Drosophila melanogaster to screen for the targets of this ubiquitin ligase under conditions of both decreased (as in AS) or increased (as in dup(15)) levels of the fly Dube3a or human UBE3A proteins. Using liquid phase isoelectric focusing of proteins from whole fly head extracts we identified a total of 50 proteins that show changes in protein, and in some cases transcriptional levels, when Dube3a fluctuates. We analyzed head extracts from cytoplasmic, nuclear and membrane fractions for Dube3a regulated proteins. Our results indicate that Dube3a is involved in the regulation of cellular functions related to ATP synthesis/metabolism, actin cytoskeletal integrity, both catabolism and carbohydrate metabolism as well as nervous system development and function. Sixty-two percent of the proteins were >50% identical to homologous human proteins and 8 have previously be shown to be ubiquitinated in the fly nervous system. Eight proteins may be regulated by Dube3a at the transcript level through the transcriptional co-activation function of Dube3a. We investigated one autism-associated protein, ATPα, and found that it can be ubiquitinated in a Dube3a dependent manner. We also found that Dube3a mutants have significantly less filamentous actin than wild type larvae consistent with the identification of actin targets regulated by Dube3a. The identification of UBE3A targets is the first step in unraveling the molecular etiology of AS and duplication 15q autism.The molecular defects associated with Angelman syndrome (AS) and 15q duplication autism are directly correlated to expression levels of the E3 ubiquitin ligase protein UBE3A. Here we used  UBE3A transcriptional regulation and imprinting UBE3A gene is primarily responsible for the autism phenotype in individuals with 15q duplication is the fact that paternal duplications, where the UBE3A gene is silent on the duplicated allele, can often be non-pathogenic or do not involve a clear ASD phenotype UBE3A gene is sufficient to produce autism-like behaviors in a mouse model of 15q duplication syndrome lending additional support to the hypothesis that elevation of UBE3A levels in the brain is the primary cause of autism in duplication 15q syndrome Angelman syndrome (AS) is a rare and severely debilitating neurological disorder with an incidence of ∼1/10–20,000 children Ube3a deficient mice show defects specifically in experience dependent plasticity Ube3a deficient and Ube3a over-expressing animals have defects in glutamatergic synaptic transmission Ube3a expression in the brain The UBE3A protein is an E3 ubiquitin ligase  electrophoresis we identified a protein that is a master regulator of actin cytoskeleton remodeling (the Rho-GEF ECT2) which physically interacts with both human and fly Dube3a proteins in vivo and may influence phenotypes related to actin cytoskeletal remodeling Our laboratory has demonstrated that over-expression of human UBE3A protein in the Here we utilize this more quantitative and reproducible preparative liquid phase isoelectric focusing (IEF) cell in combination with traditional 1-D denaturing polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins by molecular weight and quantify changes in protein levels dependent on fluctuations in Dube3a levels. This method allows us to resolve aliquots from the same fractions repeatedly until the pH comparisons are complete and the appropriate protein bands have been excised from the gels for mass spectrometry protein identification.Drosophila melanogaster heads expressing either no Dube3a protein (15bDube3a) or high levels of human or fly UBE3A proteins. By analyzing three cellular compartments we were not only able to identify proteins regulated by Dube3a involved in transcription or extracellular cellular signaling, but were also able to identify proteins that shifted from one cellular compartment to another in a Dube3a, and thus possibly ubiquitination, dependent manner. Using this approach we identified 50 proteins potentially regulated by UBE3A and implicating Dube3a involvement in neuronal homeostasis, ATP synthesis/metabolism pathways, as well as actin production or maintenance. The identification of these Dube3a targets is the first step in identifying new pathways regulated by UBE3A in humans that are responsible for the underlying defects in both Angelman syndrome and duplication 15q autism and may reveal new therapeutic targets for the treatment of these disorders.We directly compared nuclear, cytoplasmic and membrane extracts from 15bDube3a makes no Dube3a protein in the homozygous state and has been previously described Dube3a, UAS-Dube3a-C/A, and UAS-hUBE3A lines have been described elsewhere Heatshock-GAL4. The progeny of Heatshock-GAL4 crosses to various UAS transgene lines were subjected to a 37°C heat shock for 1 hr to induce transgene expression (24-fold increase in Dube3a protein and 37-fold increase in Dube3a-C/A protein) All fly crosses, with the exception of heat shock crosses which were raised at RT, were performed at 25°C on standard corn meal based media. The complete loss of function mutant Cytoplasmic Protein Extraction Buffer (CPEB from Bio-Rad). Heads were ground in ice cold buffer to release proteins and then the supernatant (cytoplasmic proteins) collected by centrifugation at 3500RPM at 4°C. The pellet was then re-extracted according to the manufacturers instructions to obtain the nuclear protein extracts. Membrane preparations were made by ultracentrifugation in a sucrose gradient using an established Drosophila protocol Heads were removed from frozen flies using molecular sieves under liquid nitrogen as previously described For each type of extract in each genotype, all protein fractions were visually analyzed across the middle 18 IEF fractions (∼pH 4.5–10.5) by loading ∼2 ug of total protein per lane on a 20 well 4–12% midi-gel in MOPS buffer (Invitrogen). Samples that were similar in pH to within +/−0.1–0.5 pH units were then run side by side on a 4–12% mini-gel for direct comparison of differentially regulated proteins. To quantify fold change in protein intensity the gels were fixed and stained with quantifiable fluorescent protein stain Sypro Ruby (Invitrogen), scanned under UV illumination using a Syngene G-box imaging system and then analyzed using software to measure intensity differences among protein bands (Syngene). Proteins which were determined to change by at least 2 fold+or – were then excised from the gels after Silver staining for visualization on a light box or directly cut from the Sypro gels under UV illumination in some cases.Peptides were identified from cut bands by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF MS) at either the UTHSC, Tufts University or Harvard Mass Spectrometry facility. Only top hits were considered for inclusion in the list and accuracy was assessed by p-value, Z-score or percent coverage. Protein identifications were performed using various parameters to determine confidence in that particular ID depending on the facility used  at the UTHSC Proteomics Core (closed 2007), the Tufts University Proteomics Core (closed 2009) and the Harvard University Proteomics Core.15bDube3a, and allowed to recover for ∼2 min. and then frozen in liquid nitrogen. Cytoplasmic and nuclear extracts were prepared from fly heads using the ReadyPrep Protein Extraction Kit (Bio-Rad). Samples were resolved on a NuPage 4–12% Bis-Tris 1.5 mm Gel using MOPS Buffer (Invitrogen) and transferred to Immobilon-FL PVDF membrane (Millipore). The membrane was blocked with 5% milk, 3% BSA, and 0.1% Tween-20 in PBS. Primary antibodies were used at 1∶5000: anti-GAPDH , anti-Arginine kinase courtesy of Glen Collier , anti-Eps15 courtesy of Hugo Bellen , and anti-ATPα (cat. #: α5) from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA. IR labeled secondary antibodies  were purchased from Li-Cor  and used at a dilution of 1∶10,000. The blot was imaged and analyzed using the Odyssey Infrared Imaging System . Samples were normalized using the signal from anti-GAPDH as the reference channel and signal intensity was then adjusted based on the calculated normalization factor assigned to each lane in the channel. Normalized intensities were used for statistical analysis from at least three technical replicates.Transgenic and control flies, no more than 3 days post eclosion, were subjected to a 37°C heat shock for 1 hr, or no heat shock in the case of 680 and no loading control was used since quantification was not possible by this method.Ubiquitinated proteins were purified using a polyubiquitin affinity resin (Thermo Scientific) in accordance with the manufacturer’s instructions. In brief, lysates of the appropriate genotype and cellular fraction were incubated with the resin at 4°C overnight on a rotating wheel. The resin was then washed and ubiquitinated proteins were eluted in SDS-PAGE sample buffer for Western blot analysis. Antibody concentrations and conditions were identical to those described above except all secondary antibodies were IRIn vitro ubiquitination assays were performed as described 2 and 4 mM of ATP) containing 50 ng of E1 ubiquitin-activating enzyme, 500 ng of E2 ubiquitin-conjugating enzyme (UBCH7), 2 µg of Dube3a-FLAG fusion protein, and 6 µg of bovine ubiquitin. The reaction was incubated at 30°C for 2 h. The reaction was terminated by the addition of SDS-sample buffer, samples were boiled and proteins resolved on a 4–12% Bis-Tris 1.5 mm acrylamide gel using MOPS buffer (Invitrogen) and transferred to an Immobilon-FL PVDF membrane for Western blot analysis. The blots were probed with both anti-Ubiquitin (Millipore) or anti-ATPα . Imaging and analysis was performed on an Odyssey Infrared Imaging System . The mean intensity values were measured from the bottom of the ∼100 kDa ATPα band to the top of the gel for three independent experiments. These values were converted to a percentage of the value for LANE 2 (Dube3a+E1/E2 proteins) in order to compensate for any Dube3a auto-ubiquitination signal. The percentages were then imported into Prism version 5.0 for statistical analysis (GraphPad).Both Dube3a and ATPα were purified from fly head cytoplasmic extracts using affinity purification protocols. N-terminally FLAG tagged Dube3a was purified using EZview™ Red anti-FLAG M2 Affinity Gel to purify the Dube3a-FLAG fusion protein following manufactures instruction (Bio-Rad). ATPα protein was purified using anti-ATPα monoclonal antibody  through immunoprecipitation with immobilized Protein G beads (Pierce) in accordance with the manufacturers instructions. http://tinyurl.com/7u6s5bh). Although some of the genes have multiple splice variants, all primers were designed as common assays that would detect all splice-forms, if possible. Three technical replicates were performed for each gene and normalized to the expression of the housekeeping TATA-binding protein (TBP) gene. Gene expression was quantified for each cDNA sample in triplicate at 40 ng/reaction using the default cycling parameters of the Roche Light Cycler 480 system. The crossing point (Cp) value for each sample was calculated using Roche absolute quantitation algorithm. The average Cp value among three technical replicates was calculated and the average sample Cp values were normalized for loading by comparison to the average Cp value of TATA-binding protein TBP. The fold change (Fc) in gene expression in fly heads was then calculated by comparing the difference of the normalized mean Cp values between 1118w and the 15bDube3a mutant or between Heatshock-GAL4 and each over-expression construct using the equation Fc = (2)Dcp.Total RNA was extracted from ∼100 ul of fly heads using 200 µl of