Drosophila, we performed chromatin immunoprecipitation-sequencing analysis with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a) within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling.Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in  dawdle, the Activin-like ligand in fruit flies. We show that down-regulation of Activin signaling in muscle, but not in adipose tissue, leads to extended lifespan. In part it does so when it alleviates the negative transcriptional repression of its Smox transcription factor (a Smad transcription factor) upon a keystone autophagy gene, Atg8a. This double signaling cascade autonomously improves muscle performance  and nonautonomously extends lifespan as it reduces the secretion of insulin peptides from the brain. The work develops the emerging model for interacting autonomous-nonautonomous roles of insulin/IGF signaling as a systems integrative mechanism of aging control.It is widely known that reduced insulin/IGF signaling slows aging in many contexts. This process requires the forkhead transcription factor (FOXO). FOXO modulates the expression of many genes, and the list of those associated with slow aging is impressive. But there are few data indicating the mechanisms or genes through which FOXO actually slows aging. Here, we identify a novel FOXO target,  Caenorhabditis elegans, mutants in insulin-like receptor daf-2 live twice as long as wild type InR and insulin receptor substrate (chico) increase adult lifespan in the fruit fly Drosophila melanogasterIgf1r) extend lifespan Reduced insulin/IGF-1 signaling increases the lifespan of nematodes, flies and rodents C. elegans lifespan extension of daf-2 and age-1(PI3 kinase) mutants requires daf-16, a FOXO homolog in worms DrosophilaFoxO3A to associate with exceptional longevity Genetic evidence places the forkhead transcription factor FOXO as the downstream effector of insulin/IGF-1 signaling Drosophila aging. Systemically reducing insulin signaling by mutations of the insulin receptor (InR) and insulin receptor substrate (chico) slows the decline in cardiac performance of aging flies while a similar outcome is produced by overexpressing FOXO and PTEN just within cardiomyocytes Drosophila fat body modulates lifespan by inducing fat body dilp6 transcription, which in turn suppresses neuronal DILP secretion One aspect that is clear is that insulin/FOXO signaling operates both nonautonomously and autonomously to control C. elegans have been used to probe how daf-16 controls lifespan in response to insulin signaling. Microarray analyses have identified many mRNA that are affected directly or indirectly by daf-16, and reducing some of these genes by RNA interference (RNAi) increases longevity daf-16 upon aging daf-16 in the long-lived daf-2(e1370) mutant, and three out of 33 tested target genes were found to increase lifespan when gene expressions were reduced by RNAi: lin-2, egl-10 and sca-1.Genome-wide studies with Drosophila, work to date has identified binding targets of dFOXO primarily from wild type adults. Alic et al identified 1423 dFOXO binding sites in wildtype adult female Drosophila using a ChIP-on-chip approach In Drosophila lifespan by identifying genes transcriptionally regulated by dFOXO in long-lived insulin signaling mutants. We conducted ChIP analysis with a dFOXO antibody followed by Illumina high-throughput sequencing from chico heterozygous mutants, which are long-lived and normal sized, and from adult flies with ablated insulin producing cells (IPCs), which are also long-lived dawdle (daw), an Activin-like ligand in TGF-β superfamily. In genetic trials, reducing daw or its downstream transcription factor Smox increases lifespan, preserves muscle function and reduces poly-ubiquitinated protein accumulation. The muscle specific benefits of activated dFOXO are mediated through the control of autophagy by Smox, which we find to bind and transcriptionally repress Atg8a/LC3, a reported longevity assurance gene of DrosophilaAtg8a in muscle was also sufficient to increase lifespan. Furthermore, reducing daw in muscle decreased DILP2 peptide secretion from the brain while peripheral insulin/IGF signaling was correspondingly reduced. Our results suggest that insulin/IGF signaling controls lifespan in part through dFOXO-mediated repression of muscle Activin signaling and its downstream functions including muscle autophagy, muscle proteostasis and subsequent remote control of systemic insulin/IGF signaling.Here we aim to understand how reduced insulin/IGF-1 signaling extends Drosophila lifespan we sequenced promoters derived from chromatin-immunoprecipitation with antibody against dFOXO in two genotypes of long-lived flies with reduced insulin signaling. Heterozygotes of 1chico live 36% longer than co-segregating wildtype sibs chico+/− adults have normal development time, body size and fecundity. Aging is likewise retarded by partially ablating adult IPCs by inducing apoptosis with a cell specific inducible driver  chico and IPCs ablated flies respectively, corresponding to 2042 and 1012 candidate genes (To understand how dFOXO extends te genes .puckered (puc) in both longevity assurance genotypes (puc is a negative regulator of JUN kinase basket (bsk), and mutation of puc extends Drosophila lifespan We identified 273 genes common to both longevity-assurance genotypes . Biologienotypes . In the To determine how candidate dFOXO targets affect longevity we selected 23 genes for further analyses  based onchico null mutant (chico−/−) and chico; foxo double mutant  , oxo −/−) . Transcrdaw, Glyp and Tsp42Ef) extended lifespan by consistently reducing age-specific mortality -Gal4 driving either UAS-RNAi or UAS-transgene. The effect of RNAi on lifespan was assessed for all 23 candidates. Knockdown of three genes  by consistently reducing mortality rate   and Activin (ligands: Daw and Act-β). These ligands signal through subfamily-specific Type I receptors  and shared Type II receptors . BMP-like ligands and Activin-like ligands activate distinct downstream signaling cascades leading respectively to phosphorylation of the Smad transcription factors Mad and Smox , the shared Type-II receptor (Punt and Wit) and two other TGF- β ligands (Myo and Mav)  .Drosophila are homologous to C. elegans TGF-β/dauer and Sma/Mab. Recent reports clarify that the TGF-β/dauer pathway can regulate somatic aging, while the Sma/Mab pathway appears to modulate reproductive aging C. elegans, Drosophila and mouse  and mouse , while the Sma/Mab ligand in C. elegans, DBL-1, is similar to BMP-like ligands in Drosophila and mouse. Together these results suggest that Activin may be a conserved longevity pathway.The TGF-β signaling pathways of nd mouse . SimilarDrosophila aging we determined which tissues produced this control. daw mRNA is highly expressed in muscle and fat body, a tissue with both liver and adipose-like activities , and this effect was reversed in chico; foxo double mutants  (babo-Act) reduced the number of autophagosomes  . In contagosomes . Since ADrosophila encode 18 autophagy genes Smox mRNA produces elevated autophagy in aged muscle, we studied the phosphorylation of this transcription factor in old flies. Smox phosphorylation is increased in aging muscle  and measured its interaction with biotin-labeled Atg8a oligonucleotide probes containing Smad box (5′-AGAC AGAC-3′). Smox-MH1 strongly bound to the Atg8a probe, and this interaction was blocked by addition of unlabeled wildtype cold probes  (Atg8a. This Smad binding element contains a direct repeat of two Smad boxes (AGAC). Upon activation, Smox, the Drosophila homologue of Smad2/3, binds to the Smad box located within the promoter of Atg8a. Activin signaling represses autophagy via direct transcriptional regulation on the key autophagy gene Atg8a.An electrophoretic mobility shift assay (EMSA) confirms that Smox binds directly within d probes . To defid probes . FurtherAGAC-3′) . TogetheAtg8a expression within muscle is sufficient to promote lifespan, we over-expressing Atg8a using a muscle-specific driver . Lifespan was modestly but significantly increased, suggesting that Atg8a gene is a specific instance of a longevity assurance genes that that functions through muscle downstream of Activin signaling   .4eBP in longevity mutants with reduced insulin, some but not all insulin/IGF pathway mutants have reduced fecundity. Here we found normal fecundity in females with reduced muscle Activin signaling  mutants) can be suppressed by daf-16 mutants, suggesting that TGF-β signaling intersects with the insulin/IGF-1 pathway for longevity in C. elegansInsulin/IGF-1 signaling modulates longevity in many animals. Genetic analysis in C. elegans were resolved only when the matricide or ‘bagging’ (due to progeny hatching within the mother) was prevented by treating daf-7(e1372) mutants with 5-fluorodeoxyuridine (FUdR) to block progeny development C. elegansDrosophila. These parallels between reproductive and somatic aging among invertebrate models and humans suggest that unopposed Activin signaling is pro-aging while favoring reproduction.Longevity benefits of reduced Activin (TGF-β/dauer) in Drosophila fly lifespan while decreasing IPC production of systemically secreted DILP2 Reduced insulin/IGF signaling extends lifespan through interacting autonomous and non-autonomous actions. Reducing IIS in some distal tissues has been shown to slow aging because this reduces insulin secretion from a few neurons: reducing IIS by increasing dFOXO in fat body or muscle extends Atg8a in Drosophila CNS is reported to extend lifespan by 56% Drosophila while lysosome activity and macroautophagy decline. Muscle performance with age  was preserved by inactivating Activin within this tissue. This genetic treatment also reduced the accumulation of protein aggregates. These effects are mediated by blocking the transcription factor Smox, which otherwise represses Atg8a. Smox directly regulates Atg8a through its conserved Smad binding motif (AGAC AGAC). These results, however, contrast with an observation where TGF-β1 promotes autophagy in mouse mesangial cells Aging muscle may produce different myokine-like signals in response to their physiological state. Aged muscles degenerate in many ways including changes in composition, mitochondria, regenerative potential and within-cell protein homeostasis Atg8a. If extrapolated to mammals, pharmaceutical manipulations of Activin may reduce age-dependent muscle pathology associated with impaired autophagy, and potentially increase healthy and total lifespan through beneficial signaling derived from such preserved tissue.Insulin/IGF-1 signaling is a widely conserved longevity assurance pathway. Our data indicate that reduced insulin/IGF-1 retards aging at least in part through its FOXO-mediated control of Activin. Furthermore, affecting Activin only in