesartan treatment significantly reduced serum levels of inflammatory markers; h-CRP, h-TNFa, IL-6, MCP-1 after 6 weeks of therapy .Blocking angiotensin-converting enzyme induces potent regulatory T cells and modulates TH1- and TH17-mediated autoimmunity .Blocking angiotensin II receptor increases bone mass , 169.Consequently, renal control of 1,25(OH)2D production is restored and extra-renal production of 1,25(OH)2D is reduced. A decrease in elevated 1,25(OH)2D means less systemic inflammation, as these studies of olmesartan indicate:Olmesartan acts in a manner similar to 1,25(OH)2D to reduce inflammation and, by inference, improve immune system function. VDR and RAS receptors are distributed in almost the same tissues. The endogenous VDR ligand 1,25(OH)2D down-regulates the RAS by repressing renin gene expression to reduce inflammation via the nuclear factor-kappa B pathway . OlmesarIn patients with autoimmune disorders and inflammatory symptoms, olmesartan is noted to provoke an increase in inflammatory symptoms indicative of a Jarisch–Herxheimer reaction (JHR). JHR is a cascade of reactions including inflammation, cytokine release, and endotoxin release as part of the immune response to the disintegration of infected cells . This imHajishengallis and Lambris  concludeOlmesartan is being used as a novel VDR ligand in the clinical setting . ImmunotAccumulating case reports now support the observation that a number of complex, chronic conditions can be improved by restoring VDR function using this type of immunotherapy , 182. ItVitamin D is essential for many important biological processes and most people get an adequate supply from exposure to sunlight Table . Long-teHuman cells live in harmony with many types of microbes but some microbes may become pathogenic under commonly experienced conditions. The innate immune system is designed to kill pathogens via 1,25(OH)2D-mediated VDR transcription of anti-microbial peptides but microbes may use strategies which down-regulate the VDR in order to live and reproduce within nucleated host cells. Studies using more advanced cell culture and molecular techniques are confirming the presence of previously undetected intracellular bacteria. Defense mechanisms that intracellular bacteria use to persist and proliferate need to be investigated. Pathogen-induced VDR dysfunction which causes the release of pro-inflammatory cytokines appears to be at the root of chronic disease and low 25(OH)D. Improving VDR activation may be the key to reducing inflammatory diseases. Treatments that up-regulate the VDR to restore normal immune function, reduce inflammation and eradicate persistent bacterial infections require further research. An immunotherapy which has demonstrated efficacy in reversing vitamin D metabolism dysfunction and reducing inflammatory symptoms is currently being used by clinicians and warrants formal study.In summary, elevated 1,25(OH)2D, often accompanied by reduced 25(OH)D, is a clinical sign of dysregulated vitamin D metabolism and evidence that the immune system is competing with parasitic microbes for VDR dominance. Failure of the immune system to mount an effective anti-microbial response results in persistent intracellular infection. This induces relentless inflammation (immunopathology) which causes tissue damage and disease symptoms.


Cardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated.By Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15–30%.We identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain. These antibodies were preabsorbed by the GST affinity column, then affinity-purified using a CNBr-activated Sepharose 4B column (GE) conjugated to the recombinant. Hybrid peptide  [cMLCK(42-55/93-102)] was synthesized and coupled with KLH. Theses amino acid sequences in cMLCK were conserved among human, bovine, dog and rodents. This rabbit anti-cMLCK antibody was prepared and affinity-purified using Epoxy-activated Sepharose 6B column (GE).3VO4, 1 mM NaF, 10 mM Na4P2O7 and proteinase cocktail inhibitor ]. After centrifugation, equal volume of SDS sample buffer was added to the supernatant and heated . The samples were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The three kinds of anti-cMLCK, anti-smMLCK (Sigma), anti-ZIPK (Abcam) and anti-myosin phosphatase target subunit 2 (MYPT2)  greatly reduced the basal twitch tension. In the presence of ryanodine, phenylephrine induced an inotropic action in C57BL/6J and C57BL/6N  the relationship was positively correlated between the phenylephrine-induced inotropic response and MLC2v phosphorylation levels (within 15–30%).We found the followings: (i) a new isoform of cMLCK (cMLCK-2) with molecular mass of 61-kDa was identified in heart from various mouse strains. (ii) C57BL/6N expressed only cMLCK-2 while other mouse strains  expressed both cMLCK-2 and 86-kDa cMLCK. (iii) The MLC2v phosphorylation levels in papillary muscle were significantly lower in C57BL/6N than in C57BL/6J. (iv) The αcMLCK was identified as the third MLCK in the MLCK family, 7. The in vitro [The levels of MLC2v phosphorylation in papillary muscles were lower in C57BL/6N (31.8%) than in C57BL/6J (47.4%). It was found that smMLCK and ZIPK each phosphorylate MLC2v in vitro , 17. Thein vitro  and of sin vitro . In addiin vitro . Taken t1-adrenoceptor agonists increase MLC2 phosphorylation in electrically paced human atria [Twitch tension induced by electrical field stimulation in papillary muscle was smaller and the time required to the peak and the half relaxation were both longer in C57BL/6N than in C57BL/6J. Phenylephrine more strongly increased the twitch tension in C57BL/6J muscle than in C57BL/6N but it had no effect on MLC2v phosphorylation level in each mouse strain. Thus, we speculate that the positive inotropic response to phenylephrine depends on the levels of basal MLC2v phosphorylation: i.e., the level of MLC2v phosphorylation under the basal conditions is higher the stronger inotropic action to phenylephrine is provoked. The results are not consistent with the previous findings that αan atria –21. Howean atria –21. Thes2+ channel blocker and activator, respectively) each blocked phenylephrine-induced inotropic response in both C57BL/6J and C57BL/6N, suggesting that the fine tuning of L-type Ca2+ channels may be responsible, in part, for the phenylephrine-induced inotropic response. In the presence of ryanodine (an inhibitor of Ca2+-induced Ca2+ release in SR), phenylephrine still induced an inotropic response in both strains. α1-adrenoceptor activation was reported to be able to increase Ca2+ influx through phosphatidylinositol turnover [2+ release from SR, but to Ca2+ infux at least in part through L-type Ca2+ channel. In a case of vascular strip, noradrenaline was reported to be able to induce arterial contraction by activating voltage-dependent Ca2+ channel [Nifedipine and BAY K 8644 , indicating that cMLCK is dependent on Ca2+/calmodulin. The regulation of cMLCK in vivo remains to be determined.BAY K 8644 and ryanodine did not modify papillary muscle MLC2v phosphorylation in either strains, suggesting that Caoversial , 6. Our 2+ sensitivity of force [Calyculin A (an inhibitor of protein phosphatase type1 and 2A) increased MLC2v phosphorylation in cardiomyocytes . It was of force .To understand the physiological roles of MLC2v phosphorylation in this study, the difference in the levels of MLC2v phosphorylation between the two kinds of sample should be considered. The phosphorylation levels of MLC2v in prepared papillary muscle samples was lower than those in whole heart immediately isolated from sacrificed mice  and 6G. 1-adrenergic stimulation exacerbates cardiac dysfunction and decreases the expression of β1-adrenergic receptors, thus leading to heart failure [1-adrenoceptors remain stable or rather increase in heart failure [1-adrenoceptor agonists [1-adrenoceptors play an adaptive and protective role, depending on the levels of MLC2v phosphorylation. Thus, a new therapeutic strategy for heart failure could find out the way to increase and maintain MLC2v phosphorylation levels (over certain ranges).In heart failure, the level of MLC2 phosphorylation decreases , 30 and  failure , 32. On  failure , 34, thuagonists . The levagonists , 30, and1-adrenoceptor agonist in heart under physiological conditions.In summary, we identified the expression of a new isoform of cMLCK with a molecular mass of 61-kDa in mouse heart. In C57BL/6J strain both 86-kDa and 61-kDa cMLCKs were expressed but only 61-kDa cMLCK was detected in C57BL/6N strain. The inotropic response to phenylephrine was closely correlated with MLC2v phosphorylation levels (within the range of 15–30%). Thus, we believe that MLC2v phosphorylation within such ranges plays an important role to induce the inotropic response by an αHowever, whether the phenomenon observed in our models, namely the MLC2v phosphorylation level-dependent inotropic response, is specific for phenylephrine or common observed in other inotropic agents including β-adrenoceptor stimulation is remained to be investigated.S1 FigPhosphorylation and expression were shown by Western blot analysis.