RNABee solution (AMS biotechnology). RNA was quantified by spectrophotometry (NanoDrop Technologies) and the integrity of the 18S and 26S rRNA verified using an Agilent 2100 Bioanlyzer (Agilent Technologies). Two µg of total RNA were used as input for each cDNA reaction synthesized using random hexamer primers via the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Intron spanning primers were designed for all 49 potential targets using the Roche Universal Probe Library . Fixed larvae were then treated with a 10 min incubation in PBS +0.01% Triton-X 100 (PBSTx) followed by a 20 min incubation in a 500 ul solution containing 3 ul Phalloidin594 (Invitrogen) in PBSTx. Larvae were washed in PBST twice and then mounted with Prolong Gold Hardset plus DAPI (Vector Labs) for image capture using a 63X lens under oil immersion on a Zeiss LSM 710 confocal microscope with identical imaging conditions for 1118w and 15bDube3a genotypes. The tile feature was used to capture the entire A2 muscle region from either the left or right side of the animal (overlap 10%). Z-stacks of 6 µm in depth were used to capture images from the muscle nuclei layer to the top of the muscle surface. A stitched Z-stack section from the middle of this stack was then used for intensity analysis using the Zen image analysis package (Zeiss). Intensity was measured in 150 µm segments perpendicular to the muscle striations in both muscles 6 and 7 from each of four animals per genotype (n = 4). Statistical analysis was performed using a Student’s t-test in the Prism 5.0 software package (Graphpad Software).Wandering 3Dube3a and a Dube3a mutant that has a single mutation at the active site eliminating the ubiquitin ligase function of the enzyme 15bDube3a which makes transcript, but no protein Heatshock-GAL4 driver line to induce global high-level gene expression in all tissues. Adult flies no more than 3 days post eclosion were subjected to heat shock to induce transgene expression in the brain and then frozen in liquid nitrogen. In the case of the 15bDube3a mutant line, flies were grown at 25°C and homozygous 15b/Dube3a15bDube3a flies were used for all experiments. Frozen fly heads were then used to isolate cytoplasmic proteins (soluble fraction), nuclear proteins (re-extracted pellet) and membrane proteins (membrane preparation). Using the fly GAL4/UAS expression system Heatshock-GAL4 alone (top) and Heatshock-GAL4>UAS-Dube3a cytoplasmic fraction proteins . Using this approach we identified 137 bands to cut from these gels comprising some 76 individual proteins identified by mass spectrometry (www.flybase.org) in order to compile a comprehensive list of protein identifications, the cellular fraction in which they were identified, pH, relative fold change, apparent molecular weight as well as database identifiers (see Table S1 for complete details).Proteins from each genotype were extracted as described in the methods section. Proteins from each fraction were then separated by isoelectric point using a liquid phase isoelectric focusing cell (IEF). The actual pH and protein concentration for all twenty fractions from IEF cell were recorded and then each fraction run in a second dimension separation by molecular weight. trometry Table 1.Dube3a. In order to visualize the effects of changes in Dube3a expression on individual proteins identified we constructed a heat map for the fold change of each protein under each condition of Dube3a over/under expression and in cytoplasmic, nuclear and membrane fractions , ATPα (CG5670), Cuticular protein 72Ec (CG4784), Fat-body protein (CG17285), Fimbrin (CG8649), and Prolyl endopeptidase (CG5355). Elevated protein expression in the mutant may indicate that these particular targets are de-repressed by loss of Dube3a, and thus could be direct ubiquitin targets degraded by the ubiquitin proteasome system. The most likely direct ubiquitin targets on this list are ATPα and ArgK since these proteins have previously been identified as a proteins ubiquitinated in the developing fly nervous system Six proteins increased by >20 fold in various cellular fractions from the Punch and Pebble targets Six proteins only showed differential protein expression in the presence of elevated human UBE3A, which was used previously to identify both Table S2). A heat map analysis of all of the transcripts is illustrated in . Thirteen genes showed a >1.5-fold increase in gene expression when the Dube3a-C/A protein was expressed, indicating that the ubiquitin ligase function of Dube3a is not required for increased expression of these particular proteins. Eleven of these genes also responded in a positive fashion to increased wild type Dube3a levels. Eight of these genes responded in a positive direction to increased expression of Dube3a-C/A and a negative manner to loss of Dube3a (i.e. 15bDube3a), or not at all, including the GTP cyclohydrolase I gene which we previously described Gene expression was analyzed for all 49 proteins identified during the proteomic screen using primer sets common to all splice forms of these genes. Seventeen genes did not demonstrate appreciable changes in gene expression (≥1.5-fold up or down) under any conditions of varying Dube3a protein levels and one gene, Pro-phenol Oxidase 1, could not be analyzed using several different primer sets  showed a >25-fold increase in gene expression in the 15bDube3a mutants ( and Table S2), while a much smaller 2.6-fold increase in gene expression was observed for the heatshock related gene Heatshock Cognate 4. Since neither the 1118w line or the 15bDube3a line were subjected to heatshock, these results indicate that heatshock proteins may be indirectly regulated by Dube3a through a transcription factor intermediate or that Dube3a itself can confer transcriptional co-repression.Three Heatshock protein encoding genes  we identified significant enrichments  for transcription factor modules shared by these eight genes: Apolipophorins, Crystallin, Eps15, Fasciclin 1, Fat-body protein 1, glyceraldehyde-3-phosphate dehydrogenase, neither inactivation nor afterpotential C and PuTable S2. TFM-Explorer analysis revealed 17 clusters with a significant enrichment for 12 different transcription factor binding sites in the promoter sequences of these eight genes . The top five hits included binding sites for transcription factors glial cells missing , Chorion Factor II , Heatshock Factor , Abdominal B  and Ultrabiothorax .We next searched for the presence of transcription factor binding sites in the eight genes that showed increased transcription when we expressed Dube3a-C/A but either were not affected by or showed decreased expression in Drosophila brain and the developing embryo The glial cells missing transcription factor is both necessary and sufficient for the production of glial cells versus neurons in both the central complex of the adult inset). Finite analysis of the GO terms for each protein indicated that the major cellular functions regulated by these proteins include carbohydrate metabolism (11%) as well as energy metabolism (15%), actin cytoskeletal structure (7%), development (4%), and nucleotide binding (6%)  classification term culled from Flybase for each hit. A general root classification of the proteins we found indicates that 18% of the proteins from the screen are cellular components, 36% are involved in a molecular function and 46% are part of a specific biological process (ing (6%) Figure 3in vivoSeveral proteins on the list of regulated candidates were immediately of interest, as they are specifically known to be involved in neuronal development and activity. These proteins include the dopamine regulatory protein Punch, which we previously showed to be regulated by Dube3a Heatshock-GAL4 induction in these cases since all flies containing Heatshock-GAL4 were subjected to the same heatshock protocol and the control flies contained the Heatshock-GAL4 driver for comparisons of band intensities. In addition, we identified at least one Heatshock protein (Hsp70Ba (CG31449)) that decreased in 15bDube3a mutants as compared to wild type outside the context of the heatshock protocol. We also found on our list 4 proteins that make up the actin cytoskeleton or muscle including Actin5c (CG4027), Actin57B (CG10067), and myosin heavy chain (CG17927) as well as Tropomyosin I (CG4898). Finally, there were 11 proteins involved in more basic metabolic processes such as dehydrogenases, isomerases and kinases. Since the over-expression of human UBE3A only resulted in changes to 6 proteins that were not also regulated by changes in Dube3a, we conducted more in depth functional analysis using just the 43 proteins in the Dube3a regulated set.The next most interesting groups of proteins were Heatshock and Heatshock cognate proteins. The identification of changes in Heatshock proteins is not an artifact of the Table S3). These included an enrichment for nucleotide binding proteins (Es = 4.1), Heatshock proteins (Es = 3.8), glycolysis (Es = 3.7), oxidation/reduction (Es = 3.3), actin cytoskeleton (Es = 2.9), homeostasis (Es = 2.1), actin binding (Es = 1.8) and mitochondrial oxidative phosphorylation (Es = 1.6). This same set of 43 proteins was then used as an input list for STRING analysis (http://string.embl.de/) in order to identify relationships among proteins that may reveal protein networks regulated by Dube3a. Confidence relationships among these proteins revealed a tight cluster of 13 proteins, some of which are involved in actin cytoskeleton remodeling and some in cellular trafficking, as well as a wider network comprising 27 proteins including the neurotransmitter regulator Punch (CG9441) and the axonal transport protein shibire (dynamin: CG18102) that are interrelated by direct physical interaction or functional association , Pro-phenol Oxidase 1 (Bc: CG42639), Cuticular protein 72Ec (CG4784) and apolipophorins (Rfabg: CG11064) are involved in fly specific structures and functions and thus do not have any appreciable homology to humans and other model organisms. Our gene ontology and phylogenetic analysis indicates that Dube3a may be a key regulator of multiple proteins that regulate cellular trafficking through actin cytoskeletal proteins requiring ATP synthesis and metabolism to function in both flies and humans.A more formal analysis of these 43 proteins identified from the screen was done using pathway enrichment analysis tools within the Database for Annotation, Visualization and Discovery suite (DAVID) , top). Changes in the expression of Dube3a did cause fluxuations in the normalized intensity levels for ATPα, but these changes were variable among the four independent technical replicates and did not reach significance .In order to investigate direct effects of Dube3a ubiquitin ligase function on potential protein substrates identified in our screen we looked for changes in protein expression or ubiquitination state under differential Dube3a expression conditions. Although antibodies were available for Eps15, Arginine kinase, and ATPα for Western blot analysis, only the Eps15 and ATPα antibodies revealed appreciable protein stability/expression changes that were Dube3a dependent  resulted in a decrease in the amount of ubiquitinated ATPα detectable in the elution as compared to wild type  head extracts . The addition of purified Dube3a plus E1/E2 proteins did produce a Ub signal slightly above background . The signal in LANE 2 was considered the detection threshold above background (100%) since recombinant UBE3A is known to trans-ubiquitinate itself in vitro, LANE 3) and a strong smear appeared with the addition of Dube3a protein . This experiment was repeated three times using