muscle is sufficient to slow its functional decline as well as to extend lifespan. Autophagy within aging muscle controls these outcomes, and we now find that Activin directly regulates autophagy through Smox-mediated repression of Flies were reared and maintained at 25°C, 40% relative humidity and 12-hour light/dark. Adults were maintained upon agar-based diet with cornmeal (0.8%), sugar (10%), and yeast (8% unless otherwise noted). RU486  used to activate GeneSwitch-Gal4 was dissolved in ethanol to a concentration of 200 µM and added to the food.MHC-Gal4Tub-GS-Gal4da-GS-Gal4S106-GS-Gal4dilp2-GS-Gal4 (provided by H. Jasper); dilp2-GS-Gal4,UAS-dicer2 (provided by S. Helfand). RNAi lines for dFOXO target genes and TGF-β pathway are from Bloomington stock center (TRiP line) and Vienna Drosophila RNAi Center . UAS-lines are: UAS-hairyUAS-pucUAS-RhoGAP18BUAS-vriUAS-witUAS-cv-2UAS-babo-Act  UAS-Atg8-CherryFly stocks included: Chico mutants were made in our lab as describe previously 1; cn1; ry506y (wildtype), 1; cn1 chico1/cn1; ry506y (−/+chico), 1; cn1 chico1/cn1 chico1; ry506y (−/−chico), 1; cn1 chico1/cn1 chico1; foxo21 ry506y . Adult on-set IPC ablation flies were made by crossing Dilp2-GS-Gal4 to UAS-rpr and inducing the cell death in IPC cells by feeding flies with RU486 for 15 days.chico−/+ and IPC ablation. Chromatin immunoprecipitation (ChIP) was performed according to previously published methods with modification Two insulin mutants were used in ChIP-Seq experiments, Drosophila reference genome using Bowtie short read aligner To map the dFOXO binding sites, we pooled the raw reads (about 20 million reads per sample) from two replicates into one data file and aligned it to Act5C) and sry genomic region were used as negative controls.For ChIP-PCR analysis, the binding enrichment was calculated as the fold change of ChIP DNA versus input DNA. The binding to the coding region of Actin gene (http://flybase.org/) and de novo motif analysis was performed using MEME Suite The DAVID functional classification tool was used for pathway and molecular function analysis on the dFOXO target genes RpL32, also known as rp49) by the method of comparative CT. Primer sequences are shown in Total RNA was extracted from 10 whole flies or from tissue of 15 flies in Trizol reagent . DNase-treated total RNA was quantified with a NanoDrop ND-1000. About 50–100 ng of total RNA was used for quantification with SuperScript One-Step RT-PCR reagent  and measured on an ABI prism 7300 Sequence Detection System . Three biological replicates were used for each experimental treatment. mRNA abundance of each gene was normalized relative to ribosomal protein L32 , flies were first tapped down to the bottom of a standard (empty) food vial, and the percentage of flies that climbed up 8 cm within 20 seconds was recorded. A total of 80 females  were scored for each genotype.Antibodies for immunostaining included: anti-polyubiquitin FK2 (1∶200) , and anti-rabbit IgG-DyLight 488 (1∶300) . F-actin was visualized by Alexa Fluor 488-conjugated Phalloidin . Lysosome was monitored by LysoTracker Red DND-99 at the concentration of 100 nM . DNA was stained with Hoechst 33342 (1 µg/ml) . Samples were processed as described in Smox polyclonal antibody was generated against the peptide sequence (DSIVDYPLDNHTHQ) corresponding to amino acids 143–156  and affinity purified  . Empty vector was used as a negative control.cDNA for Smox-MH1 DNA binding domain (1–420 nt) was cloned into pFN29K-His6HaloTag protein expression vector . After expression in Biotin-labeled DNA probes were generated using 3′-end biotin labeling . The binding reactions were carried out in a 10 µl of assay mixture containing 10 mM Tris·HCl (pH 7.5), 150 mM KCl, 5 mM MgCl2, 10 ng/µL poly(dI-dC), ∼50 ng labeled probe and 20 µg purified recombinant protein. After incubation at room temperature for 20 min, the mixtures were electrophoresed on 0.8% agarose gels in 0.5× Tris/borate/EDTA buffer. Biotin-labeled DNAs were transferred to a positive-charged nylon membrane  and detected using GelShift Chemiluminescent EMSA .10-day-old mated female flies were maintained on standard food (2% yeast) for five days at three females per vial and 8–10 vials per group. Flies were passed daily to new vials over five days and eggs were counted daily.We followed our recently reported EIA assay to measure hemolymph DILP2 Data are presented as mean ± SEM from three independent biological replicates, unless otherwise noted. Statistical significances were evaluated by t-test and one-way ANOVA analyses using GraphPad Prism Software.Figure S1−/−chico  mutants. (B). Binding of dFOXO to 4ebp promoter is enhanced in chico mutants, which is rescued by the removal of dFOXO. Primers for the coding region of Actin gene (Act5C) were used as negative control. (C). Pathway analysis of 273 identified dFOXO target genes. (D). Motif analysis on the promoters of dFOXO target genes. (E–F). ChIP-PCR validation of the binding of dFOXO to its target genes in −/+chico  and IPC ablation mutants. Primers for the coding region of Actin gene (Act5C) and sry genomic region Summary and verification of dFOXO ChIP-Seq. (A). The nuclear localization of dFOXO is promoted in (PDF)Click here for additional data file.Figure S2daw RNAi females (RU induced) relative to self-control (no RU) shows strong and consistent reduction. (B) Mortality rate of Glyp RNAi females (RU induced) relative to self-control (no RU) shows generally consistent reduction across ages where mortality rate increases with age. (C) Mortality rate of Tsp42Ef RNAi females (RU induced) relative to self-control (no RU) shows reduced rate across the final 30 of 40 days where mortality increases with age.Mortality rate for survival plots of three dFOXO target genes in (PDF)Click here for additional data file.Figure S3dpp RNAi females (RU induced) relative to self-control (no RU) shows proportionally elevated death across ages where mortality increases with age, suggesting that loss of dpp (BMP) in muscle accelerates aging. (B) Muscle specific daw RNAi (RU induced) relative to self-control (no RU) shows strong and consistent reduction in mortality, except at oldest ages. (C) Mortality rate of Mad RNAi females (RU induced) relative to self-control (no RU) shows elevated death across ages where mortality increases with age, suggesting that loss of Mad (BMP) in muscle accelerates aging. (D) Muscle specific Smox RNAi (RU induced) relative to self-control (no RU) shows consistent reduction in mortality across intervals where mortality increases with age, although the rate fluctuates at oldest ages.Mortality rate for survival plots of TGF-β pathway in (PDF)Click here for additional data file.Figure S4daw by RNAi in muscle (A) consistently lowers mortality rate while daw RNAi expressed in fat body (B) increases mortality rate. Reduction of Smox by RNAi in muscle (C) consistently lowers mortality rate while Smox RNAi expressed in fat body (D) increases mortality rate. Reduction of babo by RNAi in muscle (E) consistently lowers mortality rate while babo RNAi expressed in fat body (B) increases mortality rate at the oldest ages but otherwise does not affect mortality.Mortality rate for survival plots of tissue-specific RNAi of Activin pathway in (PDF)Click here for additional data file.Figure S5Drosophila Smox protein. (B). This Smox antibody can also specifically recognize recombinant Smox proteins expressed in E. coli. (C). Fat body-specific knockdown of Smox results in reduced protein level visualized with this Smox antibody.Verification of Smox antibody. (A). Smox antibody generated in this study recognizes a 51KDa band that presents in all four tissues tested, which is close to predicted molecular weight for (PDF)Click here for additional data file.Figure S6daw, Smox and babo RNAi. (D). The comparison of age-dependent climbing activity between control and daw RNAi flies. Asterisk indicates significant difference between treatment and control (p<0.05). (E). dFOXO shows less binding to the promoter of Atg8a. (F). Fat body-specific daw RNAi increased the level of circulating DILP2.(A–C). Knockdown efficiency of (PDF)Click here for additional data file.Figure S7Atg8a in muscle by the constitutive driver MHC-gal4 consistently reduces mortality rate across intervals where the death rate increases with age. There is high variability in mortality rate after age 70 days including an interval where no deaths were observed in the MHC; UAS-Atg8a genotype. (B) Mortality rate for survival plots of the genetic epistasis between daw and Atg8a. Reducing daw by RNAi lowers mortality rate relative to wildtype control after age 40 days. The net survival benefit of daw RNAi in this trial is muted because this cohort shows somewhat high mortality rate across the early intervals when death rate does not yet increase with age in the control cohort. Atg8a RNAi alone shows nearly identical mortality rate as control. Mortality rate of the daw and Atg8a RNAi double is similar to or slightly greater than that of control, indicating that Atg8a RNAi rescues the mortality benefit conferred by daw RNAi. Statistics of Cox proportional hazard for this epistasis are presented in Mortality rate for survival plots of the impact of Atg8a upon aging, text (PDF)Click here for additional data file.Figure S8Proposed model for the autonomous and non-autonomous roles of Activin signaling combine to control aging. Activin signaling targeted by insulin/dFOXO negatively regulates muscle autophagy, protein homeostasis and muscle functions in a cell autonomous manner, while it could also nonautonomously modulate longevity, the secretion of insulin peptides from the brain and peripheral insulin signaling. InR: insulin receptor; IPC: insulin producing cells. Myokine refers to unknown muscle-derived hormonal factor.(PDF)Click here for additional data file.Table S1Summary of lifespan analyses for 23 dFOXO-target genes reduced by RNAi.(DOCX)Click here for additional data file.Table S2Summary of lifespan analyses for seven dFOXO-target genes increased by over-expression.(DOCX)Click here for additional data file.Table S3Summary of lifespan analyses for Drosophila TGF-β pathway.(DOCX)Click here for additional data file.Table S4Atg8a over-expression.Summary of lifespan analyses for tissue-specific knockdown of Activin signaling and of (DOCX)Click here for additional data file.Table S5dawdle and Atg8a.Mean lifespan and sample size of muscle-specific knockdown of (PDF)Click here for additional data file.Table S6dawdle and Atg8a.Cox proportional hazard survival analysis for the effects of muscle-specific knockdown of (PDF)Click here for additional data file.Table S7chico and IPC ablation mutants.dFOXO direct target genes in (XLSX)Click here for additional data file.Table S8Primers for mRNA analyses.(XLSX)Click here for additional data file.