(PDF)Click here for additional data file.S2 FigCardiomyocytes were isolated from ventricles of 1-day-old Sprague—Dawley rat pups with the digestion of collagenase IV and trypsin. Following incubation for 24 h in serum-containing medium, the cardiomyocytes were incubated for 24 h in serum-free medium prior to stimulation with each agonist for 24 h. Phosphorylation levels were determined by glycerol-urea PAGE and Western blot analysis. n = 4–5.(PDF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


Felis catus) APOBEC3Z3 (A3Z3), the ortholog of human APOBEC3H, potently suppresses the infectivity of vif-defective feline immunodeficiency virus (FIV). Although a recent report has shown that domestic cat encodes 7 haplotypes (hap I to hap VII) of A3Z3, the relevance of A3Z3 polymorphism in domestic cats with FIV Vif has not yet been addressed. In this study, we demonstrated that these feline A3Z3 variants suppress vif-defective FIV infectivity. We also revealed that codon 65 of feline A3Z3 is a positively selected site and that A3Z3 hap V is subject to positive selection during evolution. It is particularly noteworthy that feline A3Z3 hap V is resistant to FIV Vif-mediated degradation and still inhibits vif-proficient viral infection. Moreover, the side chain size, but not the hydrophobicity, of the amino acid at position 65 determines the resistance to FIV Vif-mediated degradation. Furthermore, phylogenetic analyses have led to the inference that feline A3Z3 hap V emerged approximately 60,000 years ago. Taken together, these findings suggest that feline A3Z3 hap V may have been selected for escape from an ancestral FIV. This is the first evidence for an evolutionary “arms race” between the domestic cat and its cognate lentivirus.Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3  DNA cytosine deaminases can be incorporated into progeny virions and inhibit lentiviral replication. On the other hand, viral infectivity factor (Vif) of lentiviruses antagonizes A3-mediated antiviral activities by degrading A3 proteins. It is known that domestic cat  if its activity is not counteracted by the viral Vif protein. Here we investigate the ability of 7 naturally occurring variants of feline APOBEC3, APOBEC3Z3 (A3Z3), to inhibit FIV replication. Interestingly, one feline A3Z3 variant is dominant, restrictive, and naturally resistant to FIV Vif-mediated degradation. Phylogenetic analyses revealed that the ancestral change that generated this variant could have been caused by positive Darwinian selection, presumably due to an ancestral FIV infection. The experimental-phylogenetic investigation sheds light on the evolutionary history of the domestic cat, which was likely influenced by lentiviral infection. During the evolution of viruses and their hosts, cross-species transmission can lead to the emergence of a nascent lineage of viruses , 2. On tApobec3 (A3) gene, primates, including humans, have 7 distinct APOBEC3 (A3) genes designated A3A to A3H  with chronic AIDS-like disorders (Felis catus) encode multiple A3 proteins and that feline A3Z3, the ortholog of human A3H, potently impairs FIV infection through incorporation into the nascent virions , a lentivirus related to HIV-1, was first isolated in 1987 from domestic cats  expression plasmid (based on pcDNA3.1), which was used in our previous paper (vif-deficient infectious molecular clone (IMC) of FIV, a termination trinucleotide (TAA) was inserted into the vif ORF of pFIV-14  -tagged feline A3Z3 hap II to VII and a series of substitution mutants at position 65 were constructed by using a GeneArt site-directed mutagenesis system (Life Technologies). HA-tagged feline A3Z3 hap I   by usingetaluma) , 29, 30.nls lacZ gene [293T(LacZ) cells] , CRFK cells , and a HEK293T cell line transduced with the ) cells]  were culvif; a replication-incompetent vif-deficient FIV packaging construct derived from clone FIV-34TF10 [GenBank accession number M25381]) (800 ng) (5 cells) with or without each feline A3Z3 expression plasmid (800 ng) and/or Flag-tagged FIV Vif expression plasmid  (Transfection was performed by using Lipofectamine 2000 (Life Technologies) or PEI Max  in accordance with the procedures recommended by the manufacturers. To analyze the anti-FIV activity of feline A3Z3 see  and B, p(800 ng) , pTiger-,000 ng) . To dete,000 ng) , pVR1012,000 ng)  and pcDN,000 ng)  and C, pcis from FIV IMC  was cotransfected into CRFK cells (1.5 × 105 cells) with or without feline A3Z3 expression plasmid (100 ng). To analyze the activity of feline A3Z3 against feline leukemia virus (FeLV) and RD-114 virus (see 5 cells) with or without each feline A3Z3 expression plasmid . To analyze the activity of feline A3Z3 against a long interspersed nuclear element-1 (LINE-1) retrotransposition (see 5 cells). Twenty-four hours after transfection, the cells were treated with or without 10 μM MG132 (Sigma) for 16 h prior to harvesting. To analyze the sensitivity of feline A3Z3 mutants to FIV Vif-mediated degradation (see 4 cells) with or without Flag-tagged FIV Vif expression plasmid . In these assays, the cells and supernatants were harvested 2 days after transfection and were used for immunoblot analysis and their respective reporter assays as described below.To analyze the sensitivity of feline A3Z3 to Vif expressed in  IMC see , pFIV-14irus see  and B, pirus see  or pCRT1irus see  was cotrtion see , pL1-enhtion see  was cotrtion see . To analtion see , each fetion see  to D, feg for 1 h at 4°C using a TL-100 instrument (Beckman), and the pellet was lysed with 1× SDS buffer. For the transfected cells, the cells were lysed with radioimmunoprecipitation assay (RIPA) buffer  with protease inhibitor cocktail (Roche).SDS-PAGE/immunoblot analysis was performed as previously described , 30 by uhttp://imagej.nih.gov/ij/), and the following formula was used: percent degradation = 100 − {[A3Z3-HA (+Vif)]/[TUBA (+Vif)]}/{[A3Z3-HA (−Vif)]/[TUBA (−Vif)]} × 100.To calculate the percentage of feline A3Z3 degradation  to produce a virus solution. The infectivity of the virus solution was measured by luciferase assay. Briefly, 100 μl of the virus solution was inoculated into HEK293T cells in a 96-well plate (Nunc), and the firefly luciferase activity was measured by using a BrillianStar-LT assay system (Toyo-b-net) and a 2030 ARVO X multilabel counter instrument (PerkinElmer) according to the manufacturers' procedures.The culture supernatant harvested at 2 days posttransfection was centrifuged and then filtered through a 0.45-μm-pore-size filter (Millipore) to produce virus solution. The infectivity of virus solution was measured by LacZ marker rescue assay as previously described . Brieflyvif-deleted virus was prepared by cotransfection of pFP93, pMD.G, and pTiger-luc with or without feline A3Z3 expression plasmid into HEK293T cells. The culture supernatant at 2 days after transfection was centrifuged and then filtered through a 0.45-μm-pore-size filter (Millipore) to