three different purifications. One Way ANOVA analysis with Tukey’s multiple comparison testing among the four groups indicated that there is a significant difference among the groups  and that significantly less poly-Ub is detected in the lane containing no E1/E2 proteins  while significantly more poly-Ub is detected in the lane with all three components  as compared to the control lane which lacks the ATPα substrate . These results indicate that ATPα may act as a direct substrate for ubiquitination by the E3 ubquitin ligase Dube3a in vitro.To determine if ATPα can act as a direct substrate for the Dube3a E3 ubiquitin ligase we performed actin5c, actin57B, Mhc, and Tm1 we investigated the effects of decreased Dube3a levels on larval muscle. Crawling 3rd instar larvae have a large area of easily accessible body wall muscle that can be visualized with the filamentous actin (F-actin) toxin phalloidin. We dissected and fixed four wild type and four 15bDube3a mutant larvae and stained the body muscles with fluorescently labeled phalloidin (Ph594). The mean fluorescent intensity was 37% lower in the 15bDube3a mutants  than in wild type animals and the range of signal intensity was lower as well  of the unique proteins identified by our screen did not change in any cellular fraction in the 15bDube3a mutant and of those that did, only 12 out of 24 proteins (50%) changed intensity by >10-fold validating our prediction that the proteomic analysis of complete loss of function mutants alone would not have revealed the majority of proteins that are actually regulated by Dube3a in a temporal specific manner. More importantly, we have now uncovered sets of proteins involved in the process of actin cytoskeletal dynamics as well as ATP driven metabolic and catabolic pathways which will stimulate new avenues of research in existing fly and mouse models of AS and autism.Changes in 15bDube3a loss of function animals, we also found that Tropomyosin I (Tm1) levels decreased by 3.3-fold in the absence of Dube3a protein (Table S2). This may be the cause of the decrease in filamentous actin we found in larvae since Tm1 physically interacts with and stabilizes actin filaments Dube3a loss of function mutations in flies the authors found that adult flies showed locomotor defects, specifically climbing ability Dube3a deficient larvae and most likely in adult muscle as well, suggesting that muscle defects may also contribute to the locomotor defects observed in these animals. Second, the upstream regulation by Dube3a of actin cytoskeleton through fly Pebble or UBE3A regulation of ECT2 may explain the changes in actin cytoskeletal components we found in our screen since Pbl/ECT2 is a Rho-GEF which is a master regulator of the cytoskeleton. Although we did not identify Pbl by liquid phase isoelectric focusing in the current screen, we previously showed that Pbl/ECT2 physically interact with both fly and human Dube3a/UBE3A proteins Ube3a protein have significant defects in experience dependent synaptic plasticity in the visual cortex Ube3a deficient animals Ube3a can result in a decrease in dendritic spine density on basal pyramidal neurons Although we found that both actin and myosin transcripts were elevated in et al. , and failed axon connections et al. Table 2.15bDube3a loss of function mutant (Table S2). A link between the ubiquitin proteasome system and the heat shock response has been known for some time In this study we show a >25 fold increase in gene expression for Hsp70Ab, Hsp70Ba and Hsp70Bb in a homozygous in vivo, the downstream effects of this ubiquitin modification remain somewhat puzzling since there are no appreciable changes in ATPα stability of the protein by Western blot . This growing list of transcriptionally regulated Dube3a genes opens a new avenue of research into the mechanisms by which Dube3a can act as part of a transcription factor complex and which transcription factors bind to Dube3a in the nucleus.We previously showed that Dube3a can regulate dopamine levels in the fly brain through the transcriptional (ubiquitin independent) regulation of GTP cyclohydrolase I (Punch) It should also be noted that we observed changes in the cellular compartmentalization of two proteins as a direct result of over-expression of either wild type Dube3a or the presumed dominant negative form Dube3a-C/A . In the cis regulatory regions responsible for these changes in gene expression. Here we propose a new model for UBE3A regulation in the nervous system that involves not only regulation by direct ubiquitination of target proteins but also transcriptional regulation and perhaps even cellular trafficking.Finally, although we have exhausted this particular approach to the identification of direct protein targets of Dube3a in fly heads, the use of the bio-Ub method will enable a deeper investigation into the identification of Dube3a dependent ubiquitin targets in the fly nervous system. Finding additional transcriptionally regulated Dube3a genes will require a different set of tools including whole genome chromatin immunoprecipitation approaches to identify the Table S1Complete list of proteins identified in our screen, fraction where they were identified, molecular weight of the protein excised from the gel, pH, fold change, CG identifying number and FBgn identifying number.(PDF)Click here for additional data file.Table S2Complete qRT-PCR data with error calculations from triplicate replicates for all genes identified in our screen. Genes with an asterisk (*) were used for transcription factor binding site analysis.(PDF)Click here for additional data file.Table S3value and fold enrichment for a given group in the cluster as compared to the entire set of proteins.DAVID analysis of proteins identified in the screen. Clusters of proteins that act in similar pathways or biological processes are listed along with an enrichment score for the cluster as well as a p(PDF)Click here for additional data file.File S1This Microsoft PowerPoint file contains images of gels used for either direct comparison of experimental and control lanes or to excise a previously identified band for proteomic identification. The band numbers and gel numbers can be found in (PPTX)Click here for additional data file.


Mll5 is currently a member of the Mll family of SET domain histone methyltransferase proteins but studies have also showed that it could be part of the SET3 branch of proteins. Recently, constitutive knock out animal studies have shown that Mll5 is required for proper haematopoietic stem cell differentiation, and loss of Mll5 results in synthetic lethality for genome de-methylation. Mll5 deficient male mice are infertile and here we analyse the consequences of Mll5 deficiency for spermatogenesis.Mll5 is expressed in the germ cells of the testes of wild type mice. Consistent with the expression of Mll5, we demonstrate by electron microscopy, video microscopy and in vitro fertilisation techniques that Mll5 deficient mice have defects in terminal maturation and packaging of sperm. The defects seen include detachment of the acrosomal cap and impaired excess cytoplasm removal. Functional tests of sperm motility show a lack of progressive motility of spermatozoa from Mll5 deficient animals. None of these defects could be rescued by in vitro fertilization. Using microarray analysis we show that transcripts implicated in spermatogenesis are dysregulated.Mll5 deficient male mice, but not female mice, are infertile. Here we show using RNA in-situ hybridization that Tlk2, Utx, Gpr64, Sult4a1, Rap2ip, Vstm2 and HoxA10 as possible Mll5 targets that together may account for the observed spermatozoa maturation defects.Our data demonstrate a clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation providing further support for its classification in the SET3 branch of proteins. Moreover, this study identifies  Spermatogenesis occurs in most male mammals throughout their lifetime. Successful self-renewal of the spermatogonial stem cells underpins this process Mll5 have a post meiotic spermatogenic phenotype mixed lineage leukemias) are structurally and functionally homologous to the Drosophila Trithorax proteins It has been reported that independently generated knockout mouse models of S. cerevisiae SET3; Mll5 was found to be part of the NCOR complex which is believed to be functionally similar to the SET3C complex MLL5, consistent with the hypothesis that they may be part of the same complex Mll5 (KMT2E) was initially assigned to this family in part due to the sequence similarity of its PHD and SET domains to those of Mll. However, recent studies suggest that both human MLL5 and mouse Mll5, and the murine paralog, Setd5, have SET domains that are closer in sequence to the yeast SET3 and SET4 proteins Mll5. Mll5 appears not be essential for embryonic development Recently, our group and two others have described mouse knockout models for Mll5 knockout mice, however male infertility was noted in two of the initial reports set3 (Δset3), resulted in normal vegetative growth and development Gametogenesis has not been examined in detail in the in vitro fertilisation techniques that male fertility is impaired in tm1ApaMll5 mice, due to multiple defects in terminal spermatozoa differentiation / maturation. Thus providing further experimental evidence supporting that idea that Mll5 is that functional homolog of yeast SET3. We also show specific deregulation of several important gene transcripts in the testis, which may be putative targets of Mll5 regulation.In this report we show using electron microscopy, video microscopy and Mll5 is described in detail elsewhere tm1ApaMll5 by insertion of a β-galactosidase  reporter cassette and neomycin resistance cassette under an independent promoter (MC1) in coding exon 3 of the murine Mll5 locus (exon 4 in ENSMUST000000094962) in 129S6 (129SvEv) embryonic stem cells resulting in the deletion of 180 bp of coding sequence. This disrupts the 5′-most coding exon generating a frame shift. This allele (with the resistance cassette in place) was passed to the germline and backcrossed twice to 129S6 wild type mice before intercrossing. The loss of full-length mature Mll5 protein was verified by Western blotting tm1ApaMll5 male and female mice can survive through adulthood but display pleiotropic haematopoietic and maturation defects tm1ApaMll5 intercrosses result in Mendelian ratios of homozygous, heterozygous and wild type pups at embryonic day 16.5, although non-Mendelian ratios were observed later in development, likely due to loss of homozygous pups between birth and weaning secondary to an immune defect The generation of a loss of function allele for tm1ApaMll5 homozygous mating pairs produced offspring, or evidence of pregnancies. To determine whether one or both sexes were affected, we first analyzed reciprocal matings of wild type males with homozygous females and wild type females with homozygous males over a 4-month period. tm1ApaMll5 female mice are fertile but exhibit a possible rearing defect . Heterozygous littermates were able to plug and impregnate female mice with a frequency indistinguishable from wild type  reporter within exon 3 downstream of an internal ribosome entry site (IRES), allowing for expression of the β-Gal enzyme when the tm1ApaMll5 transcript is synthesized from the endogenous Mll5 promoter. Whole-mount β-Gal staining confirmed the presence of the knock out allele  and early spermatids (st) (Mll5 in developing germ cells.As current antibodies against Mll5 do not work well on tissue sections (data not shown), we used RT-qPCR, β-Galactosidase  staining and RNA in-situ hybridization to assay and visualize t allele  