The preloading of these poised complexes in this inactive state is now apparent in many different organisms across the evolutionary spectrum and occurs at a broad and diverse set of genes. In this paper, we discuss the genetic and biochemical efforts in S. cerevisiae to describe the conversion of these poised transcription complexes to the active state for productive elongation. The accumulated evidence demonstrates that a multitude of coactivators and chromatin remodeling complexes are essential for this transition.The initial discovery of the occupancy of RNA polymerase II at certain genes  Drosophila [RNA Polymerase II (RNAPII) is a 12-subunit enzyme that binds promoter DNA and catalyzes the synthesis of messenger RNA in eukaryotes. Although the recruitment of RNAPII to a promoter is necessary for productive gene expression, it is not sufficient in many cases. Early studies in osophila . These posophila , 7. Indeosophila –10. S. cerevisiae is also supported by differences in 5′ to 3′ RNAPII occupancy and the frequent pausing of RNAPII within coding regions of genes [CYC1 gene [In yeast cultured to stationary phase, approximately 40% of the genes in the genome show association of RNAPII in their inactive state . These pof genes , 15. In of genes  and at tYC1 gene  providesCYC1 gene encodes iso-1-cytochrome c, a nuclear-encoded protein involved in the electron transport chain in the mitochondria [CYC1 gene expression is extremely low [CYC1 is activated and transcript levels increase 10-fold. In contrast to the dramatic changes in transcriptional output, the occupancy of RNAPII [CYC1 promoter contains preloaded RNAPII, the general transcription factors TATA-binding protein (TBP) and TFIIH, the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex, and Spn1, a highly conserved chromatin-associated transcription factor [CYC1 prior to activation is consistent with TFIIH occupancy, since TFIIH has CTD kinase as well as DNA unwinding activities [CYC1, a number of new factors are recruited to the promoter including the Mediator complex, and the chromatin regulatory factors the Swi/Snf complex and Spt6 [The yeast chondria . In the mely low , 20. Whef RNAPII , 21, as f RNAPII , 23, is n factor , 23. Intn factor , 25. Thetivities –30. Undeand Spt6 . This poand Spt6 .CYC1, where studies indicate that they appear to be functionally distinct. The SAGA complex is a large multisubunit coactivator that facilitates gene expression at multiple steps within the transcription cycle , 33, incCYC1 promoter requires SAGA for the transition from a preloaded complex to an actively transcribing unit since deletion of SAGA-integrity subunits blocks activated transcription [The poised cription . Severalcription –38. Sinccription . SAGA alcription –42 and acription , 51. Yetcription .In summary, the preloaded promoter is not dependent on the traditional well-characterized roles of SAGA, and yet SAGA integrity is required for the transition to an actively elongating complex after the recruitment of the PIC. These elongation activities may also be important at other genes, but difficult to observe because those genes require SAGA for recruitment of the general transcription machinery. The functions of numerous components within the SAGA complex remain to be elucidated and the preloaded promoter provides an excellent archetype for further investigations. Despite the necessity for SAGA function, SAGA is not sufficient for activation and another coactivator is critical for induction of the poised promoter.CYC1 activation and is recruited after the transfer to inducing conditions [CYC1 since RNAPII is present at the poised promoter prior to activation. Mediator has also been shown to stimulate TFIIH-dependent phosphorylation of the CTD [CYC1 is observed prior to activation when Mediator is absent from the promoter, although subsequent rounds of transcription may be impacted. Mediator has also been shown to be involved in the isomerization of the PIC into a transcriptionally competent conformation [The Mediator complex is a large coactivator that is conserved from yeast to humans  and actsnditions . Mediatonditions , 58, alt the CTD , 59. Howormation . This fuormation  are invoormation , 57, 61.CYC1 under noninducing conditions leads to a failure to recruit Spt6 under inducing conditions [The transition of the poised promoter to its actively elongating form is highly dependent on a number of chromatin regulatory factors, including the Spn1/Spt6 , 63 compnditions , consistnditions , 78. Mutnditions , 79 or Snditions  suppressCYC1, the absence of promoter binding by Spn1 results in constitutive recruitment of the Swi/Snf complex [CYC1 expression is that, in order for the transition to an actively elongating state to occur, the Swi/Snf complex evicts nucleosomes and the Spn1/Spt6 complex reassembles them. Precisely how these activities are related to the poised RNAPII in the uninduced state remains to be investigated, but it is tempting to speculate that the interplay between RNAPII and the nucleosomal architecture contributes to the inactive state. The involvement of the chromatin context and inactive RNAPII complexes has also been observed at particular silent loci in the yeast genome [Swi/Snf is an ATP-dependent chromatin remodeler that disassembles nucleosomes resulting in a loss of histones from DNA . Swi/Snf complex . Thus, tt genome . CYC1 is an incredibly robust phenomenon: single deletion of dozens of different transcription factors and coactivator complex subunits has not resulted in RNAPII occupancy defects . It coueviously , MediatoHow related is the RNAPII poising observed in yeast to that in metazoans? Studies in flies and human cells have clearly established that in many cases the polymerase has started transcribing and is paused just downstream of the start site. In contrast, there is no evidence for initiated transcripts that are stalled in yeast , 90. Whe


NOD2, ATG16L1, and recently the β-catenin–dependant Wnt transcription factor TCF7L2 have been linked to impaired PC antimicrobial function. The Wnt pathway influences gut mucosal homeostasis and PC maturation, besides directly controlling HD-5/6 gene expression. The herein reported candidate gene study focuses on another crucial Wnt factor, the co-receptor low density lipoprotein receptor-related protein 6 (LRP6). We analysed exonic single nucleotide polymorphisms (SNPs) in a large cohort  and prospectively tested 2 additional European sample sets . We revealed an association of a non-synonymous SNP  with early onset ileal CD  and additionally with penetrating ileal CD behaviour . In contrast, it was not linked to adult onset ileal CD, colonic CD, or ulcerative colitis. Since the rare variant is known to impair LRP6 activity, we investigated its role in patient mucosa. Overall, LRP6 mRNA was diminished in patients independently from the genotype. Analysing the mRNA levels of PC product in biopsies from genotyped individuals , we found particularly low defensin levels in ileal CD patients who were carrying the variant. In addition, we confirmed a direct relationship between LRP6 activity and the transcriptional expression of HD-5 using transient transfection. Taken together, we identified LRP6 as a new candidate gene in ileal CD. Impairments in Wnt signalling and Paneth cell biology seem to represent pathophysiological hallmarks in small intestinal inflammation and should therefore be considered as interesting targets for new therapeutic approaches.Ileal Crohn's Disease (CD), a chronic small intestinal inflammatory disorder, is characterized by reduced levels of the antimicrobial peptides DEFA5 (HD-5) and DEFA6 (HD-6). Both of these α-defensins are exclusively produced in Paneth cells (PCs) at small intestinal crypt bases. Different ileal CD–associated genes including  Crohn's Disease (CD) is to date incurable and is characterized by severe, reoccurring inflammations that can affect different intestinal locations. The complicated and multifactorial pathogenesis is not completely understood but involves disturbed epithelial barriers and immune reactions against the commensal flora in genetically predisposed individuals. Some inherited disease mechanisms are specific for small intestinal CD and are often connected to impaired Paneth cells. These specialized cells are critical for epithelial defences in the small intestine, and their most abundant antimicrobials (HD-5 and HD-6) are primarily diminished in patients who suffer from CD at this location. In this context, we previously identified a primary role of the Wnt pathway factor TCF7L2, which regulates the expression of both these antimicrobial peptides. We have now studied a functional mutation in the Wnt co-receptor LRP6 that was more frequent in early onset ileal CD. It was also associated with severe penetrating behaviour and linked to especially low HD-5 expression in ileal CD patient biopsies. Independently, we found that a reduced epithelial expression likely represents an additional impairment of the Wnt co-receptor in the disorder. LRP6 is a new player in small intestinal CD and underlines the importance of Paneth cell antimicrobial defences in the disease pathogenesis. NOD2, ATG16L1, as well as TCF7L2  have been specifically associated with small intestinal, but not colonic CD TCF7L2 promoter region variants specifically with ileal CD Ileal Crohn's disease (CD) belongs to the group of inflammatory bowel diseases characterized by chronic intestinal inflammation, ulceration and consequent diarrhoea LRP6  of analysed ileal CD patients shared the phenotype of early onset. However it should be noted that the first found result could be prospectively tested and confirmed in two additional cohorts. Other limitations might apply to the mucosal tissue expression studies. For assessing genotype-depending expression levels we had no intestinal tissue from homozygous C-allele carriers and we only could test tissue from adult patients. Both factors are due to restricted biopsy material resources. A potentially even stronger effect in homozygous C-allele carriers or a cause-effect-relationship in paediatric ileal CD remains therefore to be investigated. Since healthy controls featuring the C-allele heterozygously showed almost normal levels of HD-5, it is clear that other factors must be involved, especially to explain the specific effect of the mutation in ileal CD patients. Patients who carry the rare variant might only in combination with other underlying disturbances be prone to an early symptom development. It is quite conceivable that these could be the same mechanisms which explain diminished HD-5 expression in ileal CD in general. The combination of different defects, including the mutation in LRP6, might add up to the strong decrease of HD-5 and HD-6 which is specifically seen in this subgroup. One potential additive mechanism could be the diminished transcriptional expression of LRP6, which was seen independently from the patient's genotype. However, the mechanisms explaining reduced LRP6 expression still needs to be determined. In addition to LRP6, the described decrease of TCF4 (TCF7L2) and this Wnt transcription factor is likely another additive mechanism LRP6 genetic variances have so far been done in the context of variability in bone mass density, bone disorders LRP6 might contribute to the occurrence of such extraintestinal manifestations but further studies are required to analyse a potential role in detail. Finally, it is important to acknowledge the context of the LRP6 association as it provides further evidence for the significance of the Paneth cell in the development of small intestinal CD. Multiple genetic CD variants have already been specifically associated with small intestinal involvement and most of the involved genes are important in the specialized cell's biology TCF7L2NOD2ATG16L1XBP1KCNN4Other studies on All patients and healthy controls included in the present studies gave their written and informed consent after the study purpose, sample procedure, and potential adjunctive risks were clarified. All studies were approved by the ethics committees of the Medical University Vienna, Austria, the University Hospital Tübingen, Germany, the University of Leuven, Belgium and the Oxford Radcliffe Hospital Trust, Great Britain. Sub-grouping of included patients was done according to phenotype data which was based on clinical, radiological, endoscopic and histopathological diagnoses at the respective IBD centres . For genWe included all exonic LRP6 SNPs which were documented in the NCBI SNPdb . DNA was isolated by standard procedures. Multiplex genotyping was performed with the MassARRAY Compact System from Sequenom  All primers were designed using reference sequences as denoted by the NCBI SNPdb and Sequenom software  and are provided in Genotyping for the relevant NOD2 mutations was performed in patient samples using TaqMan technology , as previously described 1 µl of total RNA isolated from snap frozen ileal tissue biopsies were checked for quality before transcribed into cDNA using oligo (dT) primers and the AMV- reverse transcriptase (RT) kit according to the manufacturer's protocol (Promega). Real-time PCR with cDNA corresponding to 10 ng total RNA was subsequently performed with a LightCycler 480  using materials from the LightCycler 480 SYBR Green I Master kit according to the manufacturer's protocol (Roche). Specific plasmid standards for the selected products were utilized to calculate exact copy numbers and primers are provided in HD-5 luciferase reporter constructs have been kindly provided by Béatrice Romagnolo and Pauline Andreu  as described previously http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). Linkage disequilibria and haplotype blocks were calculated with Haploview. To avoid statistical bias due to multiple testing between different subgroup in the overall association analysis, we calculated Bonferroni adjusted p-values for the comparison between early onset ileal CD and controls.mRNA levels were normalized to β- actin and evaluated by GraphPad Prism Ver. 5.0. To analyse the effect of Ile1062Val between the groups, as well as the cell culture experiments, we performed the non- parametric statistical Wilcoxon-Mann-Whitney-Test. Spearmen rank analysis was used to test for correlation. For genetic analysis we used Finetti specialized software Click here for additional data file.Table S1st- respectively 2nd-PCRP. Primers used for genotype specific elongation are termed PEXP.Genotyping Primers: Oligonucleotides used for multiplex PCR approaches are termed 1(DOCX)Click here for additional data file.Table S2Real-time PCR Primers: Oligonucleotides which were used for the creation of standards and mRNA quantification.(DOCX)Click here for additional data file.


