Oryzias latipes) a duplicated copy of dmrt1, designated dmrt1bY or DMY, on the Y chromosome was shown to be the master regulator of male development, similar to Sry in mammals. We found that the dmrt1bY gene has acquired a new feedback downregulation of its expression. Additionally, the autosomal dmrt1a gene is also able to regulate transcription of its duplicated paralog by binding to a unique target Dmrt1 site nested within the dmrt1bY proximal promoter region. We could trace back this novel regulatory element to a highly conserved sequence within a new type of TE that inserted into the upstream region of dmrt1bY shortly after the duplication event. Our data provide functional evidence for a role of TEs in transcriptional network rewiring for sub- and/or neo-functionalization of duplicated genes. In the particular case of dmrt1bY, this contributed to create new hierarchies of sex-determining genes.Control and coordination of eukaryotic gene expression rely on transcriptional and posttranscriptional regulatory networks. Evolutionary innovations and adaptations often require rapid changes of such networks. It has long been hypothesized that transposable elements (TE) might contribute to the rewiring of regulatory interactions. More recently it emerged that TEs might bring in ready-to-use transcription factor binding sites to create alterations to the promoters by which they were captured. A process where the gene regulatory architecture is of remarkable plasticity is sex determination. While the more downstream components of the sex determination cascades are evolutionary conserved, the master regulators can switch between groups of organisms even on the interspecies level or between populations. In the medaka fish ( dmrt1 transcription factor gene, dmrt1bY, has become the sex master regulator. Its ancestor dmrt1a, in contrast, has a downstream position in the sex determination cascade. We show that after the duplication of the dmrt1 gene, the new hierarchy has been established by the insertion of a transposable element into the regulatory region of the dmrt1bY gene on the sex chromosome. This transposable element, harboring a Dmrt1 binding site, enables the self- and cross-regulation of dmrt1bY expression by Dmrt1 proteins. Our study therefore provides strong evidence for the important role of transposable elements in the rewiring of gene regulatory networks.Evolutionary innovations and adaptations often require rapid changes in gene regulation. Transposable elements constitute the most dynamic part of eukaryotic genomes. Insertions of transposable elements can influence the expression of surrounding genes by donating new regulatory elements. A longstanding hypothesis postulates that the dispersal of transposable elements may rewire regulatory links between genes, thereby changing regulatory networks and shuffling regulatory cascades. A regulatory hierarchy of remarkable plasticity is the sex determination cascade. In the course of animal evolution, new master regulators frequently replace the sex determination gene on top of the hierarchy. In the medaka fish, a duplicate of the  While such alterations may occur in any cis-regulatory module, they will have fundamentally different effects depending on where in the structure of the network they occur  have recently been attributed an important role in shaping the gene regulation landscape Such rewiring is especially important when rapid adaptation of existing regulatory networks or new networks become necessary. One system where fast changes obviously regularly occurred during evolution is the genetic control of sexual development Drosophila doublesex and C. elegans mab-3 sex regulatory genes, is the Dmrt1 (Doublesex and Mab-3 Related Transcription factor 1) gene of vertebrates DMRT1 and its paralogs DMRT2 and DMRT3 leads to XY male to female sex reversal Dmrt1 is located on the Z chromosome, but absent from W, making it an excellent candidate for the male sex-determining gene of birds The most conserved downstream component characterized to date, a gene with homology to both the Oryzias latipes), which has XY-XX sex determination, a duplicated copy of dmrt1, designated dmrt1bY or DMY, on the Y-chromosome was shown to be the master regulator of male development Sry in mammals. Interestingly, also in Xenopus laevis a W-specific duplicate of dmrt1 was shown to participate in primary gonad development dmrt1bY acts, like Sry, as a dominant male determiner Sry have been characterized, as well as a number of genes involved in these processes , with those of the available dmrt1 orthologs from other teleosts , chicken and human. This phylogenetic footprinting approach should point to the conservation of regulatory elements being putatively essential for vertebrate Dmrt1 gene expression  as middle segment (“repeat 2”)  .5′-CAATGAGTTATATCACTAGAGGAGACA-3′) assigning