and RT-qt allele  in the ttm1ApaMll5 male mice and the expression of Mll5 in the developing germ cells of wild type mice, it was conceivable that loss of Mll5 generated by the tm1ApaMll5 allele might impair spermatogenesis. However, we found no gross morphological differences  from wild type mice (Pearson χ2(1) = 107, p = 3×10−25, The presence of sperm but absence of gross morphological defects in the testis suggested that defects in spermatogenesis might lie in terminal maturation stages and / or capacitation events. We examined periodic acid Schiff (PAS) stained sections of testes and epididymides by light microscopy and observed significant differences between wild type and homozygous ld types . In additm1ApaMll5 mice that were not seen in the wild type testes. When surveyed at similar locations of the caput epididymis , in some cases with several tail assemblies pooled into one cytoplasmic droplet  = 0.33, p = 0.62; tm1ApaMll5 mice, 41.8% showed apparently normal morphology under high-powered light microscopy. This raised the question of whether morphologically normal sperm were nevertheless impaired for sperm motility capacitation or the ability to penetrate the zona pellucida and fuse with the egg membrane. We therefore decided to test whether the infertility of homozygous tm1ApaMll5 mice could be rescued by sperm capacitated using an in vitro fertilization procedure. The sperm heads could be observed binding to the oocyte in vitro  = 67, p = 3x10−15) and severe impairment of their ability to fertilize eggs from superovulated wild type females when identical numbers of sperm were incubated with wild type eggs  level probeset summarization   and 7.9 fold (p = 2.4×10−7) , while transcript Utx, encoding a H3K27-specific histone demethylase that associates with Mll3/4 complexes at Hox genes Mll5 was lost (Mll is responsible for the maintenance of Hox gene expression ype mice . Hence w 0.0034) . Express altered . In addi.4×10−7) , respectanalyses , also vaanalyses . These ge testes . In addiwas lost .Mll5 mouse in functional and molecular detail. In addition to the obvious sequences similarities of the SET domain of Mll5 to SET3/4 at critical residues  as well as somatic (Sertoli) cells of mice Mll5 knockout mice also show the late stage spermatogenic impairment We also show by RT-qPCR and in-situ RNA hybridization that the ype mice . Furtherype mice  very clorm cells . In additm1Apahomozygous Mll5 male mice are infertile , although detection of premature or delayed expression could be overlooked in our expression analyses of whole testes. Corroborating our conclusion, post-meiotic spermatogenesis has also been observed in an independently generated knock out of Mll5tm1ApaMll5 mice, it appears that the loss of Mll5 from developing germ cells results in defects predominantly in the post-meiotic stages of male gametogenesis, although subtle meiotic defects cannot be completely ruled out.Our breeding experiments show with high statistical confidence that nfertile . This isnfertile , apoptos- testes , nor wer- testes  or teste- testes  significd bodies  and sperd bodies  and motid bodies  in the tMll5 -/- binds to but is unable to fertilize oocytes in in vitro capacitation and fertilization experiments   & 4. Fureriments . Such deMll5 by constitutive knockout in a mouse exhibits male infertility due to defects in late stage spermatogenesis or spermiogenesis resulting in spermatozoa, which are unable to fertilize oocytes in vitro as well as in vivo.Thus we conclude that Mll5 is important for normal spermatogenesis as functional inactivation of Mll5 in mice and concordantly found that functional inactivation of Mll5 results in a hematopoietic stem cell defect in vivo, microarray was performed on flow cytometry-sorted murine bone marrow cells to purify hematopoietic stem cells (HSCs) since that was the population of cells which was found to be defective in the Mll5 -/- mice Hoxb2 and Hoxb5Mll5 -/- mice and hence assayed differential expression of genes from the testes of wildtype and Mll5 -/- mice using microarray. Unlike differential expression in hematopoietic stem cells, we were able to find over 900 differentially expressed transcripts between wildtype and Mll5 -/- testes. Several genes were found to be differentially expressed in whole mouse testes from wildtype and Mll5 -/- mice by RT-qPCR analyses on independent batches of mice  and is associated with differentiation of the reproductive organs of plants Tlk2 mRNA is expressed at high levels in testes, leading to speculation about a role in gametogenesis in mammals, as in plants tm1Apamll5 mice, implicating the Ras-signalling pathway Sult4a1, which was up regulated over 2 fold in the testes of tm1Apamll5 mice in the microarray  were maintained as an inbred stock on a 129S6 (129SvEv) genetic background on a high-fat sterile diet. Mating pairs were supplemented with dough diet and sunflower seeds. Genotyping of mice was done by PCR using the primers as described elsewhere All mice were bred and maintained as approved by the University of British Columbia Animal Care Committee (A05-0699) or under the authority of a U.K. Home Office Project License (PPL80/1503). The transgenic mice , incubated with hyaluronidase  and washed in M2 or Human Tubal Fluid (HTF) .For experimental matings, male mice were singly housed overnight before the female was introduced and monitored for the presence of a plug. Mice were euthanized by raising COMll5 RNA probe within exon 3 using the protocol described before Testes from euthanized mice were dissected, fixed in 4% paraformaldehyde and stained overnight at 30°C with X-Gal. RNA in situ hybridization was performed on 10–15 µm sections of frozen testes using a 150-bp Embryo staining was performed as previously described 5 sperm (diluted to 1−25×106 / ml in capacitation buffer for 30 min before mixing with oocytes) from epididymides and vasa deferentia of wild type (Mll5 +/+) and homozygous tm1ApaMll5 (Mll5 -/-). . For assessment of fertilization, the number of two cell embryos with an extruded second polar body, 24 hours after fertilization was recorded.Female ICR mice were superovulated with an intraperitoneal (i.p.) injection of 1.5U pregnant mare serum , followed 48 hr later by an ip injection of 1.5U human chorionic gonadotropin . Thirteen hours after hCG administration, the superovulated female mice were euthanized and the oocytes were harvested and incubated for 4–6 hour with 2.5×102 for 30 min to allow for capacitation of the sperm. An aliquot was then taken and non-motile sperm was counted using a haemocytometer while another aliquot taken to observe sperm using time-lapse microscopy on a Zeiss Colibri AxioObserver.Z1. A third aliquot was fixed in 4% paraformaldehyde and the total number of sperm counted. The number of non-motile sperm was then expressed as a percentage of the total number of sperm. At least 100 single sperm per mouse were analyzed. For imaging, propidium iodide was used in the mounting media and the sperm heads imaged using a Nikon C1 TE2000E2 confocal microscope with a 63x objective.Testes from euthanized male (n = 3 per genotype) mice were removed and the epididymides and vasa deferentia dissected into 3 ml of DMEM and incubated at 37°C / 5% COFreshly obtained tissues were fixed in 2.5% glutaraldehyde in a 0.1 M Cacodylate buffer at pH 7.3 for 2 hr and washed in the same buffer lacking glutaraldehyde three times. Next they were fixed in 1% osmium tetroxide and potassium ferrocyanide 1% in the same Cacodylate buffer for one hour followed three rinses in distilled water before being dehydrated through a graded series of Acetone to 100% starting at 30%. After two changes in propylene oxide they were infiltrated with epon 812 and then embedded in the same epoxy resin. 60 to 70 nm thin sections were viewed in the FEI Tecnai 12 Transmission Electron Microscope.tm1apaMll5 testes using QIAzol™ lysis reagent . RNA was extracted according to the manufacturer's instructions  was set up in 384 well plates on the 7900HT Fast Real-Time PCR system  with the respective probes (Roche) and primers . Relativt test for continuous variables, and Pearson's chi squared test or Fisher's exact test for categorical variables. Results with a 2-sided P-value less than 0.05 were considered significant. For birth rate variables with a Poisson distribution, birth counts conditional on the total births have a multinomial distribution, from which exact p-values were obtained Pairwise comparisons were performed using Student's Figure S1Gametogenesis is grossly normal in homozygous tm1ApaMll5 mice. Representative sections of haematoxylin and eosin (H & E) stained seminiferous tubules in testis of wildtype (A–B) and Mll5 -/- (E–F) mice showing various different cell types. 1 – spermatogonia; 2 – spermatocytes; 3 – round spermatids; 4 – elongating spermatids; 5 – Sertoli cells, 6 – Leydig cells. Representative sections of H & E stained epididymides of wild-type (C–D) and Mll5 -/- (G–H) mice showing (1) coiled tubules of the epididymis which are (2) lined by columnar epithelium and (3) contain mature spermatozoa. The original magnification was X400 for all panels except for C and G, where the magnification was X100.(TIF)Click here for additional data file.Figure S2Representation of Microarray data showing outliers in yellow. Plot of fold change (FC) of transcripts against their respective p-values of the transcriptomes of homozygous tm1ApaMll5  (KO) to Mll5 +/+ (WT) testes as determined by microarray. The yellow dots show the outliers and transcripts with FC>1.05 and p<0.05.(TIF)Click here for additional data file.Figure S3Filtered results from the analysis of the Affymetrix Microarray and status of RT-qPCR validation. Comparisons between genes identified as differentially expressed by microarray and their location on the mouse genome according to Mus musculus, NCBI build 37, 2007-07 from the Core Gene Level analysis between Mll5 -/- (KO) and Mll5 +/+ (WT) testes are shown.(TIF)Click here for additional data file.Figure S4Validation of short listed genes by RT-qPCR. This table shows the transcripts, primers and probes used (SYBR = SYBR green used without UPL probes) as well as the relative expression of transcripts in testes of homozygous tm1ApaMll5  [KO] mice vs wild type [WT] mice. Up regulated transcripts are highlighted in red while down regulated transcripts are highlighted in green. P values that are significant are in red text.(PDF)Click here for additional data file.Figure S5Mll5 appears to have similar residues in the SET domain to yeast SET3/4. CLUSTAL 2.0.8 multiple sequence alignment of the SET domains of Mll1(KMT2A) (P552200), Mll4 (O08550), Mll2 (KMT2B) (Q6PDK2), Mll3(KMT2C) (QBR4H), Mll5(KMT2E) (Q3UG20), yeast SET3 (P36124) yeast SET4 (P42948) and Setd7 (Q8VHL1). Conserved structural residues are marked in green and residues important for catalysis in red. Critical residues not conserved are marked in purple.(TIF)Click here for additional data file.Table S1Mll5 -/- female mice are fertile.(DOC)Click here for additional data file.Table S2Mean testes weights from Mll5 +/+ and -/- mice.(DOC)Click here for additional data file.Table S3Mean testosterone levels in Mll5 +/+ and -/- mice.(DOC)Click here for additional data file.Table S4Apoptosis in Testes from Mll5 +/+ and -/- mice.(DOC)Click here for additional data file.Data S1Supplemental data.(DOC)Click here for additional data file.Methods S1Supplemental methods.(DOC)Click here for additional data file.Video S1Mll5 -/- female mice are fertile. Time-lapse video microscopy showing differences in motility of sperm from Mll5 +/+ and Mll5 -/- mice.(MP4)Click here for additional data file.