The conserved Paf1 complex (Paf1C) carries out multiple functions during transcription by RNA polymerase (pol) II, and these functions are required for the proper expression of numerous genes in yeast and metazoans. In the elongation stage of the transcription cycle, the Paf1C associates with RNA pol II, interacts with other transcription elongation factors, and facilitates modifications to the chromatin template. At the end of elongation, the Paf1C plays an important role in the termination of RNA pol II transcripts and the recruitment of proteins required for proper RNA 3′ end formation. Significantly, defects in the Paf1C are associated with several human diseases. In this paper, we summarize current knowledge on the roles of the Paf1C in RNA pol II transcription. The RNA pol II transcription cycle can be divided into three primary stages: initiation, elongation, and termination. During transcription initiation, the binding of the TATA-binding protein (TBP) subunit of TFIID to the promoter triggers the assembly of a preinitiation complex, which contains RNA pol II and the general transcription factors, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in ). The geThe regulation of transcription initiation is an important aspect of controlling gene expression and has thus been studied for many years. More recently, the regulation of postinitiation stages has been shown to be equally important for ensuring proper gene expression. Furthermore, eukaryotic cells have evolved many mechanisms to overcome the barriers imposed by chromatin on all three stages of the transcription cycle. One highly conserved protein complex that lies at the intersection between chromatin modification pathways and transcription is the Paf1C. This complex associates with RNA pol II –7 and inSaccharomyces cerevisiae [CTR9 gene (Cln three (CLN3) requiring 9) was originally identified by its genetic interaction with CLN3 [RTF1 (Restores TBP function 1) was originally discovered in a genetic selection for mutations that suppress transcriptional defects caused by TBP mutants with altered DNA binding specificity [The search for accessory proteins that cooperate with general transcription factors and regulate transcription initiation prompted experiments that led to the identification of Paf1 (polymerase-associated factor 1) in revisiae , 17. As revisiae , 18–20. ith CLN3  and subsith CLN3 . Additiocificity . Cdc73 (cificity  and Leo1cificity  may not S. cerevisiae strains lacking Paf1C subunits exhibit sensitivity to 6-azauracil (6-AU) and mycophenolic acid (MPA) [in vivo [rtf1Δ and cdc73Δ mutations on in vivo elongation rates or RNA pol II processivity, as measured on an inducible long gene, led to the conclusion that the Paf1C influenced cotranscriptional processes. Indeed, the Paf1C has been implicated in several cotranscriptional processes, including the phosphorylation of RNA pol II during elongation and the recruitment of a chromatin-remodeling enzyme, Chd1, to open reading frames [Paf1C subunits have been implicated in transcription initiation by influencing the phenotypic effects of a TBP-altered specificity mutant  and in tid (MPA) , 26. Theid (MPA) . Consistid (MPA) , 20, 26.[in vivo . Despite[in vivo . The lacg frames , 30, 31.g frames –12. Togertf1Δ or cdc73Δ cells, the remaining Paf1C subunits dissociate from the polymerase and chromatin, even though these subunits remain associated in a subcomplex [S. cerevisiae RTF1 defined a central region of the Rtf1 protein (amino acids 201 to 395), termed the ORF association region (OAR), that is required for the physical association between the Paf1C and active genes [in vivo [The Paf1C accompanies the polymerase from the transcription start site to the poly(A) site , 32. Rtfbcomplex , 31, 33.ve genes . Althougve genes , the man[in vivo . Beyond [in vivo . In this[in vivo .The interaction between Paf1C subunits and elongating RNA pol II is modulated by other transcription elongation factors. Several reports demonstrated that the Spt4-Spt5 complex promotes recruitment of the Paf1C to chromatin , 37–39. The C-terminal domain (CTD) of the largest subunit of RNA pol II, Rpb1, consists of tandemly repeated copies of a heptapeptide sequence (YSPTSPS) that can be phosphorylated on the serines at positions 2, 5, and 7 of the repeat. Importantly, the phosphorylation state of the CTD changes throughout the transcription cycle and is important for recruiting the appropriate regulatory factors during each stage of transcription (reviewed in ). Duringin vitro [in vivo, transcription rates inversely correlate with nucleosome occupancy within open reading frames (ORFs) [in vivo.During transcription elongation, RNA pol II encounters obstacles in the form of nucleosomes, the basic units of chromatin. Nucleosomes consist of two copies of each of the four histone proteins, H2A, H2B, H3, and H4, in a globular arrangement, wrapped by 147 base pairs of DNA , 65. A lin vitro , 67. Furs (ORFs) . In a res (ORFs) . Paused in vitro assay using purified factors revealed a direct interaction between the Paf1C and Bre1 [paf1Δ cells have greatly reduced levels of Rtf1 protein, Rtf1 is probably the primary subunit that promotes H2B monoubiquitylation and subsequent methylation of H3 on K4 and K79 [S. cerevisiae Rtf1 are required for these histone modifications, leading to the definition of a histone modification domain (HMD) in Rtf1 [Histone proteins are subject to a wide variety of posttranslational modifications, including the acetylation, ubiquitylation, and methylation of lysine residues. These modifications regulate RNA pol II activity at all stages of the transcription cycle. Members of the Paf1C are required for several histone modifications that are associated with active transcription . Specifiand Bre1 ; therefo and K79 . In fact in Rtf1 , 73. in vitro [In vivo, H2B K123 ubiquitylation facilitates transcription of galactose-inducible genes in yeast by promoting nucleosome-reassembly in the wake of RNA pol II in cooperation with the histone chaperone, Spt16 [H2B K123 ubiquitylation and H3 K4 and K79 methylation are enriched on the coding regions of active genes , 74, 75.in vitro . In vivoe, Spt16 . Additioe, Spt16 . S. cerevisiae, the silencing of genes near telomeres, and at the HMR, HML, and rDNA loci, is mediated by silent information regulator (Sir) proteins, which preferentially bind to hypomethylated histones . The geiewed in ). Paf1 aiewed in , 34, 73.ccupancy , 87. Bashtb1-K123R substitution) has shown that H2B K123 ubiquitylation represses many genes throughout the yeast genome [htb1-K123R cells, indicating that this modification predominantly acts to repress transcription [GAL1, GAL10, and SUC2 [ARG1 gene, by facilitating H2B K123 ubiquitylation [Microarray analysis of transcript levels in cells lacking the H2B ubiquitylation site  and histone deacetylase complexes (HDACs). H3 K4 trimethylation recruits the NuA3 HAT complex, resulting in increased H3 K14 acetylation , 105. Di regions . These r regions , yet dim regions . TherefoARG1 transcription can be only partially explained by a loss of histone modifications [in vitro transcription elongation assays have revealed a role for the Paf1C in stimulating transcription elongation of naked DNA templates by both RNA pol I and pol II [in vitro transcription of a chromatin template independently of histone modifications [ Given its important roles in modulating several histone modifications, the Paf1C likely regulates gene expression by promoting histone modifications. However, while genome-wide analysis identified numerous genes that require the Paf1C for proper expression , only a ications . Furtherd pol II , 112. Thications . Furtherin vitro and promote transcription in vivo [in vitro transcription using a naked DNA template has shown that the Paf1C and DSIF can also stimulate transcription elongation independently of chromatin [Beyond influencing gene expression through establishing histone modifications, the Paf1C interacts physically and genetically with several factors that influence chromatin structure and transcription elongation, including the elongation complex Spt4-Spt5, the histone chaperone FACT, and the chromatin remodeler Chd1. Spt4-Spt5 is required for the recruitment of the Paf1C to the elongation complex and for H2B K123 ubiquitylation , 37–39.  in vivo . A recen in vivo . However in vivo –118. Alt in vivo , 119, an in vivo . Spt4-Sp in vivo . Therefohromatin . in vitro transcription of a chromatin template [In vitro binding of an H2A-H2B dimer by FACT has led to the conclusion that FACT displaces a single H2A-H2B dimer to allow RNA pol II passage [in vitro hydroxyl radical accessibility and endonuclease cleavage experiments showed that FACT creates more accessibility than could be explained by the loss of an H2A-H2B dimer, yet partially protects nucleosomal DNA [The yeast Paf1C physically and genetically interacts with Spt16-Pob3/FACT , 26. Contemplate . FACT watemplate , 122. Betemplate . It is ntemplate –127. In  passage , 129. Hoomal DNA . These rIn addition to its physical interactions with Spt4-Spt5 and FACT, the Paf1C interacts with Chd1, a conserved ATP-dependent chromatin remodeling enzyme , 132. ChIn addition to its critical functions during transcription elongation, the Paf1C is important for proper transcription termination and RNA 3′ end formation –16, 31. Together, these observations suggest that the Paf1C plays an important role in coordinating transcription with RNA 3′ end processing. Given that the Paf1C is required for the recruitment of 3′ end processing factors to chromatin , 31, yetThe Paf1C is also required for proper termination and 3′ end formation of nonpolyadenylated transcripts . The losThe contribution of the Paf1C to transcription termination has yet to be assessed on a genome-wide scale. However, given the essential roles of transcription termination, which include regulating transcript stability and RNA pol II recycling (reviewed in –148), PaDrosophila Paf1C [As mentioned above, the known functions of the Paf1C, including RNA pol II-association  and rolela Paf1C . Howeverla Paf1C , 154. HOX genes [Drosophila and zebrafish [The Paf1C has evolved critical functions in higher eukaryotes, including promoting proper development, maintaining pluripotency in stem cells, and preventing cancer. Consistent with an important role in development, the human Paf1C is required for proper transcription of Wnt target genes  and HOX OX genes . Additioebrafish , 157. Giebrafish , 158. Foebrafish . In addiebrafish  and thosebrafish . Members of the human Paf1C have also been implicated in cancer. Pancreatic differentiation factor 2/Paf1 is overexpressed in pancreatic cancer cell lines and overexpression in cell culture results in transformation . AdditioHOX gene expression [Like the yeast counterpart, the human Paf1C influences gene expression by facilitating histone modifications. For example, the Paf1C, H2B monoubiquitylation, and H3 K4 and K79 methylation promote pression . H2B monpression , 168. Cepression , 168 andpression . Interespression , which rpression . β-catenin [In addition to its effects on histone modifications, the human Paf1C regulates gene expression through direct interactions with gene-specific activators. Human Ctr9 associates with Stat3 and recruits it to the promoters of interleukin-6 responsive genes . Cdc73 i-catenin . Additio-catenin .The Paf1C performs multiple functions during RNA pol II transcription, and these functions are conserved from yeast to humans. Previous studies have provided a wealth of knowledge about the roles of the Paf1C in transcriptional regulation; however, many important questions remain. While it is known that the Paf1C associates with RNA polymerase during elongation and dissociates near the poly(A) site, the details of the Paf1C-RNA pol II interaction, and its regulation, remain undefined. Current information suggests that the association of the Paf1C with RNA pol II is facilitated by multiple interactions. Cdc73 and Rtf1 play nonredundant roles in tethering the Paf1C to RNA pol II, but it is unclear whether these proteins make direct or indirect contacts with the polymerase or whether, like Leo1 , interacA requirement for the Paf1C in regulating transcription termination and RNA 3′ end formation has been observed at specific genes. However, determining the scope of this effect on RNA 3′ end formation or other steps in RNA maturation will require additional genome-wide studies on Paf1C-deficient cells. Existing data indicate that the Paf1C mediates transcription termination in several ways. Its physical association with RNA processing factors suggests that the Paf1C coordinates the exchange of transcription elongation factors for transcription termination and 3′ end processing factors , althougAlthough it has been shown that the Paf1C is required for proper expression of numerous genes throughout the yeast genome , a role