produce virus solution. The virus solution was inoculated into HEK293T cells (0.75 × 105), and the cells were washed with phosphate-buffered saline and harvested at 2 days after infection. DNA was extracted from the cells by the use of a QIAamp DNA blood minikit (Qiagen). To remove the contaminated plasmid DNA, the extracted cellular DNA was treated with DpnI (New England BioLabs). The partial luciferase gene was amplified using PrimeSTAR GXL DNA polymerase (TaKaRa) with the primers listed in http://www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html), and the results were summarized   structure model of feline A3Z3 hap I was constructed using the SWISS-MODEL server –45 on th–3VOW) . The ene3VOW) . The sol3VOW) –50.dN/dS ratio) to vary among both sites and branches, which enabled us to infer positive selection along a particular lineage or clade (prespecified as foreground branches)  . In thisranches)  was alsohttp://tree.bio.ed.ac.uk/software/tracer), and only parameter estimates with an effective sampling size of >200 were accepted for all runs. The maximum clade credibility tree was generated by using TreeAnnotator in the BEAST package after a 10% burn-in, and the final tree was displayed using FigTree v.1.4.2 (http://tree.bio.ed.ac.uk/software/figtree).To infer the date of emergence of feline A3Z3 hap V, a Bayesian Markov chain Monte Carlo (MCMC) method, BEAST v 1.7.5 , was empt test. Statistically significant correlation was determined by analysis using the Spearman rank correlation coefficient (rs).Data were expressed as averages with SD or standard errors of the means (SEM), and significant differences were determined by Student's vif has been submitted to DDBJ (accession number LC079040).The sequence of FIV (strain Shizuoka) A3Z3 haplotypes  at codon 65 was greater than 1 with posterior probability greater than 0.95 . Codon 65 was detected to be maintained under conditions of positive selection by using the random-effect-likelihood analysis as well, with a Bayes factor greater than 50 . These findings indicate that feline A3Z3 had evolved under conditions of positive selection and, particularly, that codon 65 had been a positively selected site during the evolution.Here we designate the reported 7 feline plotypes  hap I toplotypes . Four noplotypes . In ordehan 0.95  . As shown in To elucidate the relationship of feline A3Z3 polymorphism with FIV infection, we investigated the anti-FIV activity of the 7 A3Z3 haplotypes. In the absence of FIV Vif, the expression levels of these 7 feline A3Z3 haplotypes were similar, and the incorporation efficacies of these A3Z3 proteins into FIV particles were comparable . In addignitudes . We thengnitudes , C36 , a feline endogenous retrovirus (RD-114 virus), and a non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element-1 (LINE-1). As shown in To address whether the resistance of feline A3Z3 hap V to FIV Vif-mediated degradation is related to proteasome-dependent protein degradation, the cells expressing feline A3Z3 were treated with MG132, a proteasome inhibitor. As shown in P = 0.038; rs = −0.79). However, there was no correlation between the hydrophobicity and the degradation efficacy  that included domestic cat diverged around 131,000 years ago  that included domestic cat split from other wildcat lineages around 131,000 years ago  interval  infection in mouse (Mus musculus). Previous studies have revealed that the levels of FrMLV pathogenicity are different among mouse strains (A3 gene, of which the feline A3Z3 gene and the human A3H gene are orthologs (A3 gene of the genus Mus (A3 gene is influenced by its genotype (A3 polymorphism and that the A3 polymorphism in the genus Mus might be the result of the evolutionary pressure during the evolution of the genus Mus (A3Z3 polymorphism, the prevalence of FIV and/or FeLV infection in the cats with genes encoding A3Z3 hap V appears to be lower than that in the cats with the other haplotypes (In terms of natural resistance to FIV, hap V may be beneficial for cats expressing this A3Z3 enzyme. However, its frequency is rather low compared to those of other haplotypes . Similar15–f humans , suggest strains  and are rthologs . Moreoveenus Mus , 73 and genotype . These fenus Mus . In the plotypes . Howeverplotypes . In contplotypes . Taken tIn summary, we demonstrated that feline A3Z3 hap V is maintained under conditions of positive selection and is resistant to FIV Vif-dependent degradation. Although the evolutionary arms race between primate A3 and PLV Vif has been rigorously investigated 11–, 15, 74,


In contrast, expression of embryo development genes is repressed by PRC1-catalytic core subunits AtBMI1 and AtRING1 in common with PRC2-catalytic enzymes CLF or SWINGER (SWN). This context-dependent role of CLF corresponds well with the change in H3K27me3 profiles, and is remarkably associated with differential co-occupancy of binding motifs of transcription factors (TFs), including MADS box and ABA-related factors. We propose that different combinations of PRC members distinctively regulate different developmental programs, and their target specificity is modulated by specific TFs.Polycomb repressive complexes (PRCs) play crucial roles in transcriptional repression and developmental regulation in both plants and animals. In plants, depletion of different members of PRCs causes both overlapping and unique phenotypic defects. However, the underlying molecular mechanism determining the target specificity and functional diversity is not sufficiently characterized. Here, we quantitatively compared changes of tri-methylation at H3K27 in Arabidopsis mutants deprived of various key PRC components. We show that CURLY LEAF (CLF), a major catalytic subunit of PRC2, coordinates with different members of PRC1 in suppression of distinct plant developmental programs. We found that expression of flower development genes is repressed in seedlings preferentially  Polycomb group proteins (PcGs) are essential for development in both animals and plants. Studies in plants are advantageous for elucidation of specific effects of PcGs during development, since most PcG mutants are viable in plants but not in animals. Previous efforts in genetic study of plant PcGs revealed that different PcGs have both common and unique effects on plant development, but the mechanisms underlying the specific regulation of different developmental programs by PcGs are still far from clear. In this study, we quantitatively compared the change in H3K27me3 and gene expression profiles between mutants of key PcG members on a genome-wide scale in Arabidopsis seedlings, and successfully unraveled different developmental programs that are specifically regulated by different combinations of PcGs. This context specific effect of PcGs is closely associated with different sets of transcription factor binding motifs. Together, we revealed on a genome-wide scale that different combinations of PcGs, as well as their association with the binding sites of different TFs, serve to explain the specific regulation of different developmental programs by PcGs. The evolutionarily conserved Polycomb group proteins (PcGs) are the major epigenetic machinery regulating differentiation and development –4. PcGs clf swn show a much more severe phenotype than each of the single mutants [CLF, including early flowering and curly leaf [AtRING1 or AtBMI1, the core catalytic factors of PRC1, leads to depression of embryonic traits in seedlings, while only the atring1a atring1b double mutant displays severely fused flower phenotype [lhp1 displays some similar phenotypic defects to those of the PRC2 mutant clf[Study of the functional specificity of PcGs in both plant and animals is non-trivial. Firstly, the majority of PcG components are ubiquitously expressed, and do not have sequence-specific DNA recognition properties. Secondly, members of PcGs generally have functional redundancy and diversity, and it is difficult to distinguish the specific effect of individual members. Plants are advantageous for studying