UCHL1, COL1A2, THBS1 and TNFRSF10D were inversely correlated with promoter methylation. For THBS1 and UCHL1 the effect of this methylation on expression was confirmed at the protein level. Identification of these candidate TSGs and future research designed to understand how their silencing is related to melanoma development will increase our understanding of the etiology of this cancer and may provide tools for its early diagnosis.Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. By integrating gene expression and methylation array analysis we identified novel candidate genes frequently methylated in melanoma. We validated the methylation status of the most promising genes using highly sensitive Sequenom Epityper assays in a large panel of melanoma cell lines and resected melanomas, and compared the findings with those from cultured melanocytes. We found transcript levels of  Aberrant epigenetic modifications are a feature of several human diseases, including cancer. While several forms of epigenetic modification are known to exist, so far DNA methylation is the only one shown to directly target DNA and to be frequently aberrant in many tumor types PTEN, CDKN2A/p16INK4A and RASSF1A often have expression reduced through CpG island methylation TSPY, HOXB13 and SYK as novel TSGs in melanoma Melanoma genomics studies have identified a large number of chromosomal loci that show repeated loss of heterozygosity (LOH), highlighting widespread chromosomal instability PPP1R3C  subunit 3C), ENC1 , RARRES1  and TP53INP1 (tumor protein p53 inducible nuclear protein 1), had mRNA levels that were inversely correlated with promoter methylation (>40–60% of CpG sites) in 35–59% of melanoma cell lines and 6–25% of the fresh tumors.In a previous study, we combined 5AzadC treatment with Trichostatin A (TSA), an inhibitor of class I and II histone deacetylase enzymes, and conducted a microarray-based analysis on a panel of melanoma cell lines identifying eight highly ‘reactivated’ genes (expression fold change >4), not previously known to be epigenetically silenced in melanoma In order to identify additional epigenetically silenced genes implicated in melanocytic neoplasia, we have generated new data from 11 melanoma cell lines using Illumina Infinium Methylation27 arrays PPP1R3C, ENC1, RARRES1 and TP53INP1 that were not previously known to be silenced by DNA methylation in melanoma In our previous study, we used a microarray-based strategy in a panel of 12 melanoma cell lines treated with 5AzadC and TSA as an initial screening approach. Select candidate genes were followed up using the Epityper assay in a much larger panel of melanoma cell lines, as well as a panel of fresh-frozen melanoma samples, normal melanocyte cultures, and cell lines from other cancer types. We identified four genes, In order to use more robust criteria to select the genes epigenetically silenced during the development of melanocytic tumors, we have generated new data on 11 melanoma cell lines using Illumina Infinium Methylation arrays. The Infinium Methylation chips interrogate 27,578 CpG loci covering more than 14,000 genes. We screened 11 melanoma cell lines from our pilot study ADM, ENPP2, RAC2, SERPINE1), genes we had previously identified (PPP1R3C), those without a described function e.g. annotated as “orf” , and false positives on the Infinium Methylation chips .These new data were then integrated with previous data-sets of global mRNA expression COL1A2, CRABP2, CRIP1, FAM46B, GATA2, IGFBP4, LOX, RGC32, THBS1, TNFRSF10D, UCHL1 and VAMP8). The genes were further filtered by validating the differences in their expression levels pre- and post-treatment. Transcript levels of these 12 genes were assessed by qRT-PCR in the 5 cell lines with the highest expression differences before and after treatment. Selecting only the genes with a >4–fold average qRT-PCR expression change and the presence of CpG islands in the promoter gave a final list of 10 genes: COL1A2, CRABP2, CRIP1, GATA2, IGFBP4, LOX, RGC32, THBS1, TNFRSF10D and UCHL1. While the absolute value of the fold-changes varied somewhat between the two methodologies, there was generally good agreement between the microarray and qRT-PCR results . Importantly, for each of these 10 genes both techniques showed a >4–fold average change in expression after drug treatment.These additional filters resulted in 12 genes remaining for initial follow up (able S1) and subjected to the Epityper assay. Three genes were not considered further as they either failed to give any analysable data, probably due to the high CpG density of the region (CRABP2), or showed no differences in methylation profiles between melanocytes and the melanoma cell lines (LOX and RGC32).We then quantitated the degree of methylation of these 10 candidate genes using mass spectrometry of base-specific cleaved amplification products . These informative CpG sites were then scored in each amplicon defining the CpG island for each gene. Only the CpG sites presenting high methylation ratios (as defined below) in melanoma cell lines were averaged for the final percentage of methylation (% of methylation). This allowed grouping of the melanoma cell lines following their % of methylation: no/low methylation (0–20%), medium (20–50%) and high methylation (>50%). We then defined the average % of methylation for each gene as the average value across the melanoma cell line panel which was then compared to melanocytes.For each gene, the amplicons included in the analysis were those with different average methylation values of >20% in the entire panel of melanoma cell lines and with <10% methylation in melanocytes . For TNFRSF10D, 5 cell lines showed a 5-fold average increase in expression.Following 5AzadC+TSA treatment, In melanoma cell lines, the average % of methylation for these genes was 24%, 31% and 66% respectively. The methylation levels of these genes in the melanocyte pools were close to background (between 2 to 9%).COL1A2 and THBS1 were both divided into 5 amplicons, while 4 amplicons were designed to cover the 5′UTR and first exon of TNFRSF10D . For each of the three genes, the amplicons closest to the transcription start (within 1000 bp) showed high levels of methylation in the melanoma samples, which inversely correlated with mRNA expression (Table S1). Table S4 summarizes the number of CpG sites scored for the definition of the % of methylation for each gene .The 5′UTR regions around the transcription start sites of COL1A2 mRNA expression, which correlated with a high degree of COL1A2 promoter methylation in 67% of this subset. The other 19 cell lines with detectable COL1A2 mRNA expression all showed <20% of methylation, with the exception of 3 lines . .Over fifty percent (21/40) of the melanoma cell lines had no TNFRSF10D, 31 of 43 (72%) melanoma cell lines had no mRNA expression. Of the 31 lines not expressing TNFRSF10D, 28 (90%) showed >60% of methylation . For THBS1, 15 of 43 (35%) melanoma cell lines had no mRNA expression. Eight of the 15 (53%) lines with no THBS1 mRNA showed a high >50% methylation .For UCHL1 expression was increased by an average of 18-fold in 10 out of 12 cell lines. For the Epityper assay, the region around the 5′UTR and transcription start site of UCHL1 was divided in to 4 amplicons, also covering both exons 1 and 2 . While amplicons 2 and 6 did not show any differential methylation between melanoma cell lines and melanocytes (Table S1), amplicons 4 and 8 showed specific profiles of methylation in melanoma cell lines inversely correlated with mRNA expression (Spearman = −0.53). A majority (56%) of the melanoma cell lines had no UCHL1 mRNA expression, which correlated with a high degree of UCHL1 promoter methylation in 79% of them . The melanoma cell lines with high UCHL1 mRNA expression have <20% methylation .Following 5AzadC+TSA treatment, Next, we assessed protein levels for THBS1 and UCHL1  by western blot analysis Figure 3COL1A2, THBS1 and TNFRSF10D . THBS1 promoter methylation levels were 3.5 and 2 times higher in melanoma cell lines and tumors respectively.Simultaneously, in the Epityper assay, we included 30 fresh-frozen melanoma tumor samples and found that 13%, 15% and 30% of them respectively were methylated for NFRSF10D Table 1.UCHL1 promoter appeared to be 8-fold more methylated in melanoma cell lines compared to melanocytes, and 4-fold more methylated in the 30 fresh tumors compared to the same control. Since mRNA levels were not assessed in the tumors, we are unable to correlate the proportion of methylation with expression in these samples. The observed methylation rates in tumors are lower than in the melanoma cell lines, as expected due to stromal contamination, which will likely have the effect of decreasing the observed overall percentage of methylation of the DNA assessed.The COL1A2, THBS1, TNFRSF10D and UCHL1. Using the same cut off as for the melanoma cell lines and tumors, the percentage methylation for the COL1A2 and UCHL1 CpGs islands was 65% and 82% in the esophageal cancer cell lines respectively and 92% and 90% in the colon cancer cell lines respectively. While THBS1 was only methylated in the colon cancer cell lines (43% methylation), TNFRSF10D appeared to be the only gene methylated in the glioma lines (33%) . These different cancer types were assessed to determine whether the candidate genes might play a more general role in tumor suppression. Two cell lines from each tumor type were assessed for their methylation status for es (33%) Table 1.COL1A2, THBS1, TNFRSF10D and UCHL1 respectively.The objective of this study was to combine different array platforms to strengthen the identification of novel TSGs inactivated by promoter methylation in melanoma. Selection of candidate genes was based on reduced expression in a panel of melanoma cell lines which correlated with a high methylation profile, and lack of the same observation in melanocyte cultures. Using these criteria we identified and subsequently confirmed four genes silenced by DNA methylation in melanoma. We found 24%, 31%, 66% and 42% of cell lines and 13%, 15%, 30% and 21% of tumors were methylated for et al. and Koga et al.COL1A2 as methylated in 35% (7/20) to 89% (16/20) of melanoma tumor samples respectively. In their systematic methylation profiling of several human cancer cell lines, Paz et al. reported reactivation of THBS1 expression following 5AzadC treatment in all 18 melanoma cell lines analysed et al.TNFRSF10D promoter methylation in 85% of their melanoma cell lines (17/20) and 80% of their fresh melanoma tumor samples (32/40). In their study of the molecular effects of low dose of 5AzadC (Decitabine) on 8 melanoma cell lines, Halaban et al.COL1A2, TNFRSF10D, THBS1 and UCHL1. Here, we further document the link between these genes and melanoma by confirming the correlation between methylation and expression of these genes in a larger panel of melanoma cell lines. In our study, we assessed 45 melanoma cell lines, 30 fresh tumor samples and correlated methylation status and mRNA expression levels to observations made in pooled melanocytes. Furthermore, while the majority of the previous methylation studies were based on the analysis of gene re-expression post-5AzadC treatment but not on assesment of promoter methylation per se, we present here a study design which goes beyond 5AzadC treatment to include a precise CpG methylation profile via the Epityper assay, a sensitive and high-throughput method for DNA methylation analysis.Each of these four genes had previously been linked to melanoma. Muthusamy COL1A2 aberrant promoter methylation has been described in different cancer cells such as breast cancer, medulloblastoma, hepatoma, colorectal cancer et al. describe the possible advantages a decrease in collagen synthesis may confer on cancer cells, including faster cell growth and increased tumorigenic potential Within the large collagen family, collagen type I is the most abundant, structural component of healthy connective tissue and consists of a heterotrimer of two α1 (COL1A1) and one α2 (COL1A2) chains. Cellular p53 negatively regulates COL1A2 through TGF-β signalling in normal dermal fibroblasts THBS1  encodes the glycoprotein thrombospondin, which is generally considered a tumor suppressor and mediates cell-to-cell and cell-to-matrix interactions important for platelet aggregation and angiogenesis THBS1 by methylation has been described in several cancer types such as neuroblastoma Down-regulation of TNFRSF10D  is a member of the TNF-receptor superfamily containing an extracellular TRAIL-binding domain and a truncated cytoplasmic death domain. This receptor does not induce apoptosis but has been shown to play an inhibitory role in TRAIL-induced apoptosis TNFRSF10D expression is directly regulated by p53 and regulates cellular chemosensitivity TNFRSF10D promoter hypermethylation has been described as a mechanism of inactivating this gene in several cancer types The protein encoded by UCHL1  inactivation by promoter methylation in melanoma. This gene encodes a peptidase activator of the ubiquitin-dependent protein degradation pathway. Originally identified in neurons and in cells of the diffuse neuroendocrine system, mutations in this gene have been associated with Parkinson disease UCHL1 methylation has been reported in multiple tumors UCHL1 methylation as a biomarker for diagnosis and prognosis of certain tumors et al. recently showed in nasopharyngeal carcinoma cell lines that UCHL1 was a member of the p53/p14ARF/MDM2 complex UCHL1 regulatory region.The current study details COL1A2, THBS1, TNFRSF10D and UCHL1) we identify here as methylated in melanoma, encode components that fit within the p53 ontology pathway. Moreover, two of the genes we had identified in a previous methylation study are also associated with p53 function, either being induced by p53 (ENC1) or interacting with p53 (TP53INP1) Interestingly, all four genes , one common methylated gene, A panel of 12 melanoma cell lines derived from primary cutaneous melanomas or their metastases were used http://www.atcc.org/).The 2 colorectal cancer cell lines Co115 and LIM 2405, esophageal cancer cell lines OE19 and OE33, glioma cell lines T46 and T50 were purchased from the American Type Culture Collection . All samples were run on an Agilent Bioanalyzer  using a DNA 12000 LabChip kit to check for DNA integrity, purity and concentration. 