it to DNA transposons (class II transposable elements). However, it does not contain a transposase gene or any other open reading frame and thus constitutes a non-autonomous class II element. Only few diagnostic motifs are available to classify such elements dmrt1bY promoter has a 8 bp target site duplication (5′- GTGTGGCT-3′) and other copies of this element in the medaka genome have target site duplications of the same length. Repeat 1 is found in multiple copies in the medaka genome  also represent repetitive sequences with multiple copies in the medaka genome. These two repeat regions are found side by side in other regions of the genome. Thus, they together build a larger repeat element (“repeat 1”) into which repeat 2 was inserted (see below). Repeat 1 has a length of 1316 bp and is characterized by 27 bp terminal inverted repeats (TIRs)  P element element that we term Izanagi  and region IV (“repeat 4”) also have multiple copies in the medaka genome . They dodmrt1bY promoter region (9.107 Kb) was next analyzed for the presence of putative transcription factor binding sites using the MatInspector program  and the activity of the promoter was measured in a variety of cell types using transient transfection analysis. Sequential deletions of the 9 Kb promoter were generated from pBSII-ISceI::9 Kb Dmrt1bY prom::GLuc. In all three cell types basal promoter activity was detectable when using the 3 Kb proximal region (Xiphophorus A2 and medaka HN2), but not in Sertoli TM4 cells, a dramatic drop in promoter activity was observed when the region from bp −2985 to −6207 was added  are located at positions −5933 and −5524  were scanned for such target site sequences. A unique and robust Dmrt1 binding site of high prediction probability was found only in the medaka dmrt1bY promoter  (dmrt1a promoter (lower threshold set to 0). Interestingly, this predicted Dmrt1 binding site is nested within the above newly described Oryzias latipes Izanagi element in the proximal active part of dmrt1bY promoter  , S3, S4 promoter . The medpromoter . We firsequences . In totapromoter . Furtherdmrt1bY promoter by the Izanagi element has been important for the evolution of dmrt1bY function within the sex determining cascade, we asked about the timing of the Izanagi insertion in relation to the duplication of the medaka dmrt1 genes. The dmrt1 gene duplication occurred in a common ancestor of medaka (O. latipes), O. curvinotus and O. luzonensis around 10 million years ago Given that the Dmrt1 binding site donated to the dmrt1bY promoter and the Izanagi element consensus and mapped it onto a linearized neighbour joining (NJ) tree of dmrt1 genes from the genus Oryzias, which was based on neutral sites only (third codon positions). This analysis showed that the repeat 1 insertion occurred after the split from O. mekongensis but before the divergence of medaka, O. curvinotus and O. luzonensis  and embryonic stem (MES1) cells)  in comparison to the wild-type promoter  cells) . To conf created . When copromoter  whereas promoter .dmrt1bY promoter. The above-described experiments employing Dmrt1bY were repeated, this time using the autosomal Dmrt1a. When the proximal 2868 bp dmrt1bY promoter region containing the putative Dmrt1 binding site fused to luciferase was co-transfected with a plasmid expressing Dmrt1a, dmrt1bY promoter activity was considerably reduced –up to 92%- (dmrt1bY (ranging from −55% to 74%). Using the mutant dmrt1bY promoter revealed that removing the Dmrt1 target site was able to restore transcriptional activity (We next addressed the question of a possible cross-regulation of the Dmrt1a protein towards the  to 92%- . This reactivity  in medakdmrt1bY promoter (dmrt1bY Dmrt1-target sequence and in vitro translated Dmrt1a or Dmrt1bY demonstrated that both proteins are indeed able to bind to the Dmrt1 target sequence (position −2132 bp) (Electrophoretic mobility shift assays (EMSAs) were performed to show the direct interaction of Dmrt1a and Dmrt1bY proteins within the target site in the promoter . DNA bin2132 bp) . Binding2132 bp) .dmrt1bY and regulation by its paralog Dmrt1a in vitro. We next addressed whether this regulation indeed exists in vivo.Thus far we could show feed back down-regulation of in vivo dmrt1bY transcriptional regulation, a transgenic line where the dmrt1bY promoter drives dmrt1bY::GFP fusion protein expression was established with the goal of quantifying gonadal dmrt1bY promoter activity in either Dmrt1a expressing or non-expressing tissues (dmrt1bY promoter activity (5.5 times more in average) in absence of dmrt1a expression (ovary) compared to the dmrt1a expressing (testes) background  on testis tissue using GFP antibody for immunoprecipitation. An up to 7-fold enrichment compared to the control confirmed the capacity of Dmrt1bY to bind not only to the Dmrt1 promoter-nested Izanagi-target