the effect of PcGs in development since most plant PcG mutants are viable, while animal development is generally vulnerable to PcG mutations. Previous efforts in genetic dissection of PcGs’ functions provide important clues as to the specialized functions of PcG members. For example, CURLY LEAF (CLF) and SWINGER (SWN) are two highly similar enzymatic subunits of the PRC2 complex , and the mutants . Howeverrly leaf . Similarhenotype ,13. In ahenotype ,14,15 cahenotype ,13,16. Hutant clf, and morutant clf. Moreoveutant clf,18,19. Ie.g. specific recruitment of PcGs by diverse strategies for transcription repression [lhp1 mutant. The global H3K27me3 pattern in the mutant was similar to that in wild-type Col-0 plant, leading to a conclusion that LHP1 is responsible for recognizing H3K27me3 and facilitating PRC1 binding but not for depositing H3K27me3 [lhp1[lhp1 being localized to some specific genomic regions, which had been missed in detection by genome-wide profiling with relatively low resolution. Therefore, for unraveling the locus selectivity and distinguishing the specific effects of different PcGs, combining high-resolution genome-scale data with quantitative analyses methods are indispensable.Multiple mechanisms had been proposed in Arabidopsis to explain the target specificity of PcGs, pression ,20–27 anpression ,29. HoweH3K27me3 . Howeverme3 [lhp1,23. ThisArabidopsis seedlings. We revealed that CLF collaborates with different PRC1 subunits to repress flower and embryo development. We further demonstrated that the target specificity of these different combinations of PcGs is closely associated with different sets of TF binding motifs, pointing to an active interplay between particular TFs and the specific activity of different PcGs.The next-generation sequencing technology has enabled the detection of epi-genome profiles with high sensitivity and specificity –32. We hin vitro[To dissect the composition of PRC2 complex in Arabidopsis, we used the FIE-3XFLAG fusion protein in immunoprecipitation experiments to identify associated proteins from leaf explants cultured in callus-induction medium. Following mass spectrometry analysis see , we idenin vitro,13,16, nclf-29, tfl2-2, atbmi1a,b, and atring1a,b (EMBRYONIC FLOWER1 (EMF1)[To compare roles of different PcGs, we used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to characterize the genome-wide profiles of H3K27me3 in Col-0, ring1a,b . In Col-ring1a,b , 84% of ring1a,b . The topring1a,b . Notablyring1a,b . To compring1a,b  panel I,ring1a,b . Next, tR1 (EMF1), a plantR1 (EMF1). These rclf-29, we identified substantially more regions with reduced H3K27me3 modification than with increased H3K27me3 marks  of which overlapped with H3K27me3 peaks in Col-0, while those non-overlapping regions also tend to be marked by H3K27me3 in Col-0, which are below peak detection cutoff, as shown by the read intensity distribution plot  showing concerted reduction of H3K27me3 in both mutants. On the other hand, loss of AtBMI1A and AtBMI1B or AtRING1A and AtRING1B specifically reduced H3K27me3 level in 566 regions (peak set II), which represents PRC2 target sites affected by AtBMI1 and AtRING1 directly or indirectly, but is independent of LHP1. Thus, the dependence of H3K27me3 on LHP1 seems tightly correlated with the specific effect of CLF. In addition, 741 regions belonging to class III show increased H3K27me3 levels.To dissect the cooperation of these PcGs on H3K27me3 modification, we collected 3,289 H3K27me3 regions regulated by at least one PcG component, and clustered the M values of H3K27me3 change in these regions to 3 clusters . Althougin vitro,13,16, tCLF or LHP1 lead to apparent reduction of H3K27me3 in surrounding regions, but for most cases the signal at the summit is only slightly reduced , ABI4, ABF1, SPL and MYB . The firNext, we identified motifs over-represented in peak sets I and II . Of notehttp://www.ncbi.nlm.nih.gov/geo/). By querying against these processed binding sites, we found that the binding regions of some MADS box TFs are significantly enriched in peak set I but not in other H3K27me3 regions , is regula patens. InteresHP1 Figs , which patbmi1a,b,13.Our finding that PRC2 composition regulates differential transcriptional programs is analogous to a recent genome-wide study about human blood cell development . It was SEP3 repression via H3K27me3 [Target selection by PcGs in different developmental stages is critical for correct developmental regulation. Specific recruitment of PcGs by particular factors ,20–23, PH3K27me3  show gooMADS box TFs play a central role in flower development, and their family size increased explosively with the origin of angiosperm . We founn of plant telomeres [It is intriguing that telo-box, a widespread short motif identical to the repeat (AAACCCT)elomeres , is specelomeres , and wheelomeres . And a relomeres . It is pThe chromo-domain protein Heterochromatin protein 1 (HP1) and Polycomb (Pc) in animals bind H3K9me3 and H3K27me3, respectively –68. DespNotably, LHP1 has no effect on genes jointly controlled by both PRC1 and PRC2 (targets of peak set II) despite the coincident binding of LHP1 and H3K27me3 in these regions. If the requirement of chromo-domain protein for H3K27me3 maintenance is a widespread mechanism, then there may be other chromo-domain proteins functional for spreading of H3K27me3 in these H3K27me3 regions. Alternatively, PcGs employ multiple strategies for H3K27me3 maintenance at different loci, either using LHP1 or cooperating with core subunits of PRC1 to create compacted chromatin structures . There aWe constructed pFIE: FLAG-FIE transgenic plants [swn-21[lhp1-6 (SALK_011762)[tfl2-2 (CS3797)[atring1a,b[atbmi1a,b[clf-29swn-21 were grown in soil under long day (16 hour) photoperiods at 22°C in green house, and seedlings were harvested after 2 weeks. clf-29swn-21 was grown on half-strength Murashige and Skoog (MS) medium in the same long day conditions as above, and the whole plants were harvested after 2 weeks. Harvested materials were frozen in liquid nitrogen for total RNA isolation or directly vacuum-infiltrated with formaldehyde crosslinking solution for ChIP assay. Quantitative real-time PCR analysis of ChIPed DNA with specific primers (_N521003), swn-21[3)[swn-21, lhp1-6 K_011762), tfl2-2  (CS3797), atring1tring1a,b and atbmatbmi1a,b,16 in Co primers  was perfclf-29 and atbmi1a,b, or in independent lines of LHP1 mutants lhp1-6 and tfl2-2. Biological replicates of ring1a,b RNA-seq were performed. ChIP assay was performed with the antibody against H3 trimethyl-Lys 27  as previously described . Samples without antibody were used as a negative control. More than 10 ng ChIP DNA or 2 μg total RNA from each sample was used for Illumina library generation following the manufacturer’s instructions . Library construction and deep sequencing were performed by Genergy Biotechnology Co. Ltd.  using Illumina HiSeq 2000 following the manufacturer’s instructions (Illumina). Raw data comprise 50 bp of single-end sequences for ChIP-seq and 100 bp of pair-end sequences for RNA-seq.Both ChIP-seq and RNA-seq experiments were performed in biological duplicates for Arabidopsis thaliana genome (TAIR10) using BWA 0.7.5a-r405 [http://bioinfo.sibs.ac.cn/gb2/gbrowse/tair10/. The genotype for each sample was validated by RNA-seq data  and irregular GC content, cutting sequencing adaptor followed by filtering short reads. As a result, 12–36 million reads with MAPQ>20 were obtained for further analysis  to transcription end site (TES)), The gene annotation file was downloaded from the TAIR homepage (http://www.arabidopsis.org). Published data sets including FIE ChIP-seq and LHP1 ChIP-chip data were downloaded from Gene Expression Omnibus (GEO) http://www.ncbi.nlm.nih.gov/geo/ with accession numbers GSE48857 [MACS1.4  was usedGSE48857  and GSE8GSE48857 , respectGSE48857 , and ChIGSE48857 . For IGVGSE48857 , we normtfl2-2. Firstly, we shuffled LHP1 binding sites for 1,000 times, and obtained 1,000 random genomic regions with the same length distribution