500 ng of genomic DNA from 11 melanoma cell lines and a reference pool of melanocytes derived from several donors were bisulfite treated using an EZ-96 DNA methylation kit  as per the manufacturer's instructions. They were then hybridized to Infinium Methylation BeadChips  containing 27,578 CpG loci covering more than 14,000 genes http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE28356.Percent methylation (beta) was calculated from the ratio of fluorescent signal intensities of the methylated (M) and unmethylated (U) alleles, for each sample at each specific CpG site, using the equation beta = Max/[Max+Max+0]×100. On this scale unmethylated sites are represented by beta values close to zero, while heavily methylated sites show values approaching 100%. We then expressed each site specific melanoma cell line methylation value as a delta difference, compared to that of melanocytes. In this way negative values represented a cell line and site specific decrease in methylation, while positive scores indicated a relative increase in the degree of methylation. The provided manifest file linked individual methylation sites to official gene symbols, which we used to associate the methylation data to that of mRNA expression and demethylation, as described below. The data are MIAME compliant and have been deposited in Gene Expression Omnibus database (These data (GSE7127) were generated as part of previously published studies These data were generated as part of a previously published study Expression profiles generated for each cell line before and after drug treatment (expressed as a fold-change ratio) showed that across the panel of 12 cell lines a total of 8,144 non-redundant genes were re-expressed with >2-fold change after treatment . Genes reactivated in all 12 cell lines were removed from further analysis since they are likely to represent cellular stress response to 5AzadC treatment, or promoter demethylation of genes normally silenced within the melanocytic lineage.This filtering left 3125 genes to be considered from cross-analysis with the Beadarray27 methylation and U133 Plus 2.0 mRNA expression data described above.Fold-change expression profiling data (compared to melanocyte pool), fold-change demethylation data (compared to matching untreated cell line) and Illumina methylation profiling data (delta % methylation compared to the melanocyte pool) for each of the 11 cell lines for which all data were available, were imported into Microsoft Excel and linked via official gene symbol (HUGO Gene Nomenclature Committee). Data filters were then applied to each of the three data types in order to identify genes with clear evidence of methylation  and evidence of reduced expression compared to the melanocyte pool. Genes symbols were filtered to identify those in which at least 2 samples showed ≥60% methylation (Beadarray27) correlated to an average post-demethylation re-expression fold-change of >4 and 4-fold mRNA global decrease across the panel of 11 melanoma lines. From this set of 26 genes, after removing oncogenes , genes we had previously identified (PPP1R3C), those without a described function e.g. annotated as “orf” , and the presence of false positives on the Infinium Methylation chips , 16 genes remained for initial follow up . Of these, 12 genes  were subject to further validation as described below.mRNA extraction & expression array data were obtained as previously described The Sequenom EpiTYPER assay is based on in-vitro transcription and base-specific cleavage of a PCR amplicon and the subsequent analysis of the resulting RNA fragments by MALDI-TOF mass spectrometry 5′-AGGAAGAGAG-fw primer-3′, reverse: 5′-CAGTAATACGACTCACTATAGGGAGAAGGCT-rev primer-3′) to allow further in vitro transcription. One microliter of modified DNA was used for the PCR reactions carried out in a total volume of 5 µl. Unincorporated dNTPs were dephosphorylated by incubation at 37°C for 40 min in the presence of shrimp alkaline phosphatase (SAP) (Sequenom). Two microliters of this SAP-treated PCR mixture were used as a template in a 7 µl transcription reaction containing RNase A and T7 polymerase (Sequenom). Transcription and digestion were performed simultaneously at 37°C for 3 h. After the addition of 20 µl of H2O and 6 mg of CLEAN resin (Sequenom), 22 nl of the cleavage reactions were dispensed onto silicon chips preloaded with matrix . Mass spectra were collected using a MassARRAY mass spectrometer (Bruker-Sequenom) and analysed using proprietary peak picking and signal-to-noise calculations (Sequenom Epityper v1.0.5).EZ-96 DNA methylation kits  were used for bisulfite treatment of 1 µg of genomic DNA from 44 melanoma cell lines, 1 nevus cell line, 2 colorectal cancer cell lines (Co115 and LIM 2405), 2 esophageal cancer cell lines (OE19 and OE33), 2 glioma cell lines (T46 and T50), and 30 fresh-frozen melanoma tumors. DNA from pools of melanocytes was used as reference. Each gene promoter was divided into several amplicons . The tarThe relative amount of methylation (% methylation) was determined by comparing the signal intensities between the mass signals of methylated and non-methylated template.For data analysis only unique CpG units (units can contain one or more consecutive CpG dinucleotides) are included. CpG units overlapping with other cleavage fragments in the mass spectrum were excluded from data analysis. CpG methylation ratios were filtered using an uncertainty threshold of 10%. Only data values (CpG methylation ratios) with an estimated error smaller than 10% were included in the analysis. This filtering ensured that only precise data values were used for downstream calculation.For each gene, the amplicons presenting no significant difference in methylation between melanoma cell lines and melanocytes were dropped from the analysis . In each2)/n) of the Spearman coefficient.Spearman test was applied for the correlation between mRNA expression and methylation of the genomic region assessed. A t-test (t = r/Sr) was performed to obtain the significance Click here for additional data file.Figure S2UCSC browser for localisation of the amplicons used for the Epityper assays. The alignments show the localisation of the Illumina probes.(PDF)Click here for additional data file.Figure S3Distribution of the melanoma cell lines according to their methylation status for a. COL1A2 mRNA expression, b. TNFRSF10D mRNA expression, c. THBS1 mRNA and protein expression, d. UCHL1 mRNA and protein expression. The melanoma cell lines were grouped following their methylation profiles: high (>50%), medium (20–50%) and no/low (0–20%).(PDF)Click here for additional data file.Figure S4Epityper results for the COL1A2, THBS1, TNFRSF10D and UCHL1 promoters in melanocytes, 45 melanoma cell lines, 30 fresh melanoma tumors and cell lines from other tumor types . The software uses a color coding to show the range of methylation: red to yellow for 0 to 100% of methylation. While the melanocytes show no methylation across the amplicon, the melanoma cell lines and fresh tumors present different patterns of methylation.(PDF)Click here for additional data file.Table S1Primers for PCR. Each gene promoter was divided into different amplicons. The target regions were then amplified using the primer pairs and annealing temperatures defined by the MethPrimer program.(XLS)Click here for additional data file.Table S2Primers for qRT-PCR.(XLS)Click here for additional data file.Table S3Levels of gene reactivation in a panel of 12 melanoma cell lines post-5AzadC+TSA treatment.(PDF)Click here for additional data file.Table S4Informative CpG sites count. For each gene, only the amplicons presenting a significant methylation profile difference between melanoma cell lines and melanocytes were scored. In each amplicon, only the informative SpG sites were counted for the final % of methylation value for each gene.(PDF)Click here for additional data file.


GDF9) and bone morphogenetic protein15 (BMP15) mRNA expression in cumulus-oocyte complexes (COCs) of buffalo ovary during in vitro maturation (IVM). GDF9 and BMP15 are expressed specifically in mammalian oocytes and also participate in cumulus-oocyte crosstalk. Quantitative real-time polymerase chain reaction (qRT-PCR) technique was applied to investigate the relative abundance (RA) of GDF9 and BMP15 mRNA transcripts throughout the IVM process. Relative mRNA expression pattern of these specific genes were assessed in oocytes and cumulus cells at 0, 6, 12 and 24 h of in vitro culture. Our results revealed that RA of GDF9 during different hours of IVM showed significant reduction between 0 h and 24 h of maturation in oocytes and BMP15 transcript increased significantly (P<0.05) between 6 h and 12 h and decreased again between 12 h and 24. In cumulus cells, GDF9 remained stable during IVM upto 12 h of maturation and decreased significantly between 12 h and 24 h of maturation. Conversely, significant reduction of BMP15 was observed between 0 h and 6 h, stayed stable upto 12 h and became undetectable at 24 h of maturation. In conclusion, these two genes were differentially expressed during the period of oocyte maturation process and notably, BMP15 expression pattern is associated specifically with the period of cumulus cell expansion.The present study has evaluated the association of growth differentiation factor9 ( GDF9 and BMP15 have been involved in the control of ovulation rate in mammals . This stage specific expression through intra-follicular cascade at the correct time is vital for follicular growth in order to reach the ovulatory phase  play numerous role in ovarian folliculogenesis. In particular, a growing body of evidence in recent years indicated that two famous members of Transforming growth factor-β superfamily, Campbell . In thisBubalus bubalis) is a multipurpose livestock animal. It is of particular important production animal due to the source of high quality animal protein in developing countries  technique for mRNA expression of GDF9 and BMP15 genes. Accordingly, the current work will provide important leads for understanding the mRNA transcriptional level of GDF9 and BMP15 genes in oocytes during IVM in case of buffalo.The domestic Asian water buffalo  unless otherwise stated. Disposable plastic wares used were purchased from Nunc, Denmark.et al.  normal saline to the laboratory within 2-3 h. In the laboratory, ovaries were washed five times in modified Dulbecco’s phosphate buffered saline (mDPBS) containing gentamicin. Oocytes were aspirated with a 10 cc syringe using 20 gauge needle into 50 ml conical tubes from follicles (>3 mm in diameter) of buffalo ovaries. The aspirated COCs were graded based on morphological appearance of the cumulus cell investments and homogeneity of ooplasm under stereo zoom microscope as described by Nandi et al.  and the et al.The maturation of oocytes was evaluated under stereomicroscope based on the degree of cumulus expansion  were treated with 0.1 per cent trypsin-EDTA for 2–3 min and mechanically denuded of surrounding cumulus cells by repeated pipetting. The cumulus-free oocytes were observed under stereo zoom microscope to ensure that they were free of cumulus cells. The oocytes were washed twice in PBS and transferred to a fresh 1.5 ml micro centrifuge tube with minimum volume of PBS and then frozen at −80°C. After the removal of cumulus free oocytes, the remaining cumulus cells in PBS were transferred to micro centrifuge tube and centrifuged at 300 g for 2 min according to Caixeta et al. . The supin vitro matured oocytes (MO 50), cumulus cells (cells removed from 50–75 COCs) using RNeasy Plus Mini Kit  and eluted in 30 μl RNase free-water. The purity and