Epigenetic mechanisms are fundamental to understanding the regulatory networks ofgene expression that govern stem cell maintenance and differentiation.Methylated histone H3 lysine 4 (H3K4) has emerged as a key epigenetic signal forgene transcription; it is dynamically modulated by several specific H3K4methyltransferases and demethylases. Recent studies have described newepigenetic mechanisms by which H3K4 methylation modifiers control self-renewaland lineage commitments of stem cells. Such advances in stem cell biology wouldhave a high impact on the research fields of cancer stem cell and regenerativemedicine. In this review, we discuss the recent progress in understanding theroles of H3K4 methylation modifiers in regulating embryonic and adult stemcells’ fates. Stem cells have long-term self-renewing activity and can commit to multiple celltypes upon differentiation signals. Since Yamanaka and colleagues demonstrated thatthe four DNA-binding transcription factors Oct4, Sox2, c-Myc, and Klf4 transformfibroblasts into a type of pluripotent cells known as induced pluripotent stemcells, the importance of transcription factors in cellular reprogramming has beenmore recognized . HoweverHistone lysine methylation has been widely accepted as a key epigenetic modification.Unlike acetylation, the methylation does not change the charge of lysine residuesand thus has a minimal direct effect on DNA-histone association. Rather, thedifferent methylation status of specific histone lysines can serve as a uniqueplatform for recruiting methylation “reader” proteins that activate orrepress genes’ transcriptional activity. In general, histone H3 lysine 4(H3K4), H3K36, and H3K79 methylation are gene activation marks, whereas H3K9, H3K27,and H4K20 methylation are gene-repressive modifications  domain-containing methyltransferases, including mixedlineage leukemia 1–5 (MLL1−5), SET1A/B, SET7/9, SET and MYNDdomain-containing protein 1–3 (SMYD1−3), Absent, Small, or Homeotic1-like (ASH1L), SET domain and Mariner transposase fusion gene (SETMAR), and PRdomain zinc finger protein 9 (PRDM9) . MethylaSimilar to other histone modifications, histone methylation can be reversed byhistone demethylases (HDMs). The first identified lysine-specific demethylase 1, together with LSD2, belongs tothe polyamine oxidase family. LSD1 and LSD2 remove methyl groups from di- andmonomethylated H3K4 but are unable to demethylate trimethylated H3K4 -28. LSD1In this review, we summarize the recent progress in understanding the functions ofH3K4 methyltransferases and demethylases in modulating stem cells’ fates.Drosophila and several mammals, and is importantfor animal embryonic development  that are transcriptionallyinactive in ES cells [Although H3K4me3 is clearly associated with actively transcribed genes, however,studies have demonstrated that H3K4me3 is localized around the transcriptioninitiation sites of numerous unexpressed genes in human ES cells, primaryhepatocytes, and several other cell types ,37,41. IES cells ,37,42,43ES cells ,45, indiOct4[Compared with mouse thymocytes, mouse ES cells contain higher levels of totalgenomic H3K4me3 and have higher H3K4me3 occupancy at the promoter of thepluripotent gene Oct4. In agreOct4. In addiOct4. It is bMost H3K4me3-containing promoters are also occupied by H3K9/H3K14 acetylation . In tranH3K4me2 decorates genomic regions independently of H3K4me3, although most of itoverlaps with H3K4me3 near the transcription start sites . H3K4me2Drosophila, thereare three Set1 homologs: dSet1, Trithorax (Trx), and Trithorax-related (Trr).The deletion of any of their genes results in lethality in flies, indicatingthat their target genes may not be redundant. In particular, loss ofdSet1, but not Trx or Trr, leads to a globalreduction of H3K4me2/3, suggesting that Trx and Trr have morespecialized functions [Drosophila. Other SET domain-containing histone methyltransferasesthat methylate H3K4 but are not closely related to yeast Set1/COMPASS have alsobeen identified and include MLL5, SET7 , SMYD1-3, SETMAR, andPRDM9 [Some H3K4 methyltransferases are well conserved in different species. In yeast,the Set1 complex, also called Complex of Proteins Associated with Set1(COMPASS), catalyzes the mono-, di- and trimethylation of H3K4 ,8. The punctions . Human SndPRDM9 ,15,24.SET1A/1B and MLL1−4 are present in multi-protein complexes and share commoncore subunits, such as WDR5, Retinoblastoma-binding protein 5 (RBBP5), ASH2L,and Dumpy-30 (DPY-30), which are also highly conserved in yeast and flies   was initiallycloned from acute myeloid and lymphoid leukemia that contain frequentMLL1 chromosomal fusions and translocations [MLL1 gene encodes a protein of 3,972 amino acids;this protein contains several highly conserved functional domains, includingthe N-terminal AT-hook DNA binding domains, Plant homeo domains (PHD), aBromo domain, and the catalytic SET domain , a critical regulator ofneurogenesis, and is required to resolve the poised bivalent state to theactively transcribed status with predominant H3K4me3 during neurogenesis ofneural stem cells [Homozygous deletion of  defects ,70. Spec−/− mice . In addi−/− mice ,72. Mll1se brain . Mechaniem cells .Mll2-null mice die before embryonic day E11.5, withdrastically reduced expression of Hoxb2 and Hoxb5[Mll2 may be only required briefly fordevelopment, because it appears to be dispensable for mouse developmentafter E11.5 [Mll2−/− ES cells maintain pluripotency, have increasedapoptotic activity, and undergo skewed cellular differentiation along threegerm layers [Mll1 and Mll2 knockout mice aredifferent in adult tissues. For example, hematopoietic-specific loss ofMll1 showed defects in hematopoiesis [Mll2 loss did not show any aberrant bloodprofiles and notable pathology [MLL2  has a similar protein domain structure tothat of MLL1 and was found to be the MLL1 paralog . Like Mland Hoxb5. Howeverer E11.5 . Mll2−/−m layers . Therefoopoiesis ,72, wherathology .Drosophila Trr and were co-purified astranscriptional coactivator complexes [Drosophila and mammals. For example, the MLL3/MLL4 complex isrecruited to HOXC6 gene and activates its transcription in anestrogen receptor-dependent manner [MLL3 and MLL4 genes in human cancers, includingcolorectal cancer, non-Hodgkin B-cell lymphoma, and medulloblastoma [trr geneproduct suppresses cell growth in Drosophila eye imaginal discs. Ofinterest, trr mutation markedly reduced H3K4 monomethylation levelswithout significantly changing H3K4 di- and trimethylation levels [Drosophila enhancers [Mll3 homozygous mutant mice, which have an in-framedeletion of a 61-aa catalytic core of the SET domain, exhibited reducedwhite adipose tissue, stunted growth, and slow cellular doubling rate [MLL3  and MLL4  are mammaliancounterparts of omplexes ,78-80. Mt manner . Frequenblastoma -85. Consn levels , in agrenhancers . Mll3 hoing rate ,89. Duriing rate .NESTIN andkey developmental genes HOXA1–3 are activated by MLL4 duringRA-induced differentiation. Intriguingly, the tandem PHD4-6 ofseven PHD motifs in MLL4  and myogenticregulator genes  [Mll5 knockout male mice are sterile, at least in partbecause of deregulated expression of genes that are required for terminaldifferentiation during spermatogenesis [Independent studies have demonstrated that MLL5 is required for hematopoiesis -94. Moreyogenin) . Mll5 knogenesis . Of inteogenesis ,95, GlcNogenesis .Cfp1−/− ES cells displayed aberrant H3K4me3 peaks atnumerous ectopic sites , suggesting that CFP1 recruits the SET1 complex to CpGisland-containing promoters and consequently prevents it from generatingH3K4me3 to inappropriate chromatin locations [Human SET1A and SET1B have an N-terminal RNA recognition motif and aC-terminal enzymatic SET domain Figure . TheSETocations ,98,99.A protein sequence analysis revealed that SET1A shares 39% identity with aSET domain protein named SET1B . AlthougDrosophilaTrithorax group protein that is essential for expression of severalHOX genes. Some reports have indicated that ASH1L primarilyacts as a H3K4 methyltransferase [HOX gene activation andis required for the myelomonocytic lineage differentiation of hematopoieticstem cells [HOX gene expression, suggesting that ASH1L’scatalytic activity is dispensable for hematopoietic stem celldifferentiation [ASH1L  is the human homolog of Ash1, a nsferase ,100,101,nsferase -104. ASHem cells . Of intentiation .Myogenin) and reduced gene expression bydecreasing H3K4me1 levels at its target genes. Intriguingly, SET7antagonizes Suv39h1-mediated H3-K9 methylation at the myogenicdifferentiation gene promoters [SET7  is an H3K4 mono- and di-methytransferase ,106-108.romoters .Smyd1 catalytic mutant, which lacks H3K4methyltransferase activity [Smyd2 has nophenotype during mouse heart development [Smyd1  is essential for mouse cardiac differentiation . Consistactivity . SMYD2 mactivity ,111-113.activity . Unlike elopment . SMYD3 ielopment ,115. It elopment .SETMAR  encodes a chimeric proteinthat contains an N-terminal SET domain and a C-terminal mariner transposasedomain [edomain   is a PR/SET domain-dependent histonemethyltransferase that is required for meiotic prophase progression . Deletio of mice . Mechani of mice -120.WDR5, a core subunit of the SET1 and MLL1−4 complexes, plays an importantrole in ES cell self-renewal and somatic cell reprogramming . WDR5 isDPY-30 and RBBP5 are other core components of the SET1/MLL methyltransferases. Incontrast to ASH2L and WDR5, DPY-30 and RBBP5 were not required for ES cellself-renewal . DPY-30 Differing biological outcomes for ASH2L and WDR5 from DPY-30 and RBBP5 aresurprising because these four proteins are core components of the sameSET1/MLL1−4 methyltransferases. These unexpected findings might beexplained by the following possibilities. Besides the known SET1/MLL1−4complexes, some of these subunits may be present in other complexes in the samecells so that they may exert different biological functions fromSET1/MLL1−4 complexes. In fact, gel filtration analysis of ES cell nuclearextracts showed that elution profiles of WDR5/OCT4 did not overlap with those ofWDR5/ASH2L/RBBP5, suggesting that WDR5 also belongs to another new complexcontaining OCT4 . AnotherThe reversibility of histone methylation was not clear until the discovery of thefirst histone demethylase LSD1 in 2004 . SubsequLSD1 protein contains an N-terminal SWIRM domain and a long C-terminalFAD-dependent amine oxidase domain (AOD). The AOD is divided by an insertionknown as the tower domain Figure . LSD1 alLsd1-null mice are embryonic lethal aroundE6.5, and Lsd1-deficient mouse ES cells demonstrate increased celldeath and impaired differentiation, such as embryoid body formation defects [p21, in neural stem cells [Lsd1 deletion results inprogressive loss of DNA methylation [Lsd1−/− ES cells had a strong potential to generateextraembryonic tissues from the embryoid body [Numerous studies in ES cells and neural stem cells strongly suggest that LSD1is a key histone methylation modifier in transcriptional regulation for stemcell fate determination.  