of LHP1 binding sites. Secondly, we calculated and recorded the numbers of these random sites overlapping with LHP1 binding. Since none of the 1000 overlapping numbers exceeds the actual overlap number between LHP1 and regions with reduced H3K27me3 in tfl2-2., the P value is thus lower than 1e-3.Permutation test was used to test the significance of peak overlap between LHP1 binding sites and regions with reduced H3K27me3 in clf-29, lhp1-6, tfl2-2, atring1a,b and atbmi1a,b) were collected, resulting in 2,438 genes, which were further classified to 3 groups via k-means clustering. The clf-29swn-21 double mutant has effect on much larger range of genes, and only the expression change profile for 2,438 genes are shown here. Transcriptomic data for different tissues were downloaded from GENEVESTIGATOR [Differentially expressed genes were detected by DESeq , based oSTIGATOR . Gene Se2 (fold-change) of read intensity between Col-0 and PcG mutants  of 269 motifs identified from both mammals and plants collected by JASPAR database , and perhttp://www.ncbi.nlm.nih.gov/geo/) and processed the data to peak lists as described above. Next, we calculated the enrichment of the overlap between peak set I or peak set II with each of these peak lists using Fisher’s exact test, and the top enriched TF bindings are shown in To identify TFs whose binding enriched in different peak sets, we collected ChIP-seq data sets from 695 publicly available studies (till May 2015) from Gene Expression Omnibus (GEO http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE67322. Tracks for all sequencing data and related public data can be visualized through our local genome browser: http://bioinfo.sibs.ac.cn/gb2/gbrowse/tair10/The ChIP-seq and RNA-seq data were deposited in Gene Expression Omnibus (GEO S1 Text(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Figclf-29 (B) tlf2-2 (C) atring1a,b and (D) atbmi1a,b. Each dot represents a peak. X-axis is the A value, which represents the average intensity. Y-axis is the M value, which represents the difference of the intensity. The color range represents -log10 P value associated with normalized peaks. Here, positive M value indicates higher H3K27me3 level in PcG mutants as compared to that in Col-0, and negative M value represents lower H3K27me3 level in PcG mutants. The numbers of regions with elevated or depressed H3K27me3 levels in each mutant as compared to wild type are labeled based on combined criteria |M| > 1 and P value < 1e-3.(A-D) represent comparisons between Col-0 and (A) (PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Figclf-29swn-21 not depicted in (A) Heatmap includes another 1,290 genes whose expressions are only affected in (PDF)Click here for additional data file.S6 FigK-means clustering is used.(PDF)Click here for additional data file.S7 FigThe microarray expression data were downloaded from GENEVESTIGATOR. Tissues are partitioned to 8 tissue clusters as shown in (PDF)Click here for additional data file.S8 FigX-axis represents the distance of given motifs to peak summit (bp), y-axis represent the fraction of motifs located in a given position.(PDF)Click here for additional data file.S9 Fig(A) IGV screenshots showing examples of co-occupancy between H3K27me3 in seedlings and the bindings of MADS-box TFs in inflorescence. The positions of MADS box TF binding motif CArG-box are indicated by pink bars at bottom of the screen shots, and are highlighted by grey area. (B) Those peak set I targets also occupied by floral organ identity TFs show significant inflorescence biased expression.(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 Fig(PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.S13 Figclf-29 and tfl2-2 as revealed by ChIP-seq data. Top panel is qPCR result. Shown are mean ±s.d. For each loci, input, ChIPed, and negative control samples were repeated for 3 times. Y-axis represents % input = 2(Ctiput—CtIP) -2(Ctinput-Ctneg). CtIP: cycle threshold (Ct) value of samples immunoprecipitated using H3K27me3 antibody; Ctneg: Ct value of negative control, which is the samples immunoprecipitated with beads but without antibody; Ctinput: Ct value of input DNA without immunoprecipitation. Bottom panel shows read count of regions where ChIP-qPCR validations were performed. (C-D) ChIP-qPCR validations of regions with decreased (A) or increased (B) H3K27me3 in atring1a,b and atbmi1a,b.(A-B) ChIP-qPCR validations of regions with decreased (A) or increased (B) H3K27me3 in (PDF)Click here for additional data file.S14 FigCorrelation between M values of ChIP-seq replicates. Left panel: scatter plots in lower left triangle showed the correlation of M values between replicates, and numbers in upper right triangle showed Pearson correlation coefficients, with larger numbers having bigger font sizes; right panel: heatmap showing the correlation coefficients across samples. (A) Boxplots showing high correlation between replicated ChIP-seq data in terms of the M value distribution of peak set I and peak set II shown in (PDF)Click here for additional data file.S15 Fig(A) Distribution of H3K27me3 changes  for 108 genes and 164 genes in PcG mutants. (B) Distribution of expression changes for 108 genes and 164 genes in PcG mutants.(PDF)Click here for additional data file.S16 FigRegions in each PcG mutant with reduced H3K27me3 were identified, followed by motif enrichment analysis. The heatmap shows the enrichment P values in each mutant for motifs shown in (PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(A) ChIP-seq and RNA-seq read statistics for each sample. (B) 5,055 H3K27me3 peaks identified in Col-0, as well as list of peak target, which is defined as the nearest gene whose gene body is located within 1 kb upstream or 1 kb downstream of the peak region. (C) Biological functions enriched in Col-0 H3K27me3 peak targets, including GO terms of biological process (BP), molecular function (MF), cellular compartment (CC), and Interpro domains. (D) Motifs enriched in 5,055 Col-0 H3K27me3 peaks with P<1e-5, as well as the enrichment statistics of CArG box motifs.(XLSX)Click here for additional data file.S3 Table(A) Genomic coordinates, motifs and target genes information for 3,289 H3K27me3 regions regulated by at least one PcG component with cutoff: |M|>1 & P<1e-3. (B) 51% regions with reduced H3K27me3 in clf-29 show less reduction in summit regions. (C) 53% regions with reduced H3K27me3 in tfl2-2 show less reduction in summit regions.(XLSX)Click here for additional data file.S4 Table(A) 2,438 differentially expressed genes in PcG mutants with the following criteria: |FC|>1 and P <0.05 in at least one mutant. (B) Biological functions enriched in Col-0 H3K27me3 peak targets, including GO terms, Interpro domains, and SP_PIR keywords. (C) Biological functions enriched in Col-0 H3K27me3 peak targets, including GO terms, Interpro domains, and SP_PIR keywords.(XLSX)Click here for additional data file.S5 Table(A) 108 target genes whose expression and H3K27me3 are controlled by CLF and LHP1. (B) 164 target genes whose expression and H3K27me3 are controlled by RING1 and BMI1.(XLSX)Click here for additional data file.S6 Table(A) 108 genes ranked by their extent of inflorescence biased expression. Column B is the enrichment score of GSEA, and Column C represents whether the given gene has strong inflorescence biased expression as returned by leading edge analysis implemented in GSEA. (NOTE: low expressed genes across RNA-seq samples (read density in given gene <2) were removed before GSEA). (B) 164 genes ranked by their extent of embryo biased expression. Column B is the enrichment score of GSEA, and Column C represents whether the given gene has strong embryo biased expression as returned by leading edge analysis implemented in GSEA. (NOTE: low expressed genes across RNA-seq samples (read density in given gene <2) were removed before GSEA).(XLSX)Click here for additional data file.S7 TableThe enrichment statistics and co-occupied genomic regions are listed.(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.