concentration of isolated RNA was determined by ND1000 spectrophotometer  and purity was assessed using the A260/A280 nm ratio with expected values between 1.8 and 2.0. The cDNA synthesis was carried out with 6 μl of RNA using Superscript III first-strand synthesis system  and random hexamer primer in a final reaction volume of 25 μl. The cDNA synthesis reactions were carried at 25°C for 10 min for annealing, 50°C for 60 min for extension, followed by heat inactivation of the enzyme at 85°C for 5 min. Reactions without Reverse transcriptase served as controls. After termination of cDNA synthesis, each RT reactions were diluted with nuclease-free water to a final volume of 50 μl and stored at −80°C till further use.Total RNA was extracted from pools of immature (IM 75), Bubalus bubalis complete mRNA sequence of GDF-9 (GenBank: FJ529501.2), BMP-15 (GenBank: EF375880.1) and GAPDH (GenBank: GU324291.1). Oligo Analyzer (1.1.2 Version) software was used to determine the Tm, GC per cent, primer loops, primer dimers and primer-primer compatibility. The FastPCR Professional (6.2.45 beta 4 version) has been used for Real time PCR primer designing and the oligonucleotide primers were synthesized from Sigma-Aldrich (USA). The designed primers were optimized prior to quantification experiments using polymerase chain reaction. For these genes, the expected sizes of the products were confirmed by gel electrophoresis on a 2% agarose gel. The primer sequences, expected fragment size of amplified products and genbank accession numbers are shown in Table Oligonucleotide primers for qRT-PCR analysis were designed from Real-time PCR was performed to quantify the mRNA transcripts of GDF-9, BMP-15 in oocytes and cumulus cells using SYBR Green JumpStart Taq ReadyMix kit from Sigma-Aldrich, USA. Each cDNA sample was analysed in duplicate using real-time thermal cycler . A non template control (NTC) was prepared using DEPC water without cDNA. Reactions were performed in 20 μl volumes and loaded in the real-time qPCR 96-well plate (10 μl/well) and the plate was centrifuged in cooling (4°C) plate at 560 rpm for 2 min to remove the air bubbles and kept for reaction in real time thermal cycler.GDF9 and BMP15 genes using 10-fold dilutions of the sample and resultant slope of the curve was utilized for the calculation of the quantitative expression of both the target genes. The Ct values were recorded for both the target  and endogenous control genes (GAPDH). The data were accepted only if the NTC had no amplification.The PCR cyclic conditions were 95°C for 10 min followed by 40 cycles of denaturing at 95°C for 15 seconds and then annealing  for 1 min. The standard curve was obtained for GAPDH, Data were quantified using the method of relative quantification in qPCR Pfaffl . The thrStatistical analysis of the data was done as per the standard procedures of Snedecor and Cochran . The genTotal of 692 culturable grade oocytes were collected in a batch of experiments and 20–25 oocytes were cultured in 100 μl medium at each time-point to observe the progress of IVM. The COCs morphological changes were found at different time periods of culture  variation in the RA of GDF9 transcripts in cumulus free oocytes at between 0 h and 24 h of maturation. A significant (P<0.05) variation in the RA of BMP15 transcript was observed in cumulus free oocytes of different maturation times. It increased between 6 h and 12 h of maturation and reduced between 12 h and 24 h.The mRNA expression of s Figure A. SpecifGDF9 and BMP15 mRNA transcripts in cumulus cells during different hours of IVM were presented in Figure GDF9 remained stable upto 12 h of maturation, but decreased significantly at 24 h of maturation. However, BMP15 showed a significant reduction in the RA between 0 h and 6 h, stayed stable upto 12 h and become undetectable at 24 h of maturation.The differential expression pattern of GDF9 and BMP15 play essential roles during follicle maturation through actions on granulosa cells. GDF9 and BMP15 expression are less well characterized especially during IVM of COCs. Oocyte maturation is one of the important stages for the successful production of embryos in vitro. Moreover, it is well known that oocyte developmental potential is a reflection of proper maturation. So understanding the molecular mechanisms regulated during maturation is of great importance to optimize the IVM conditions. Since GDF9 and BMP15 are considered as important oocyte maturation factors during folliculogenesis  between 6 h and 12 h and decreased again between 12 h and 24. This increase in BMP15 transcript may be correlated with the cumulus expansion after 12 h of maturation in vitro. Similar to our finding, Li et al.  during IVM; It showed gradual increase between 6 h and 12 h and decreased again between 12 h and 24. However in cumulus cells, GDF9 remained stable during IVM upto 12 h of maturation and decreased significantly between 12 h and 24 h of maturation. In case of BMP15 significant reduction was observed between 0 h and 6 h, stayed stable upto 12 h and become undetectable at 24 h of maturation. In future, further characterization of these genes from in vivo matured buffalo COCs will help in better understanding of the GDF9 and BMP15 role in oocyte maturation of this species.To conclude, during maturation of COCs


With dipyridamol stress cardiac magnetic resonance imaging (DSCMR), we obtain high spatial resolution images that allow us to assess the transmurality of hypoperfusion but the clinical usefulness of this information has not been evaluated.We aimed to asses the usefulness of visual estimate of transmurality in relation to the coronary treeWe reviewed the CMR data base( 2008-2009) to obtain data from patients with positive DSCMR and medical records to know the coronary tree. We visually asses transmurality of hypoperfusion in each of the 17 segments in all patients studied and hypoperfusion was classified as> or <50% of the segment area.We studied, 112 consecutive patients 108 of them had performed. coronary angiography, 66 males(61%), Mean age 66±10 years 5 (4.6%) without significant coronary lesions (PPV: 95%). Patients with hypoperfusion > 50% in any segment (79 patients 73%) had a number significantly higher of affected vessels , greater number of segments with systolic dysfunction induced (SDI) and greater number of territories affected 1,7±0,7 vs 1,4±0,6 p=0.04). The number of segments with hypoperfusion> 50% was significantly higher in patients with SDI 1.the presence of hypoperfusión > 50% indicates greater severity of coronary artery disease (CAD) with more vessels and territories afected.2.The number of segments with severe hypoperfusion is higher in patients with systolic dysfunction induced. 3. The transmurality of hypoperfusion is an indicator of severity of CAD and should be reported routinely


Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutant plants exposed to high light. The high light treatment of the wild type and stn8 caused specific increase in phosphorylation of Lhcb4.1 and Lhcb4.2 isoforms of the PSII linker protein CP29 at five different threonine residues. Phosphorylation of CP29 at four of these residues was not found in stn7 and stn7stn8 plants lacking the STN7 protein kinase. Blue native gel electrophoresis followed by immunological and mass spectrometric analyses of the membrane protein complexes revealed that the high light treatment of the wild type caused redistribution of CP29 from PSII supercomplexes to PSII dimers and monomers. A similar high-light-induced disassembly of the PSII supercomplexes occurred in stn8, but not in stn7 and stn7stn8. Transfer of the high-light-treated wild type plants to normal light relocated CP29 back to PSII supercomplexes. We postulate that disassembly of PSII supercomplexes in plants exposed to high light involves STN7-kinase-dependent phosphorylation of the linker protein CP29. Disruption of this adaptive mechanism can explain dramatically retarded growth of the stn7 and stn7stn8 mutants under fluctuating normal/high light conditions, as previously reported.Photosynthetic oxidation of water and production of oxygen by photosystem II (PSII) in thylakoid membranes of plant chloroplasts is highly affected by changes in light intensities. To minimize damage imposed by excessive sunlight and sustain the photosynthetic activity PSII, organized in supercomplexes with its light harvesting antenna, undergoes conformational changes, disassembly and repair via not clearly understood mechanisms. We characterized the phosphoproteome of the thylakoid membranes from  This proceeds in photosynthetic membranes, called thylakoids, which are densely folded inside chloroplasts and heavily packed with protein-pigment complexes Chlamydomonas reinhardtiiThe recent years had witnessed discoveries demonstrating that environmentally-dependent differential phosphorylation of thylakoid membrane proteins regulates lateral migration Chlamydomonas reinhardtii involves a functional coupling of phosphorylated CP29 protein to PSI 72 or Thr74 and phosphorylation of Lhcb4.2 at Thr78 or Thr806 is known as STN7-dependent stn7 and stn7stn8 mutants lacking STN7 is severely retarded under fluctuating high/low light conditions in comparison with stn8 and wild type plants stn7, stn8 and stn7stn8 plants exposed to high light had yet been reported.The photosynthetic state 1 to state 2 transition in stn7, stn8 and stn7stn8 plants exposed to high light. We mapped the sites of phosphorylation in the membrane proteins, quantified phosphorylation of the PSII core proteins under high light and found high-light- and STN7-dependent phosphorylation of CP29 variants Lhcb4.1 and Lhcb4.2. Using immunoblotting and mass spectrometry we conducted a study on the differences in composition of thylakoid protein complexes separated by blue native gel electrophoresis from the plants exposed to normal or high light. We revealed that high light treatment caused relocation of the CP29 protein from the PSII supercomplexes to PSII monomers and dimers and this movement, as well as the phosphorylation of CP29, was lost in the mutant plants lacking the STN7 protein kinase.In this work we analyzed the phosphoproteome of the thylakoid membranes in Arabidopsis wild type, stn7, stn8 and stn7stn8, deficient in the STN7 and STN8 kinases involved in phosphorylation of several major thylakoid proteins stn7, stn8 and stn7stn8 mutants (72 or Thr74) and Lhcb4.2 (phosphorylation at Thr78 or Thr80), was STN7-dependent  at Thr6. This phosphorylation of CP29 in stn7 was 4 to 5 times lower than in the wild type  stn8 (SALK 060869) stn7stn8−2 s−1 with a photoperiod of 8 h light and 16 h dark was used and in the case of high light experiments photosynthetic flux of 900 µmol photons m−2 s−1 was applied.Arabidopsis thaliana wild type (ecotype Columbia) plants, n-dodecyl-β-D-maltoside. To separate proteins in the second dimension, single lanes were cut out and incubated with 5% β-mercaptoethanol in SDS sample buffer for 30 min at room temperature and then subjected to SDS-PAGE using 15% acrylamide gels. For immunoblotting the gels were incubated in blotting buffer  for 30 min before the proteins were transferred to a PVDF membrane . The D1, Lhcb1, PsbS and anti-phosphothreonine antibodies were described previously Thylakoid isolation was essentially done as described in 4HCO3, 10 mM NaF to a final concentration of 2.5 mg of chlorophyll/ml and incubated for 3 h at 22°C with a sequencing grade-modified trypsin from Promega  at 5 µg of enzyme/mg of chlorophyll and the released peptides were prepared and analyzed as in stn7 mutant thylakoids were esterified using either d0-methyl alcohol or d3-methyl d-alcohol (Sigma Aldrich). The isotope-labeled peptides were mixed 1∶1 before phosphopeptide enrichment using IMAC Isolated thylakoids were resuspended in 25 mM NHFigure S1Peptide identification views from MASCOT MS data analyses of phosphorylated peptides sequenced by collision induced dissociation (CID) or electron transfer dissociation (ETD) of their ions in the samples from the high-light-treated plants. The spectra and corresponding lists of singly and doubly charged fragment ions identified in the MASCOT search are shown.(DOC)Click here for additional data file.Figure S2The sums of the tandem mass spectra for CP29 isoforms Lhcb4.1 and Lhcb4.2 and for two core proteins from each of the three complexes: PSII (D1 and D2 proteins), PSI (PsaA and PsaB proteins) and LHCII (Lhcb1 and Lhcb2 proteins) counted in the gel bands corresponding to PSII supercomplex, PSII dimer, PSII monomer and LHCII trimer from plants exposed for three hours to either normal or high light.(DOC)Click here for additional data file.Figure S3stn7, stn8 and stn7stn8 mutant plants exposed for three hours to either normal or high light, as indicated. Immunoblotting was done using antibodies against the D1, Lhcb1 and PsbS proteins, as indicated.Blue native gel separation and analyses of the thylakoid membrane complexes from the (EPS)Click here for additional data file.