defects -129. Simem cells . Interesem cells ,132. In hylation . Moreovehylation . In conthylation . In addioid body .4H2C2 and CW-type zinc fingers, whichare required for demethylase activity [Lsd2 is not essential for mouse development. However,the DNA methylation of several imprinted genes is lost in oocytes fromlsd2-deleted females [LSD2 (AOF1 or KDM1B) was recently identified as a homolog of LSD1; itdemethylates H3K4me2/1 like LSD1 ,134-136.activity   was identified as a binding partner of pRB protein inearly 1990 . RBP2 cos ofRbp2.HOX genes,resulting in increased H3K4me3 levels and gene activation [Alkalinephosphatase, Osteocalcin, and Osterix[During ES cell differentiation, RBP2 is dissociated from tivation . Consisttivation . A genomtivation . Beshiritivation . Interestivation . RBP2 ind Osterix.1, and C5HC2 zinc finger in additionto the catalytic domain JmjC [Breastcancer1, early onset (BRCA1), Caveolin 1(CAV1), and HOXA5 genes as a result of increasedH3K4me3 levels on their promoters [Plu1 knockout mice, which have the phenotype of neonatallethality and neural defects [JARID1B (PLU1 or KDM5B) was shown to be overexpressed in breast cancer celllines . As a meain JmjC ,34  and JARID1D (SMCY or KDM5D). Both demethylaseshave similar domain structures and contain a conserved and functional JmjCdomain that is responsible for demethylating H3K4me2/3 -32. SMCX5HC2 zinc finger, for its full demethylaseactivity towards H3K4me3/2 [in vitro; the protein complex mediates H3K4me3 demethylation atthe Engrailed 2 gene promoter and is required for Engrailed2 gene repression [in vivo.JARID1D requires multiple domains, including ARID, JmjC, andC3K4me3/2   via the PHD finger protein 19 (PHF19),which interacts with the H3K27 methyltransferase complex PRC2;NO66-PHF19-PRC2 represses gene expression by reducing H3K36me3 andincreasing H3K27me3 [NO66 has been reported to demethylate H3K4me3/2/1 and H3K36me3/2  and to cH3K27me3 .Stem cells are indistinguishable from somatic cells at the genomic level. Incontrast, there are remarkable differences in epigenomes that may be represented bycovalent and noncovalent modifications of histones and DNA. As reviewed herein,specific epigenetic modifiers, such as H3K4 methylation modifiers, may playfundamental roles in orchestrating cellular epigenomes whose genomic sequences areidentical. Consistent with this, many H3K4 methylation modifiers and theircomponents are required for ES cell self-renewal or differentiation. In addition,some of them cooperate with transcription factors for efficient somatic cellreprogramming. For example, WDR5 is required for the efficient generation ofpluripotent stem cells that were induced by Oct4, Sox2, c-Myc, and Klf4 . TherefoRecently, small molecule inhibitors against specific histone methyltransferases,including LSD1 inhibitors, have been developed by several pharmaceutical companies,although their specificities and efficacies require improvement . CertainThe authors declare that they have no competing interests.BG prepared the initial draft of the paper. MGL initiated and modified themanuscript. Both authors read and approved the final manuscript.


Drosophila melanogaster identified the SNARE gene Syntaxin 5 (Syx5) as playing an important role in copper regulation; flies heterozygous for a null mutation in Syx5 display increased tolerance to high dietary copper. The phenotype is shown here to be due to a decrease in copper accumulation, a mechanism also observed in both Drosophila and human cell lines. Studies in adult Drosophila tissue suggest that very low levels of Syx5 result in neuronal defects and lethality, and increased levels also generate neuronal defects. In contrast, mild suppression generates a phenotype typical of copper-deficiency in viable, fertile flies and is exacerbated by co-suppression of the copper uptake gene Ctr1A. Reduced copper uptake appears to be due to reduced levels at the plasma membrane of the copper uptake transporter, Ctr1. Thus Syx5 plays an essential role in copper homeostasis and is a candidate gene for copper-related disease in humans.Copper is essential for aerobic life, but many aspects of its cellular uptake and distribution remain to be fully elucidated. A genome-wide screen for copper homeostasis genes in  Copper (Cu) is essential to aerobic organisms as a cofactor in diverse metabolic processes including cellular respiration and proliferation; formation of connective tissue, melanin and neurotransmitters; antioxidant defence; cell signalling; and angiogenesis Ctr1 is the major copper uptake protein in mammalian cells, and is thought to form a trimer containing a pore at the plasma membrane through which copper can pass Due to the varied metabolic processes for which copper is required, there are a wide variety of copper-related diseases with diverse phenotypes. For example, Menkes disease is caused by impaired ATP7A-mediated transport of dietary copper from the polarised gut epithelial cells, resulting in systemic copper deficiency and Wilson disease is caused by impaired ATP7B-mediated transport of copper from the liver resulting in copper toxicosis Drosophila melanogaster has recently proven a useful model for characterizing the role of copper homeostasis genes. Drosophila has orthologues of all major copper homeostasis proteins and several studies have demonstrated the high level of functional conservation with humans Drosophila, two homologous proteins, Ctr1A and Ctr1B, fulfil the function of mammalian Ctr1. Ctr1A is constitutively expressed Ctr1B is induced in the midgut by dietary copper limitation and is needed to boost absorption Drosophila orthologue of mammalian copper transporting ATPases, ATP7A and ATP7B The vinegar fly Drosophila to further illuminate our understanding of copper homeostasis mechanisms Syntaxin 5 (Syx5) as playing an important role in copper regulation in the fly; flies heterozygous for a null mutation in Syx5 display significantly increased tolerance to high levels of dietary copper.A genetic screen was performed in trans-Golgi network (TGN) for docking of vesicles including the COPI type Syx5–mediated transport between endoplasmic reticulum (ER) and Golgi Drosophila Syx5 has recently been shown to play a role in translocation of proteins to the apical membrane and is also required for Golgi reassembly following cell division Drosophila Syx5. Given the known roles of mammalian Syx5 in both anterograde and retrograde intracellular trafficking, Syx5 represents an excellent candidate for involvement in uptake or intracellular distribution of copper. The current study presents evidence that Syx5 is vital for efficient copper uptake in insect and mammalian cells as well as in vivo in Drosophila.SNAREs are involved in fusion of vesicles to target membranes and are therefore central to intracellular trafficking Drosophila copper tolerance had been previously mapped to a single locus on Chromosome 2 encoding Syx5AR113/CyO DrosophilaSyx5 were screened for copper tolerance r10, cn, with a deletion spanning Syx5, were included as controls. The offspring from crosses of AR113/CyOSyx5×Armenia, Df(2L)r10, 1/CyOcn×Armenia, and a double-balancer stock (IF/CyO)×Armenia were also screened to confirm copper tolerance segregated with the Syx5 mutations.Increased olerance . The SyxAR113/CyOSyx5 and 1/CyODf(2L)r10, cn had significantly higher survival to the adult stage than Armenia, 1118w or IF/CyO when reared on a copper-supplemented diet, but no difference in survival was observed on basal media or the copper chelator BCS r10 deletion  and the A113Syx5 allele , but not with any balancer or wild-type chromosomes  is consistent with copper deficiency under basal conditions metallothionein (copper sequestration) response, raising expression to similar levels in both +/−Syx5 and wild-type larvae. MtnC was the only metallothionein to show lower expression compared to wild-type . These results are consistent with direct measurements of copper content  result in a copper load sufficient to increase +/−Syx5 mortality, despite the high tolerance compared to wild-type  Syx5 were used to suppress Syx5 activity. Targeted suppression of Syx5 in the developing eye (Gmr-GAL4), nervous system (Elav-GAL4) or midgut (Mex-GAL4) resulted in larval or pupal lethality, probably due to the essential role of this gene in intracellular trafficking and Golgi reassembly following cell division The GAL4-UAS system in Syx5 in the Pannier domain (Pnr-GAL4), a band down the centre of the developing thorax and abdomen, is also normally lethal. However rare survivors raised at 18°C show abdominal hypopigmentation phenotypes typical of copper deficiency Syx5 in the same domain resulted in reduced scutellum and bristles but no change in pigmentation  or DmATP7 (copper efflux) was unable to rescue the lethality caused by strong Syx5 suppression. Together with the Syx5 hypomorph combination  was examined in human embryonic kidney  cells following  surface . A biotiDrosophila in suggesting that copper homeostasis phenotypes are observed in otherwise viable, fertile flies when Syx5 levels are only mildly reduced. Greater reductions cause additional cellular disturbances and a range of phenotypes not necessarily related to copper homeostasis.Western blot densitometric analysis showed that Syx5 levels were reduced to 24–33% of wild-type in the human cell lines  and S5. Drosophila Syx5 orthologue is required for Golgi reassembly following cell division and for translocation of proteins to the apical membrane Roles for mammalian Syx5 in both anterograde and retrograde vesicular transport have been well characterized and severe Syx5 reduction causes fragmentation and dispersal of the Golgi Drosophila leads to significantly increased tolerance to high dietary copper with no adverse impact on viability or fertility. Drosophila copper tolerance can be altered by manipulating levels of known copper uptake proteins or metallothioneins directly or via suppression of the metallothionein transcription factor, MTF-1 Syx5 specifically in the adult thorax generated a typical copper-deficiency phenotype.In contrast, results presented here have shown that a 50% reduction in Syx5 levels in Drosophila pupae only, not in cell lines, and could therefore be a systemic response to reduced copper accumulation.Reduced Syx5 levels also resulted in elevated zinc accumulation in the fly but, unlike the copper phenotype, this did not affect zinc sensitivity. A possible explanation is that, with reduced copper levels, more metallothionein is available to sequester excess zinc and the flies are able to absorb more without detrimental effects. It is notable that this change in zinc accumulation was seen in in vivo copper deficiency phenotype is due to a reduction in the efficiency of copper accumulation. Importantly, consistent results were seen in both Drosophila and human cell lines, suggesting that Syx5 plays an evolutionarily conserved role in cellular copper uptake. In human cell lines, we observed no detectible effect on copper uptake kinetics over 10 minutes, but rather a gradual reduction in copper accumulation over a period of hours. Copper turnover was unaffected by suppression of Syx5 and copper levels differed most at steady state levels.The study of copper uptake and retention in cultured cells confirmed that the Drosophila Syx5 have impaired transport of proteins to the apical membrane of epithelial cells in vivo.Consistent with the finding that Syx5 suppression affects the copper uptake pathway, hCtr1 levels at the plasma membrane were reduced to 20% of control when Syx5 was suppressed in human cells. Previous studies have found that flies lacking +/−Syx5 heterozygotes examined here are otherwise healthy, this raises the possibility that mild loss of Syx5 function may be important in copper-related disease in humans. This may be particularly relevant to conditions such as cancer and Alzheimer's disease, where subtle changes in cellular copper regulation may influence progression of the disease Although mild dispersion of the Golgi was seen in Syx5 suppression cells and it has been shown previously that the loss of Syx5 can cause severe cellular and fertility defects We have presented evidence for a key role of Syx5 in cellular copper uptake, indicating that it plays a significant role in copper homeostasis. The finding that mild loss of Syx5 function significantly influences intracellular copper levels in the absence of other obvious phenotypes at the whole organism level provides a novel candidate for etiology of diseases resulting from copper dyshomeostasis Drosophila strains were maintained on standard medium at 25°C. Armenia, 60Arm . 