site but also to the Izanagi Dmrt1bY-target site in general  gonadal expression  the decline of dmrt1bY promoter driven fluorescence was paralleled by a rise of dmrt1a promoter expression in Sertoli cells (dmrt1bY promoter expression remained only in few Sertoli cells scattered around the germ cells (dmrt1a promoter was now predominantly expressed (Stable transgenic lines expressing fluorescent reporter protein (GFP and/or mCherry) were established to monitor the expression dynamics of Dmrt1a or Dmrt1bY pression  in two dpression  as well pression . This expression . Noteworli cells . In fullrm cells  while thxpressed .C. elegans to mammals, genetic evidence has suggested that the dmrt1 gene is an important regulator of male development at a downstream position of the regulatory network. In medaka, a duplicated copy of dmrt1 has acquired the upstream position of the sex-determining cascade. The analysis presented here provides evidences that this evolutionary novelty, which is predicted to require a rewiring of the regulatory network is brought about by co-option of “ready-to -use” pre-existing cis-regulatory elements carried by transposing elements. We could show that the master sex determining gene of medaka, dmrt1bY, is able to bind to one of these elements in its own promoter. This binding leads to a significant repression of its own transcription.Sex determination involves a complex hierarchy of genes. Expression screen analyses have resulted in hundreds of candidate genes that show sex-specific expression pattern. However it has been difficult to place these genes into a network of gene regulation and function. Nevertheless, several genes encoding for transcription factors, with specific temporal and spatial expression patterns during early gonad induction, have been suggested to participate in this process. Among them, from dmrt1bY is exclusively expressed and exerts its sex determining function dmrt1a gene, with its proposed specification and maintenance function for the Sertoli cells, is expressed only when the testes are in the process of differentiation. Notably, the master sex determinator gene dmrt1bY, continues to be expressed. In adult testes, where both paralogs have been shown to be expressed, the predominant expression of dmrt1a compared to dmrt1bY  indicates a particular transcriptional context acquired by dmrt1bY during its evolution towards becoming a novel master sex determination gene.So far no putative Dmrt1 binding site could be observed within the more than 10 Kb upstream medaka cis-regulatory element containing the Dmrt1 binding site, pre-existing within the Izanagi element at the time of its insertion, was co-opted in order to confer dmrt1bY its specific expression pattern after gene duplication around 10 million years ago Izanagi elements via the Dmrt1 binding site  and dmrt1bY (LG1) region sequences were obtained from http://www.ensembl.org/; release 49, March 2008): stickleback groupXIII, scaf57; Fugu scaf4; Tetraodon chr12, scaf14966; zebrafish chr5, scaf463; chicken chrZ, supercontig194; human chr9, supercontig NT_008413.Comparative analysis of vertebrate http://www.repeatmasker.org/). Additional copies of repeat elements and their genomic environment in the medaka genome  were identified with BLASTN with >85% sequence identity over >85% of query length (Izanagi consensus was identified by comparison to the PFAM database (http://pfam.sanger.ac.uk/).Screens for repetitive elements were performed with RepeatMasker (y length . Alignmehttp://www.genomatix.de/). Binding sites for Dmrt1 in different genomes were identified using the matrix provided by http://rsat.ulb.ac.be/rsat/). MEGA4 Izanagi element consensus (0.034 +− 0.005) as well as between repeat 2 and the Rex1 element consensus (0.010 +− 0.003) using the Kimura-2-parameter model. Linearized neighbor-joining trees of dmrt1 and tyrosinase a gene were obtained as described in ref. Transcription factor binding sites were determined using MatInspector of the Genomatix portal (open reading frame (ORF) was isolated by restriction enzyme digestion (XhoI/EcoRI) from BAC clone Mn0113N21 Gaussia luciferase gene from pGLuc-basic (New England Biolabs) plasmid was inserted between EcoRI and NotI sites of pBSII-ISceI::9 Kb Dmrt1bY prom (pBSII-ISceI::9 Kb Dmrt1bY prom::GLuc plasmid). pBSII-ISceI::3 Kb Dmrt1bY prom::GLuc and pBSII-ISceI::6 Kb Dmrt1bY prom::GLuc plasmids were constructed the same way removing 5′ fragments of the 9107 bp Dmrt1bY promoter region using Eco47III and HindIII restriction enzyme digestion respectively and re-ligation.For promoter analysis, a 9107 bp fragment upstream of the Dmrt1bY AATGCATTGTTGCAG; mutated form: GCCGGCTTCCCACCA). All PCR-obtained fragments were sequenced.Mutation of the Dmrt1bY binding site was performed by PCR in the context of pBSII-ISceI::3 Kb Dmrt1bY prom::GLuc plasmid . GFP fusion protein