Influenza virus is not known to affect wild felids. We demonstrate that avian influenza A (H5N1) virus caused severe pneumonia in tigers and leopards that fed on infected poultry carcasses. This finding extends the host range of influenza virus and has implications for influenza virus epidemiology and wildlife conservation. Panthera tigris) and two leopards (P. pardus) at a zoo in Suphanburi, Thailand, showed clinical signs, including high fever and respiratory distress, and they died unexpectedly. The animals had been fed fresh chicken carcasses from a local slaughterhouse. At that time many chickens around Suphanburi were dying with respiratory and neurologic symptoms of what was retrospectively identified as H5N1 virus infection (The 2003–2004 avian influenza A (H5N1) virus outbreak in Southeast Asia resulted in 24 reports of fatal human cases  due to direct transmission of the virus from birds to humans. During the H5N1 virus outbreak in Thailand in December 2003 . The,,The virus isolates obtained from the tiger and the leopard contained a glutamine at position 222 (226 in H3) and a glycine at position 224 (228 in H3) in HA1, which were also found in other recent H5N1 isolates and which are related to preferential binding to avian cell-surface receptors  resulted in transient virus excretion and a temporary increase in body temperature but did not induce clinical signs of disease (–This report is the first of influenza virus infection causing disease or death in nondomestic felids. Generally, influenza virus is also not considered pathogenic for the domestic cat. Experimental infection of domestic cats in the 1970s and 1980s with influenza A viruses of subtypes H3N2 from humans, H7N3 from a turkey, and H7N7 from a harbor seal (,Our findings in tigers and leopards extend the host range of this virus and, together with the findings in domestic cats (


SCL6A4 gene has been the most extensively studied functional polymorphism in psychiatry, providing worldwide evidence of its importance in several disorders, such as OCD.Obsessive compulsive disorder (OCD) is a neuropsychiatric condition that has been shown to be heritable and for which a major gene effect has been reported based on segregation studies1SCL6A4), located on chromosome 17q12in vitro compared with the short form of the 5HTTLPRThe efficacy of selective serotonin reuptake inhibitors (SSRIs) in the treatment of OCD has lead to the hypothesis of a serotonergic dysfunction in this disorderet alet alet alSCL6A4 coding region has not revealed differences in OCD patients8et alA possible association between 5HTTLPR polymorphism and OCD has been studied by some research groups. Billett SCL6A4 gene should always be performed. Family-based association analysis confirmed negative linkage disequilibrium between the 5HTTLPR polymorphism and OCD. TDT and HRR analysis did not reveal preferential “l” allele transmission. These studies did not support McDougle’s initial hypothesiset alIn addition, many association studies have resulted in controversial results because population stratification cannot easily be avoided8et alNew evidence showing that some of the previous results may differ since the involvement of the 5HTTLPR (A/G) gene polymorphism modulating the 5HTTLPR, it may impose to new studies the need to assess this polymorphism. In addition, most association data come from case-control studies that do not assess population stratification instead of family based samples. The study presented in this issue by Tibrewal


We also found fewer numbers of circulating Pmels in mice that received Runx2 overexpressing Pmels when compared to mice that received control Pmels. In addition, there was decreased accumulation of Runx2 overexpressing Pmels at the tumor site when compared to the control Pmel . To further interrogate the role of Runx2 in T cells, we assessed the production of IFN-γ after stimulation in vitro with either plate bound anti-CD3 or gp100 expressing tumor cells. We found that IFN-γ production was comparable between Runx2 overexpressing Pmels and control Pmels after anti-CD3 stimulation. However, IFN-γ production was impaired in Runx2 overexpressing Pmels upon stimulation with tumor cells. Furthermore, in vitro characterization also revealed that Runx2 overexpressing Pmels have decreased proliferation and display an apoptotic phenotype. Taken together, our studies suggest that Runx2 regulates apoptosis, proliferation and IFN-γ production in tumor reactive T cells. Further studies to mechanistically understand these findings are ongoing.Adoptive T cell therapy (ACT) is a promising treatment for melanoma patients with a clinical response rate of about 50%. However, half of patients treated do not respond to this therapy, underlining the need for improvement . One of the limitations of ACT is the poor effector function of transferred T cells influenced by the immunosuppressive tumor microenvironment. In order to identify pathways which may contribute to this observation, we used a murine ACT model in which mice bearing established B16 tumors were treated with Pmel T cells which recognize the melanoma antigen gp100 in the context of H-2Db. Pmel T cells were recovered on day 6 and 13, after transfer, from the tumor and spleen of treated mice and their gene expression patterns were compared. We found that 720 genes were differentially expressed by T cells recovered from the tumor site compared to those recovered from the spleen. Amongst the differentially expressed genes were several transcription factors, including Runx2, Rora, E2F1 and Tcf7. After an initial in vivo screen, Runx2 overexpressing Pmels conferred a worse antitumor effect when compared to the control Pmels (median tumor size 30.7 vs 20.7 mm


ASF1 and SNF2 on derepression of the DDR genes in hydroxyurea-treated cells. Collectively, our results show that the DDR genes fall into a class in which Asf1 and SWI/SNF independently control transcriptional induction.The histone chaperone Asf1 and the chromatin remodeler SWI/SNF have been separately implicated in derepression of the DNA damage response (DDR) genes in yeast cells treated with genotoxins that cause replication interference. Using genetic and biochemical approaches, we have tested if derepression of the DDR genes in budding yeast involves functional interplay between Asf1 and SWI/SNF. We find that Asf1 and SWI/SNF are both recruited to DDR genes under replication stress triggered by hydroxyurea, and have detected a soluble complex that contains Asf1 and the Snf2 subunit of SWI/SNF. SWI/SNF recruitment to DDR genes however does not require Asf1, and deletion of Snf2 does not affect Asf1 occupancy of DDR gene promoters. A checkpoint engagement defect is sufficient to explain the synthetic effect of deletion of  SWI/SNF is a large multi-subunit enzyme that remodels chromatin by a mechanism requiring its DNA-stimulated ATPase activity Like SWI/SNF, the non-enzymatic histone H3/H4 chaperone Asf1 also regulates the interactions of histones with DNA ASF1 and the genes affected by deletion of components of SWI/SNF PHO5 and HO genes of budding yeast PHO5, SWI/SNF functions in advance of Asf1 to promote activator binding under low phosphate inducing conditions. Subsequent stable association of SWI/SNF with the PHO5 promoter requires Asf1 HO, SWI/SNF recruitment by activator bound to the URS1 upstream promoter element paves the way for Asf1 association at a downstream site, URS2. The latter event is necessary for SWI/SNF recruitment to the downstream URS2 element PHO5 and HO depend on Asf1 and SWI/SNF for induction, and that a SWI/SNF recruitment step at these genes depends on Asf1, strongly suggests an important role for Asf1 in SWI/SNF functions that affect transcription.Microarray analysis of mRNA expression has revealed significant overlap between the genes affected by deletion of RNR3 and HUG1. This regulation has been characterized in cells treated with genotoxins that inhibit replication: hydroxyurea (HU), which causes replication fork pausing by triggering depletion of deoxyribonucleoside triphosphates (dNTPs), and methane methylsulfonate (MMS), which modifies DNA in a way that causes fork blocking. Asf1 promotes derepression of RNR3 and HUG1 upon treatment with HU RNR3 in cells treated with MMS Asf1 and SWI/SNF have also been implicated in the regulation of two well-studied DNA damage response (DDR) genes, HODrosophilaSeparate studies of Asf1 and SWI/SNF have revealed participation of both factors in transcriptional regulation of the DDR genes HUG1 and RNR3 in cells lacking ASF1, SNF2, or both, under conditions of replication stress triggered by growth in the presence of 0.2 M HU . Hydroxyurea treatment of wild type cells elicits robust derepression of HUG1 and RNR3HUG1 is more strongly derepressed than RNR3, and its expression peaks approximately 2 hours after that of RNR3  RNR3 derepression in cells treated with HU requires SNF2 , deletion of ASF1 does not have a strong effect on Snf2 recruitment to RNR3 under conditions of replication stress. We next examined the association of another SWI/SNF subunit, Snf5, with the promoter of RNR3 in wild type and asf1Δ cells. Although Snf5 was less readily detected by ChIP than Snf2 (see ASF1 has little effect on Snf5 cross-linking to RNR3 (RNR3 and HUG1 increases after treatment with HU or MMS RNR3 and HUG1 in response to HU is unaffected by deletion of SNF2 (compare WT to snf2Δ bars). Collectively, our results support the hypothesis that Asf1 and SWI/SNF can be independently recruited to the promoters of DNA damage response genes during replication stress induced by treatment of cells with HU.At Snf2 see , it appe to RNR3 . Like SWasf1Δ, snf2Δ, and asf1Δ snf2Δ cells grown in rich medium. As expected ASF1 null mutants  asf1Δ snf2Δ haploid strain generated using one-step gene replacement was compared to the growth rates of the asf1Δ and snf2Δ single mutants under optimal culture conditions (rich medium with glucose at 30°C). The asf1Δ and snf2Δ single mutants grew at similar rates and both were slower-growing than the congenic wild type strain; the asf1Δ snf2Δ double mutant, however, was slower-growing than either single mutant  to grow after UV irradiation, or in the presence of HU or MMS; under all these growth conditions, cells must cope with elevated structural perturbation of DNA. Consistent with shared functions for Asf1 and SWI/SNF in protecting against replication stress, ve to HU . The pro culture . Therefoasf1Δ cells do not have the same MMS sensitivity as snf2Δ cells: cells lacking ASF1 are sensitive to MMS (as previously reported) SNF2 are not . SNF2 has no effect on Rad53 phosphorylation state under normal culture conditions . Rad53 is also activated normally in the snf2Δ mutant when it is cultured for two hours in 0.2 M HU  asf1Δ and snf2Δ cultures at two hours after HU addition, but are in the minority in asf1Δ snf2Δ cultures  SCR1 loading control.Total RNA was isolated by hot phenol extraction Total proteins were prepared by trichloroacetic acid precipitation Tandem affinity purification of protein complexes was performed essentially as described Chromatin immunoprecipitation was performed according to Minard et al., including wash-out of hydroxyurea prior to fixation