1118w . ‘Df(2L)r10’, Df(2L)r10, 1/CyOcn (BL1491). AR113/CyOSyx5, also referred to as +/−Syx5 (BL3645) w; IF/CyO; MKRS/TM6b, Tb . Gmr-GAL4, −}2P{Gmr-GAL4.w (BL9146). Mex-GAL4 MD237P{GawB}pnr (BL3039). ‘UAS-Syx5’, RNAi transformants 3857 and 3859 specific for Syx5 yielded the same results, and data shown are for 3857 (Vienna Drosophila RNAi Center).All 4.5H2O; Merck) as specified in the text. Survival to the adult stage was measured for five replicates of 50 first instar larvae per condition. Male AR113Syx5/CyO, Df(2L)r10/CyO or IF/CyO mutants were crossed to female Armenia and visible Chromosome 2 markers were used to monitor segregation of AR113Syx5, Df(2L)r10 and CyO. Offspring from the replicates were pooled for χ2 analysis.Standard medium was supplemented with 100–1000 µM of the copper chelator bathocuproinedisulfonic acid  or 1–4 mM Cu  pre-pulled glass needles. Integrants at both these attP sites were obtained. The 5′-TCATGGATCCGAAAAAATGGAACAAAAACTCATCTCAGAAGAGGATCTGGATCATTCCCACCATATGGG -3′) and reverse (5′-GGGCTCTAGAGAATTCAATGGCAATGCTCTGTGATATC -3′) oligonucleotides and by incorporation into the mammalian expression vector, pcDNA3. The forward oligonucleotide introduced sequence encoding the myc epitope in-frame immediately after the start codon and provided a flanking 5′ BamH1 endonuclease restriction site. The reverse oligonucleotide provided an EcoRI endonuclease restriction site 3′ to the stop codon. Template cDNA was isolated from human hepatoma HepG2 cells  using the SuperScriptTM III CellsDirect cDNA synthesis system (Invitrogen) following the manufacturer's protocol. The PCR reaction contained 1×PCR buffer, 0.2 mM of each dNTP, 2 mM MgCl2, 0.2 µM of each primer, 2.5 units Platinum Taq DNA polymerase and 3 µl of cDNA (Invitrogen). Reactions were run on an Eppendorf Epgradient S Mastercycler on the following program: one cycle of 94°C for 2min, 38 cycles of 94°C for 45s, 57°C for 60s and 72°C for 60 s, followed by one cycle of 72°C for 2 min. The resultant PCR product was digested with BamH1 and EcoR1 and cloned into pcDNA3 at the same sites. Integrity was confirmed by sequencing. Stable transfection of HEK293 cells  with the myc-tagged Ctr1 construct was performed using FuGENE® HD (Roche) following the manufacturer's instructions. The cells were recovered in Dulbecco's Modified Eagle's medium (DMEM) containing 10% (v/v) FCS and transfectants were selected with 500 µg/ml G418 for 14 days.The myc-tagged Ctr1 construct was generated through PCR amplification of cDNA using the forward  as previously reported Ctr1A or Ctr1B were generated by co-transfecting pCoHygro with either pAcCtr1A, pAcCtr1B or pAc empty vector control using Lipofectamine2000 and propagated in Schneider's Complete Media (Invitrogen) with 10% foetal calf serum (Trace Scientific) supplemented with 300 µg/ml hygromycin-B according to the manufacturer's instructions (Invitrogen). Media was replaced with SFM for all experiments and supplemented with CuCl2 (Sigma) at the concentration specified in the text. Wild-type (GM2069) and ATP7A null (Me32a) human fibroblast cells have been described previously Syx5 (cDNA bases 214–757). Control dsRNA was derived from EYFP cDNA or Adult Cuticle Protein 1 (13–558), which is not expressed in S2 cells. siRNA suppression in mammalian cells utilized Stealth RNAi duplexes (Invitrogen). 40 nM Syx5 or low GC negative control Stealth RNAi duplexes were transfected into mammalian cells with Lipofectamine2000 according to the manufacturer's instructions (Invitrogen). Cells were seeded to be 30–50% confluent on the day of transfection and growth media was replaced with Opti-MEM (Invitrogen). Opti-MEM was replaced with growth media 4–6 h after transfection 48 h before experiments. Gene suppression was confirmed using qPCR and western blot. qPCR was performed as previously described GAPDH, Actin42A and βActin were used for normalisation in Drosophila larvae, Drosophila S2 cells and mammalian cells respectively. Primer sequences are shown in dsRNAi in S2 cells was conducted as previously reported 64Cu  and non-radioactive copper at the concentrations described in the figure legends. Copper retention was measured by incubating cells with copper for 24 h, washing, and incubating for an additional 2–8 h in basal media. Radioactivity was measured with a γ-counter . Copper levels were normalized to total cellular protein, which was determined using BioRad protein reagent according to the manufacturer's instructions (BioRad). Total metal accumulation was measured using a Vista-AX Inductively Coupled Plasma Atomic Emission Spectrometer  in samples digested in 70% HNO3 and metal levels were measured as described previously Drosophila metal levels were measured in five replicates of 50 pupae and expressed as ng/pupa. S2 cell metal levels were normalized to total cellular protein.Copper accumulation was measured as previously reported Cell surface proteins were labelled with 0.5 mg/ml sulpho-NHS-SS-biotin (Thermo Scientific) and precipitated with streptavidin-agarose beads (Thermo Scientific) as previously described Statistical analysis was conducted using SPSS v16 (SPSS). A one-sample Kolomogorov-Smirinov test was used to assess whether data was normally distributed. Statistical analyses are described in Figure legends. P<0.05 was deemed statistically significant.Table S12 values comparing siblings from crosses to wild-type Armenia.χ(0.03 MB DOC)Click here for additional data file.Table S2Quantitative PCR primer sequences.(0.03 MB DOC)Click here for additional data file.Figure S1+/− DrosophilaSyx5 show no viability or fertility defects. Ten individual pairs each of +/−Syx5 heterozygous virgin females and 1118w males (Syx5 f×w1118 m), 1118w virgin females and +/−Syx5 heterozygous males (w1118 f×Syx5 m), or 1118w virgin females and 1118w males (w1118×w1118) were maintained in vials containing standard laboratory medium which was replaced every 24 h. Fertility was measured by allowing pairs 24 h to mate, then counting eggs produced every 24 h for five days (A). Male reproductive output was measured as the egg production of females inseminated by +/−Syx5 males. Twenty replicates of 50 eggs were transferred into vials containing standard laboratory medium and viability was scored as the number of adults to emerge after 20 days (B). The average number of eggs laid per female over a five day period and the average number of eggs to reach the adult state were compared among strains using a one-way ANOVA with LSD post-hoc testing. Since both −/w1118Syx5 and 1118CyO/w offspring were produced in +/−Syx5 crosses, an independent samples T-Test was used to confirm there was no difference in survival between these sibling genotypes then they were pooled for comparison to the 1118w strain. The Syx5 mutation did not adversely affect fertility: there was no significant difference in the number of eggs produced from either cross compared to those produced by the 1118w control strain . There was no adverse impact on viability of eggs from +/−Syx5 parents, in fact there was slightly higher survival of the 1118w female×+/−Syx5 (44±1 s.e.m.) male compared to the 1118w strain . Emergence from the reciprocal cross was intermediate (40±1) and not significantly different from either.(0.14 MB TIF)Click here for additional data file.Figure S2+/−Syx5 heterozygote Drosophila. Metal content was measured by ICP-AES on flies reared to the pupal stage on basal media (A). Data are mean ± s.e.m. metal content per pupa from five replicates of 50 pupae and are expressed relative to wild-type (1118w) levels. In addition to a decrease in copper accumulation (shown in detail in +/−Syx5 flies accumulated 1.5-fold more zinc than wild-type (A). Zinc tolerance was determined as described for copper tolerance in the main text by supplementing media with 0–8 mM zinc  (B). No significant differences in mortality were detected.Metal accumulation and zinc tolerance in (0.37 MB TIF)Click here for additional data file.Figure S3+/− DrosophilaSyx5. Wild-type and +/− DrosophilaSyx5 were reared to third instar on basal media (A) or 1 mM copper (B) and qPCR was used to investigate Ctr1B and MtnA-D expression levels from three replicates of 50 larvae. Ctr1A has no transcriptional response to copper levels and is included as a control. Gene expression is mean relative to wild-type. Error bars are s.e.m. Under basal conditions Ctr1B is upregulated in +/−Syx5 larvae, indicative of copper deficiency. Copper exposure alleviates the deficiency and leads to similar MtnA, MtnB and MtnD upregulation in +/−Syx5 and wild-type larvae. An independent samples T-Test was used to determine statistical significance for differences between +/−Syx5 and wild-type exceeding a two-fold magnitude, as indicated by dotted lines (*P<0.05).Copper-responsive gene expression in (0.24 MB TIF)Click here for additional data file.Figure S4Syx5 suppression in human cells does not significantly affect copper uptake kinetics. (A) Copper uptake measured over one hour in GM2069 cells treated with control (squares) or Syx5 (circles) siRNA for 48 h. 64Cu was used to measure copper accumulation in cells exposed to 2 µM copper for 5–60 minutes. Values are mean with s.e.m. of nine replicates from three experiments. There was a tendency for the rate of copper accumulation to be lower following Syx5 suppression, however linear regression analysis demonstrated that this was not significantly different. (B) GM2069 cells treated with control (squares) or Syx5 (circles) siRNA for 48 h. 64Cu was used to measure copper accumulation in cells exposed to 2–100 µM copper for 10 min. Values are mean with s.e.m. of six replicates from two independent experiments. Non-linear regression analysis demonstrated that copper uptake kinetics were not statistically significant different following knockdown of Syx5.(0.31 MB TIF)Click here for additional data file.Figure S5Syx5. GM2069 and Me32a cells treated with control or Syx5 siRNA for 48 h. Western blot analysis of whole cell lysate from these cells using anti-Syx5 antibody detected two electrophoretic species as previously reported Syx5 suppression in each of these cell lines: Densitometry analysis demonstrates that, relative to control cells, Syx5 protein levels were reduced to 27.7% in GM2069 and 31.1% in Me32a cells. 1. Subramaniam VN, Loh E, Hong WJ (1997) N-ethylmaleimide-sensitive factor (NSF) and alpha-soluble NSF attachment proteins (SNAP) mediate dissociation of GS28-syntaxin 5 Golgi SNAP receptors (SNARE) complex. J Biol Chem 272: 25441–25444.RNAi suppression in human cells reduces protein levels for (0.24 MB TIF)Click here for additional data file.Figure S6Syx5 suppression does not cause substantial Golgi fragmentation or affect early endosome localization in human cells. Golgi distribution: Immunocytochemistry in GM2069 cells utilized anti-Syx5 (1∶50) and mouse anti-Golgin 97 was used as a TGN marker . Secondary antibodies were Alexa 488 anti-rabbit and Alexa 594 anti-mouse . DAPI  was used to detect the nucleus. Images were recorded at 100× magnification using an Olympus FluoView 1000 confocal microscope with Olympus FluoView ver1.6a software (Olympus). Images at each wavelength were captured sequentially and multi-color maximum brightness stacked images were prepared using Image J . GM2069 cells were treated with control siRNA (A–C) or Syx5 siRNA (D–F). Syx5 is shown in Green , Golgin 97 is shown in Red  and DAPI is shown in Blue. Merged images are also shown . Syx5 suppression reduced Syx5 levels but did not dramatically alter the distribution of Golgin 97 (D–F). Golgi distribution was measured using Image J and was found to be 42.5±7.2 µm2 in control and 72.2±9.7 µm2 following Syx5 suppression. Thus the Syx5 RNAi suppression achieved in this study produced a mild phenotype in comparison to the effects of extreme Syx5 inhibition found in previous studies Early endosome localization: Immunocytochemistry in GM2069 cells utilized anti-Syx5 (1∶50) and mouse anti-EEA1 was used as an early endosome marker . Secondary antibodies, DAPI staining and image analysis were conducted as described above for Golgi distribution. GM2069 cells were treated with control siRNA (G–I) or Syx5 siRNA (J–L). Syx5 is shown in Green , EEA1 is shown in Red  and DAPI is shown in Blue. Merged images are also shown . The localization of early endosomes was not affected by Syx5 suppression (J–L). 1. Amessou M, Fradagrada A, Falguieres T, Lord JM, Smith DC, Roberts LM, Lamaze C, Johannes, L (2007) Syntaxin 16 and syntaxin 5 are required for efficient retrograde transport of several exogenous and endogenous cargo proteins. J Cell Sci 120: 1457–1468. 2. Diao A, Frost L, Morohashi Y, Lowe M (2008) Coordination of Golgin Tethering and SNARE Assembly: GM130 binds Syntaxin 5 in a p115-regulated manner. J Biol Chem 283: 6957–6967.(0.84 MB TIF)Click here for additional data file.