We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met–heme coordination significantly compared to the monomer. HT cyt c552 forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c551 also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.Cytochrome  Th. Thc famc551 [c551 and thus leads to an increase in high order oligomers. However, formation of dimers was minimal and no trimer was detected by the treatment of M61A PA cyt c551 with ethanol  and Thr78–Ala83 (at loop 3), respectively. According to hydrogen exchange NMR measurements, the thermodynamic property of foldons is different between PA cyt c551 and horse cyt c [GHX) loses its secondary structure at the early stage of unfolding, showing that the region corresponding to the low energy foldon has low structural stability. The region containing loop 1 and helix 2 of PA cyt c551 is low in stability [c is low [c551 and horse cyt c correspond to the low stability regions of their monomers. Unfolding simulations have suggested that the hinge loop are ‘hot-spots’, around which proteins tend to locally unfold prior to complete unfolding [It has been suggested by molecular dynamics simulations that the native topology generally determines the domain-swapped structure . Althougeins Fig  and 5D; se cyt c ,24. A fotability , whereasnfolding . These rc551. The active site structure of the dimer was formed by the same amino acids as that of the monomer, but the heme axial ligands (His and Met) belonged to different protomers. By comparison of the domain-swapped structures of cyt c family proteins, we propose that the stability of the local structure may determine the position of the hinge loop in domain swapping, and thus the knowledge on protein folding may help to predict the structural features of domain swapping.In summary, we found that the region containing the N-teminal α-helix and heme was exchanged in the domain-swapped dimer of PA cyt S1 Figc551. (C and D) Elution curves of the solution (C) before and (D) after heating purified dimeric WT PA cyt c551 at 70°C for 10 min. Absorbances at 409 nm (red) and 280 nm (blue) were detected. Measurement conditions: column, Superdex 75 10/300 GL column; flow rate, 0.5 ml/min; buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, 4°C.(A) Elution curve after an addition up to 80% (v/v) ethanol, subsequent lyophilization, and resolvation with buffer. (B) Elution curve of monomeric WT PA cyt (TIF)Click here for additional data file.S2 Figc551 are depicted for the (A) 250–800 nm and (B) 600–800 nm regions. Measurement conditions: sample concentration, (A) 7.6 μM and (B) 41 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, room temperature.Optical absorption spectra of oxidized monomeric (red) and dimeric (blue) WT PA cyt (TIF)Click here for additional data file.S3 Figc551 are depicted. Concentration of each protein was calculated from the intensity of its Soret band. Measurement conditions were the same as those for CD spectra of oxidized monomeric (red) and dimeric (blue) WT PA cyt (TIF)Click here for additional data file.S4 FigFobs—Fcalc) superimposed on the Thr20–Met22 residues (hinge loop) of dimeric WT PA cyt c551 (pink and cyan) is depicted. The Thr20–Met22 residues were omitted from the calculations of the phases and structure factors . The electron density map is shown in green at a contour level of 2.5σ. The hemes and the Thr20–Met22 residues are depicted as stick models in dark and pale colors, respectively. The oxygen and nitrogen atoms of the Thr20–Met22 residues are shown in red and blue, respectively.The difference electron density map ((TIF)Click here for additional data file.S5 Figc551 are superimposed. The hemes, Cys12, Cys15, His16, and Met61 are depicted as stick models. The Thr20–Met22 residues (hinge loop) are shown in pale colors. The hemes and Thr20–Met22 residues (hinge loop) are depicted in dark and pale colors, respectively. The sulfur atoms of the heme axial Met ligand and heme-linked Cys are shown in yellow, and the nitrogen atoms of the heme axial His ligand are shown in blue.Structures of monomeric (gray) and dimeric (pink and cyan) WT PA cyt (TIF)Click here for additional data file.S6 Figc551 are depicted. (A) Hydrogen bonds of monomeric WT PA cyt c551: Cys15CO/Gly24NH, His16Nδ/Pro25CO, Ala17CO/Tyr27NH, and Ile18CO/Lys28NH (PDB ID: 351C). (B) Hydrogen bonds of dimeric WT PA cyt c551: Cys15ACO/Gly24BNH, His16ANδ/Pro25BCO, Ala17ACO/Tyr27BNH, Ile18ACO/Lys28BNH, and Asp19AOδ/Lys28BNζ (PDB ID: 3X39). The N-terminal region (Gly1–Met22) and the rest of the protein are shown in pink and gray, respectively. The hemes, Cys12, Cys15, His16, Met61, and residues involved in the hydrogen bonds are shown as stick models. The hydrogen bonds are shown as dotted yellow lines. The nitrogen and oxygen atoms involved in the hydrogen bonds are shown in blue and red, respectively. The N- and C-termini are labeled as N and C, respectively.Major hydrogen bonds (< 3.2 Ǻ between heavy atoms) between the N-terminal region and the rest of the protein in WT PA cyt (TIF)Click here for additional data file.S7 Figc551 are depicted. Measurement conditions: sample concentration, 10 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, room temperature.Spectra of oxidized monomeric WT (red) and M61A (green) PA cyt (TIF)Click here for additional data file.S8 Figc551. Absorbances at 409 (red) and 280 nm (blue) were detected. Measurement conditions were the same as those for (A) Elution curve after an addition up to 80% (v/v) ethanol, subsequent lyophilization, and resolvation with buffer. (B) Elution curve of monomeric M61A PA cyt (TIF)Click here for additional data file.S9 Figc551 are depicted. Measurement conditions: sample concentration, 100 μM (heme unit); scan rate, 1°C/min; buffer, 50 mM potassium phosphate buffer; pH, 7.0.Thermograms of oxidized monomeric (red) and dimeric (blue) WT PA cyt (TIF)Click here for additional data file.S10 Figc551 are depicted. Measurement conditions: sample concentration, 100 μM (heme unit); solvent, 50 mM potassium phosphate buffer containing 200 mM sodium chloride; pH, 7.0; temperature, room temperature; scan rate, 10 mV/s.Voltammograms of oxidized monomeric (red) and dimeric (blue) WT PA cyt (TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 TableRoot-mean-square deviation values for the Cα atoms of the N-terminal region and the rest of the protein (excluding the hinge loop) between the structures of the monomer and protomers of the dimer are calculated.(DOC)Click here for additional data file.


Cancer therapy that specifically targets malignant cells with minimal or no toxicity to normal tissue has been a long-standing goal of cancer research. Rad51 expression is elevated in a wide range of cancers and Rad51 promoter has been used to transcriptionally target tumor cells, however, a large size of Rad51 promoter limits its application for gene therapy. To identify novel tumor-specific promoters, we examined expression levels of Rad51 paralogs, Rad51B, Rad51C, and Rad51D as well as Rad52 in a panel of normal and tumor cell lines. We found that Rad51C is significantly overexpressed in cancer cells. The expression was up-regulated by approximately 6-fold at the mRNA level and 9-fold at the protein level. Interestingly, the 2064 bp long Rad51C promoter fragment was approximately 300-fold higher in cancer cells than in normal cells. A construct containing Rad51C promoter driving diphtheria toxin A efficiently killed several types of cancer cells with very mild effect to normal cells. These results underscore the potential of targeting the homologous recombination pathway in cancer cells and provide a proof of principle that the Rad51C promoter fragment can be used to transcriptionally target cancer cells. Among numerous gene therapy tools, use of a cancer specific promoter fused with a toxic gene offers unique opportunities for selective targeting of cancer cells. However, only a few promoters were identified with a cancer specific activity. The best-characterized promoter is hTERT, which ift model . Due to Rad51 is overexpressed in a wide variety of cancer cell types . ConsequHere, we explored the utility of the HR gene Rad51B, Rad51C, Rad51D and Rad52 for transcriptionally targeted therapy of cancer. We found that expression of Rad51C was significantly elevated in the group of cancer cells. Moreover, the expression of firefly luciferase or GFP fused pRad51C was approximately 300 times higher in cancer cells than in normal cells. We then engineered a prototype targeting construct containing a diphtheria toxin A driven by 2064 bp fragment of the Rad51C promoter (pRad51C-DTA). The pRad51C-DTA specifically targeted cancer cells while it had very mild effect on all normal cell lines.To identify cancer-specific promoters we first compared the expression levels of Rad51B, Rad51C, Rad51D and Rad52 in a panel of seven normal and seven cancerous cells lines. Normal cells included three lines of normal human fibroblasts HCA2, IMR90 and WI38, and four different lines of normal human mammary epithelial cells HMEC1, HMEC2, HMEC3 and HMEC4. The cancer cell lines included four of breast cancer cells HCC1954, MCF-7, T47D and MDA-MB-231, a fibrosarcoma cell line HT1080, a cervical cancer cell line HeLa and a transformed human kidney cell line GP2-293. We extracted total RNA from the exponentially growing cells, and measured transcript levels by real-time PCR Figure .MWUP=0.3176; Rad51D, MWUP=0.2086; Rad52, MWUP=0.1282; Rad51C, MWUP=0.037; Figure All four Rad genes showed a trend towards higher expression in cancer cells Figure . ExpressMWUP=0.0006)  Figure .MWUP=0.0006)   gene generating pRad51C-DTA Figure . DTA encThe second approach we used to measure the efficiency of cancer cell killing by pRad51C-DTA was to analyze inhibition of protein synthesis triggered by therapeutic construct. pRad51C-DTA and a plasmid encoding firefly luciferase were co-transfected into the panel of normal and cancer cells, and the level of luciferase activity was compared in the cells transfected with pRad51C-DTA to the control cells that received the luciferase construct only. Protein synthesis measured by the luciferase activity was reduced by ~25% to ~28% in the seven normal cell lines Figure . In contWe have shown that Rad51C transcript and protein levels are elevated in cancer cells. This is consistent with the previous findings that the levels of partner HR protein Rad51 , as wellHere we have shown that Rad51C promoter is highly active in cancer cells and the pRad51C-DTA construct can target cancer cells with high selectivity. Targeting cancer cells with a construct encoding DTA gene driven by Rad51 promoter had previously shown promise in cultured cancer cells and animal models , 4, 23. The activity of Rad51C promoter fused to a reporter gene was dramatically higher in cancer cells relative to the increase in the endogenous levels of Rad51C transcript. A possible explanation is that the 2 kb Rad51C promoter used in the fusion constructs is missing suppressive regulatory element(s). Alternatively, the endogenous Rad51C transcript may contain binding sites for micro RNAs that are missing in the constructs. Regardless of the cause, this phenomenon could be exploited for targeting cancer cells.In summary, our study provides evidence that HR pathway is a promising therapeitic target. We demonstrated that Rad51C promoter can be used to transcptionally target cancer cells and identified a 2 kb promoter fragment that provides strong differential expression in cancer cells. This work expands the list of cancer-specific promoters that hold promise for clinical applications.2 and 5% CO2 at 37°C in a humidified incubator . The growth conditions for the 14 cell lines were as described [All cells were cultured in 3% ORad51C promoter was amplified from genomic DNA isolated from HCA2 cells using primers 5'GCTGAATTCGCATAAGCATGAAATCTCCCT GAAGATAG3' and 5'ACCGGTACCCGCTGCATTTCAAAGCGGAA CGTCTTC3', and sub-cloned into a vector with EcoRI and KpnI. After the promoter sequence was confirmed, we further subcloned Rad51C promoter into pEGFP-N1 by replacing CMV promoter with ApaLI and KpnI, generating pRad51C-EGFP. Then the promoter region was amplified with primers 5'ATTGGTACCGCATAAGCATGAAATCT CCCTGAAGATAG3' and 5'CGCGCTAGCCGCTGCATTTCAAAG CGGAACGTCTTC3', and cloned into pGL3-basic vector to create pRad51C-firefly luciferase construct using KpnI and NotI.Based on pRad51C-EGFP, we obtained pRad51C-DTA by replacing EGFP ORF with DTA ORF amplified from pROSA26KPN using primers 5'GGCGGTACCGCCACCATGGATCCTG ATGATGTTGTTGATTCTTC3' and 5'GTCGCGGCCGCTTAGAGCTTTAAATCTCTG3'.Total RNA was extracted from cells that were seeded 48 h before harvesting using TriReagent . After cDNA synthesis, real-time PCR was performed on an ABI7500 real time PCR machine . The components and settings of quantitative PCR were determined according to the manual of SYBR Green PCR Master Mix . The primers for amplifying Rad51B, Rad51C, Rad51D, Rad52 and GAPDH were as follows: Rad51B, 5'CCCAAAGATGCAAACGGCTT3' and 5'TCGTCCAAAGCAGAAAGGGT3'; Rad51C, 5' GGATTTGGTGAGTTTCCCGC3' and 5' TCTTTGCTAAGCTCGGAGGG3'; Rad51D, 5' TCTGGCCAAATCTTCCCGAC3' and 5' TCCCAAACAACAGCACAGGT3'; Rad52, 5' CGTTTGCCACCAGAAACCAC3' and 5' TTCCTGTTGTGCGTTGGTCA3'; GAPDH, 5' TGGTATGACAACGAATTTGG3' and 5' TCTACATGGCAACTGTGAGG3'.Rapidly growing cells were harvested for protein extraction. Thirty μg of each sample was analyzed by Western blot with Rad51C antibody .For examining the promoter activity, all cells were transfected using a Lonza 4D electroporation machine  with according programs: HCA2, IMR90 and WI38, DT130; HMEC1, HMEC2, HMEC3 and HMEC4, EL110; HT1080, FF113; GP2-293 and MCF-7, CM130; Hela, CN114; HCC1954 and T47D, FF150; MDA-MB-231, FF138. Cells were cultured for 72h and harvested for FACS analysis on a FACScalibur . Data was further analyzed using Flowjo software. For analyzing the survival rate and inhibitory effect on luciferase activity, cells were transfected using Fugene reagent .6 cells. Then 20 μl of cell lysates was mixed with 100 μl of luciferase substrate , and the mixture was immediately analyzed on a GloMax20/20 luminometer .At 72 h post transfections, cells were harvested and counted before being lysed with passive lysis buffer  at the ratio of 200 μL/1×10