Eucalyptus gunnii CCR and CAD2 promoters direct similar expression patterns in vascular tissues suggesting that monolignol production is controlled, at least in part, by the coordinated transcriptional regulation of these two genes. Although consensus motifs for MYB transcription factors occur in most gene promoters of the whole phenylpropanoid pathway, functional evidence for their contribution to promoter activity has only been demonstrated for a few of them. Here, in the lignin-specific branch, we studied the functional role of MYB elements as well as other cis-elements identified in the regulatory regions of EgCAD2 and EgCCR promoters, in the transcriptional activity of these gene promoters.Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the final steps in the biosynthesis of monolignols, the monomeric units of the phenolic lignin polymers which confer rigidity, imperviousness and resistance to biodegradation to cell walls. We have previously shown that the in vivo footprinting, we identified an 80 bp regulatory region in the Eucalyptus gunnii EgCAD2 promoter that contains two MYB elements, each arranged in a distinct module with newly identified cis-elements. A directed mutagenesis approach was used to introduce block mutations in all putative cis-elements of the EgCAD2 promoter and in those of the 50 bp regulatory region previously delineated in the EgCCR promoter. We showed that the conserved MYB elements in EgCAD2 and EgCCR promoters are crucial both for the formation of DNA-protein complexes in EMSA experiments and for the transcriptional activation of EgCAD2 and EgCCR promoters in vascular tissues in planta. In addition, a new regulatory cis-element that modulates the balance between two DNA-protein complexes in vitro was found to be important for EgCAD2 expression in the cambial zone.By using promoter deletion analysis and cis-elements clearly demonstrates the importance of MYB cis-elements in the transcriptional regulation of two genes of the lignin-specific pathway and support the hypothesis that MYB elements serve as a common means for the coordinated regulation of genes in the entire lignin biosynthetic pathway.Our assignment of functional roles to the identified  The most characteristic components of secondary cell walls are lignins, complex phenolic polymers, which play fundamental roles in mechanical support, water and solute conductive properties and disease resistance in higher plants ) involved in the transcriptional regulation of EgCAD2  EgCAD2 and [-119/-70] EgCCR regulatory fragments with mutated MYB sites  whereas an unlabelled non-specific DNA  did not affect the formation of the complexes. Mutation of the MYBa and MYBb sites in EgCAD2 and mutation of the MYB site in EgCCR abolished the binding of recombinant EgMYB2 to the regulatory fragments. This loss of binding occurred even when a single MYB site remained intact in the EgCAD2 promoter, indicating that both MYB sites are required for maximal binding of EgMYB2.EMSAs were performed using recombinant tes Fig. . The speEgMYB2 cDNA expressed under the control of the CaMV 35S promoter (effector construct) and wild-type or mutated -203 EgCAD2 or -119 EgCCR promoters-GUS fusions (reporter constructs) into leaf mesophyll cells . Interestingly, the endogenous activity of the MYBa-mutated promoter was notably higher than that of the wild-type promoter, suggesting that the MYBa element might bind a repressor, at least in leaf mesophyll cells. Single or combined mutations of the MYBa and MYBb elements reduced the GUS expression driven by EgMYB2 to the same level as their respective controls, thereby reflecting the loss of activation by EgMYB2. Again, this effect was observed even with a single mutated MYB site, indicating that both MYB sites are required for transactivation of the EgCAD2 promoter by EgMYB2. Transactivation of the -119 EgCCR wild-type promoter by EgMYB2 by about 2-fold was also abolished upon mutation of the MYB site  and namCompetition with a 100-fold molar excess of the corresponding unlabelled specific competitor DNA fragments blocked the formation of all the complexes , similar results were obtained as with the mutation of MYBb alone, ie loss of the H and L2 complexes. Taken together, these data suggest that MYBb is a key cis-element involved in the formation of the H and L2 complexes and that BSb, and to a lesser extent BSa, apparently influence the ratio between the H and L2 complexes. Concerning the EgCCR promoter, only the mutation of the MYB element led to an overall decrease in all the complexes  when compared to the wild-type -203 promoter. Similarly, mutation of the MYB site in the EgCCR promoter resulted in a marked decrease of GUS activity (63%) when compared to the wild-type -119 promoter  with the sequence CTGGTT and an MBSIIG consensus site for MYB proteins. A simpler organisation exists in the [-119/-70] regulatory region of the EgCCR promoter with two putative cis-elements: a G-rich box (BS1) and a MBSIIG MYB consensus site  in the sus site .cis-elements revealed the importance of the MYBb cis-element in EgCAD2 and of the MYB cis-element in EgCCR for the formation of the high and low mobility complexes and for the transcriptional activation of the EgCAD2 and EgCCR promoters in vascular tissues.Our results with EMSAs and transgenic plants with mutated EgCAD2 promoter activity. This does not exclude its participation in other promoter functions, such as the response to external stimuli  EgCCR promoter regulatory fragments used as probes for EMSA were obtained by amplification of pCambiaZ containing the wild-type or mutated cis-elements. Primers (4 pmol) common to the wild-type or mutated promoters were phosphorylated with 4 pmol of g[ 32P]-ATP  and T4 polynucleotide kinase (Promega) in 8 μl at 30°C for 30 min. The regulatory fragments were amplified by Pfu DNA polymerase with the radiolabelled common primer and variable second primers, depending on the introduced mutation . The BY2 cell protein extract was clarified by centrifugation for 30 min at 35000 g.Binding reactions were performed at room temperature for 30 min in 25 μl binding buffer containing 25 mM Tris-HCl, pH 8, 250 mM NaCl, 10% glycerol, 7 mM β-mercaptoethanol, 2 μg poly (dIdC): poly(dIdC), 75 000 cpm of labelled probe and protein extracts. Free and bound DNAs were separated on 4.8% polyacrylamide gels in 0.5× TBE, 2.5% glycerol. Gels were then fixed, dried and autoradiographed.GUS fluorimetric tests were carried out as described previously by using 4-methylumbelliferyl-β-D-glucuronide as substrate . MeasureHistochemical localization of GUS activity was performed as described by  after prE. coli strain BL21 containing the plasmid pGEX-5X-1-EgMYB2 cDNA was described in [EgMYB2 fusion protein was performed by adding isopropyl β-D-thiogalactoside (Sigma) to a final concentration of 0.1 mM. After growth at 28°C for 5 h, cells were lysed by two passages through a French press at 12000 p.s.i.  in 20 mM potassium phosphate buffer, pH 7.5, 140 mM NaCl, 0.5 mM PMSF and 10 mM β-mercaptoethanol. The fusion protein was purified by FPLC  on prepacked 1 ml GSTrap™ FF columns (Amersham biosciences) following the supplier's instructions. Proteins were eluted with 50 mM Tris-HCl, pH 8 containing 10 mM reduced glutathione protein (Sigma) and were then concentrated and dialysed by centrifugation on a Vivaspin concentrator  with 50 mM Tris-HCl, pH 8, 140 mM NaCl, 7 mM β-mercaptoethanol, 20% glycerol. Protein concentration was determined with the Bradford reagent (Bio-Rad).The ribed in . InductiEgMYB2 cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter) were as in [EgCAD2 promoter-GUS or EgCCR promoter-GUS fusions) are described above. Agrobacterium strains GV3101:pMP90 containing effector or reporter constructs were co-infiltrated into leaves of Nicotiana benthamiana at the 5-leaf stage using a 1 ml syringe. After infiltration, the plants were maintained for 2 days in a growth chamber at 28°C under a photoperiod of 16 h light. Quantitative GUS assays were carried out on the infiltrated zone.Co-transfection experiments were performed essentially according to the method of . Effectore as in . ReporteAR and PR performed the experiments. AR helped to draft the manuscript. YM participated in the histological analysis. JGP initiated and coordinated the research. NCG and VP conceived and designed the study, supervised the assays, analyzed the data, discussed the results and wrote the paper. All authors read and approved the final manuscript.EgCAD2 gene expression in Eucalyptus suspension-cultured cells.Click here for fileCAD2 and CCR promoters from several Eucalyptus speciesSequence analysis of the regulatory regions of the .Click here for fileEgCAD2 promoters containing mutated BSa, MYBa or MYBa-MYBb sitesGUS expression driven by .Click here for filePosition of MBSIIG and MBSII MYB consensus elements within the 500 bp proximal promoter regions of phenylpropanoid biosynthesis genes.Click here for fileOligonucleotide primers used for the various experiments.Click here for file