P53 and MDM2 mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the “tetraploidy checkpoint”, p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much  Tetraploid cells contain twice the normal amount of DNA and are rare in most normal tissues. However, tetraploid cells are relatively common in cancer and are thought to contribute to tumor development, aneuploidy, and therapy resistance P21) or apoptosis  P53 is a tumor suppressor and important regulator of tetraploidy Tetraploid G1-arrest is typically characterized by depletion of G2/M proteins  and increased expression of G1 arrest proteins  in 4N cells p53 wild-type and p53-null HCT116 cells , resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (NEN Life Science Products). Antibodies to p21 (H-164), Cyclin A (H-432), Geminin (FL-309), Cdk2 (M2) and p53 (AB-6) were from Santa Cruz Biotechnology; antibodies to Cyclin B1 (V152), CDC2 (POH1), and Tyr-15 Phospho-CDC2 were from Cell Signaling; antibodies to MDM2 (2A10) were from Calbiochem. Antibody to Cyclin E (HE-2) was from BD Pharmingen. Primary antibodies were detected with goat anti-mouse (Pierce) or goat anti-rabbit (Life Technologies) secondary antibodies conjugated to horseradish peroxidase, using Clarity chemiluminescence (Bio-Rad).For cell cycle analysis, cells were harvested and fixed in 25% ethanol overnight. The cells were then stained with propidium iodide . Flow cytometry analysis was performed on Gallios flow cytometer (Beckman Coulter) and analyzed with CellQuest (Becton Dickinson) and FlowJo 8.7 . For each sample, 10,000 events were collected. For live cell sorting, cells were incubated with Hoechst 33342  for 90 min at 37°C and then harvested. Cell sorting was performed based on DNA content using a MoFlo cytometer equipped with a UV excitation laser. The sorted cells were plated at low density.Quantitative real-time PCR (qRT-PCR) was performed to measure mRNA levels for Cyclin A2, Cyclin B1, CDC2, P21, Bax, Noxa, PUMA, P53R2, Actin in cells that were untreated or treated with Nutlin, Cisplatin, or IR as indicated. The quantitative real-time PCR reaction was run in a 7300 Real Time PCR System  using SybrGreen PCR master mix,  following manufacturer's instructions. Thermocycling was done in a final volume of 20 µL containing 2 µL of cDNA and 400 nmol/L of primers (Primers are listed in Cell were incubated with Colcemid (50 ng/µL) (Sigma-Aldrich) for an hour at 37°C, harvested and washed with PBS and hypotonic solution (2 parts of 0.075 M KCl and 1 part of 0.7% NaCitrate). The cells were then fixed in fixative (3 part methanol and 1 part acetic acid) and spread onto a slide. The spreads were examined under a phase contrast microscope.Cells were harvested and washed with PBS. Then incubated with hypotonic solution (2 parts of 0.075 M KCl and 1 part of 0.7% NaCitrate) for 20mins at 37°C. The cells were then fixed in fixative (3 part methanol and 1 part acetic acid). The slides were prepared and aged at 55°C for 3 mins on a thermobrite. The slides were incubated with 0.05% Pepsin (Fisher #P53-100) for 16 mins at 37°C and then washed with PBS and dehydrated in 70%, 85% and 100% ethanol for 1 minute each at room temperature. Then the probe Vysis LSI TP53 SpectrumOrange/CEP 17 SpectrumGreen (Abbott Molecular Inc # 01N17-020) was added hybridize at 73°C for 5 min and 37 degree for 18 hours. Then the slides were washed with SSC buffer (Gibco #15557-044) with 0.5% NP-40 (Fisher #P53-100) for 2 mins at 73°C and counterstained with DAPI II (Abbott Molecular Inc # 30-804818). Slides were examined under fluorescent microscope.Cells were plated 24 hours before being untreated or treated with CP, IR, or Nutlin in appropriate dilutions to form 50–100 colonies. After 2–3 weeks colonies were fixed with formaldehyde and stained with 0.05% crystal violet. The colonies were counted and normalized with the plating efficiency of untreated cells.We wished to ask if transient p53 activation by Nutlin could promote tetraploid cell formation from diploid precursors and, if yes, whether the resulting tetraploid cells would have increased resistance to therapy-induced apoptosis. HCT116 is a human colon cancer cell line that expresses wild-type p53. In our previous study, HCT116 were plated at single cell density and ten individual diploid clones were isolated (clones designated D1–D10) that varied in their relative sensitivity to cisplatin (CP)-induced apoptosis We previously showed 4N arrested cells can reinitiate DNA synthesis after Nutlin removal and replicate their DNA without an intervening mitotic division, a process known as endoreduplication P53 and chromosome 17-specific probes. This FISH analysis showed tetraploid clones have 4 copies of chromosome 17 and P53, while diploid D3 and D8 cells have 2 copies of chromosome 17 and P53 (P21), DNA repair genes (P53R2), and apoptotic genes , were also induced comparably in the CP and IR-treated diploid and tetraploid clones -induced apoptosis. To this end, D3 and D8 were Nutlin treated for 24 hrs followed by Nutlin removal for an additional 16 hrs. The cells were then labeled with the live-cell DNA stain Hoechst 33342. 2N and 8N cells were isolated by flow cytometry and replated at low density for isolation of individual clones . A total and P53 . Finally and P53  and lowe and P53 . These r and P53 , and thad clones . These rP53 gene  may depend on the P53 copy number, and that tetraploid cells may therefore express higher p53 levels in response to Nutlin and be hyper-sensitive to Nutlin-mediated growth inhibition. To test this, diploid D3 and D81B and their tetraploid counterparts T3 and TD6 were Nutlin treated for 24 hrs. P53 and MDM2 protein levels were determined by immunoblot and mRNA levels determined by qRT-PCR. The results showed that the tetraploid clones express elevated (∼2X more) p53 and MDM2 mRNA and protein than diploid clones, both basally , and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays a role in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).Tetraploid (4N) cells contain twice the DNA content of diploid (2N) cells. Tetraploid cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy 2 or M phase) cells must first assume a G1-like state from which they can enter S phase, referred to as tetraploid G1. Typically, tetraploid G1 is characterized by depletion/loss of G2/M marker proteins  and increased expression of G1 phase markers in 4N cells In order for endoreduplication to occur, 4N , CDC6 and Cdt1, and the replicative helicase MCM2-7 P53 gene copies, we considered tetraploid clones might express more p53 protein than diploid cells after IR or CP treatment and therefore be more resistant. However, we found p53 was induced to a comparable level in IR and CP-treated diploid and tetraploid cells. Thus, the increased IR and CP-resistance in tetraploid cells in our study does not appear to be associated with increased p53 levels or activity.Tetraploid clones that arose after Nutlin treatment were resistant to IR and CP compared to the diploid cells from which they came. Given that the Nutlin effects are p53-dependent, the results demonstrate that transient p53 activation can promote formation of therapy resistant tetraploid cells from diploid precursors. The basis for IR/CP-resistance in the tetraploid cells is unclear. Castedo et al. compared radiation and chemosensitivity of diploid RKO and HCT116 cells with tetraploid clones that arose after prolonged nocodazole treatment P53 gene copies (by FISH), expressed approximately twice as much P53 and MDM2 mRNA as diploid cells, and expressed approximately twice as many p53-MDM2 protein complexes (by p53 and MDM2 co-immunoprecipitation). We speculated that if tetraploid cells contain more p53-MDM2 complexes than diploid cells, they might be hypersensitive to MDM2 antagonists that disrupt these complexes. Indeed, we found p53 (and p21) were induced to a higher level in Nutlin-treated tetraploid cells vs Nutlin-treated diploid cells, and the tetraploid cells were more sensitive than diploid cells to Nutlin in apoptosis, colony formation, and cell cycle arrest assays. In sum, the results suggest therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive than diploid cells to anti-cancer MDM2 antagonists (e.g. Nutlin).Targeting tetraploid cancer cells is an important goal given the potential involvement of these cells in tumor progression, therapy resistance, and aneuploidy. The tetraploid clones that arose after Nutlin treatment in the current study contained twice as many P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).Transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells. Therapy-resistant tetraploid cells, by virtue of having higher Table S1Primers used for gene expression analysis.(DOC)Click here for additional data file.