vector (dmrt1bY::GFP and deltadmrt1bY::GFP) were constructed as described in For establishment of transgenic lines, either GFP or mCherry open reading frames were inserted between Xiphophorus embryonic epithelial A2 cells, and medaka spermatogonial (Sg3) and fibroblast like (HN2) cells were cultured as described Mouse TM4 Sertoli cells, Gaussia luciferase activity was quantified using the Luciferase Reporter Assay System from Promega and normalized against co-tranfected firefly luciferase expressing plasmid  5′-AGCTGACGGCCGCGAAGCAAGCT and respective complements were annealed by heating to 90°C for five minutes in 1X T4 PolyNucleotide Kinase (PNK) buffer (70 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 5 mM dithiothreitol); slow-cooled to 50°C; held at that temperature for 5 minutes and then cooled to room temperature. For radioactive labelling 50 pmol of the duplex 5′ termini were used together with 50 pmol of gamma-[32P]-ATP and 20 units of T4 PNK in 1X adjusted T4 PNK buffer and incubated for 20 minutes at 37°C. Unincorporated nucleotides were removed through a Sephadex G-50 spin column.(Dmrt1bY-Trgt) KpnI and then in vitro transcribed using mMessage mMachine kit (Ambion). Finally, Dmrt1a or Dmrt1bY proteins were in vitro translated using Ambion's Retic Lysate Kit from the previously in vitro transcribed capped Dmrt1a or Dmrt1bY RNAs.For producing Dmrt1a and Dmrt1bY proteins, pRN3::Dmrt1a or pRN3::Dmrt1bY plasmids were linearized using 2, 1 µg torula rRNA, 0.075% Triton X-100, 1 mM DTT, 1 µg BSA, 0.5 ng radiolabeled duplex probe and 2 or 4 µL in vitro translation mix in a total volume of 20 µL. 1/10 volume heparin (50 mg/mL) was added just before loading the binding reaction. For control reticulocyte lysate alone together with radiolabeled duplex probe was used and did not result in any shift (data not shown). Binding reactions were performed on ice for ten minutes and complexes were resolved on a 5% native acrylamide (37.5∶1) gel in 0.5 X TBE and then directly subjected to autoradiography.DNA binding reaction contained 10 mMTris-HCl (pH 7.9), 100 mM KCl, 10% glycerol, 5 mM MgCl5′-CCTTCTTCCCCAGCAGCCT-3′/eGFP3: 5′-AGTCGTGCTGCTTCATGTGGTC-3′; DMTYa2: 5′-CGACTCCATGAGCAGTGAAA-3′/eGFP3: 5′-AGTCGTGCTGCTTCATGTGGTC-3′; DMTYa2: 5′-CGACTCCATGAGCAGTGAAA-3′/eGFP5: 5′-GAACTTCAGGGTCAGCTTGC-3′. Error bars represent the standard deviation of the mean. Relative expression levels (according to the equation 2–DeltaCT) were calculated after correction of expression of elongation factor 1 alpha  and brain expression was set to 1 as a reference.Total RNA was extracted from 9KbDmrt1bYprom::Dmrt1bY::GFP::Dmrt1bY3′UTR transgenic fish (Carbio genetic background) using the TRIZOL reagent (Invitrogen) according to the supplier's recommendation. After DNase treatment, reverse transcription was done with 2 micrograms total RNA using RevertAid First Strand Synthesis kit (Fermentas) and random primers. Real-time quantitative PCR was carried out with SYBR Green reagents and amplifications were detected with an i-Cycler (Biorad). All results are averages of at least two independent RT reactions and 2–5 PCR reactions from each RT reaction using each time three set of primer combination  was utilized according to the manufacturers instructions, using testis tissue samples either from dmrt1bY::GFP or deltadmrt1bY::GFP transgenic fish (20 testes for each) and GFP antibody (Upstate) for immunoprecipitation. After immunoprecipitation  primer sets were used for enrichment quantification by real-time PCR.For SceI meganuclease was injected into the cytoplasm of one cell stage medaka embryos (Carbio strain). Adult F0 fish were mated to each other and the offspring was tested for the presence of the transgene by PCR from pooled hatchlings. Siblings from positive F1 fish were raised to adulthood and tested by PCR from dorsal fin clips as described For the generation of stable transgenic lines the meganuclease protocol in vivo Chromatin Immunoprecipitation. Similarly, for in vivo Dmrt1bY promoter activity quantification another transgenic line expressing a 9Kbdmrt1bYprom driven dmrt1bY::GFP fusion protein was created. Dmrt1a prom::GFP transgenic medaka was generated following the BAC transgenic method 5′ -tctgacatgagcaaggagaagcagggcaggccggttccggagggcccggcTCAACCGGTCGCCACCATGG-3′ Reverse:5′-ttcagcggagacacgaagccgtggttccggcagcgggagcacttgggcatcGTCGACCAGTTGGTGATTTTG-3′.Identically to the transgenic line expressing the Dmrt1bY protein fused to GFP Figure S1Dmrt1 upstream regions. Medaka Y chromosomal dmrt1bY region (upper part) and autosomal dmrt1a region  are used as references. Regions I–IV contribute to length differences between the medaka dmrt1 upstream regions. Dark blue and green indicate exons of genes and pseudogenes, respectively, light blue and green untranslated regions. Red indicates conserved non-coding sequences. Conservation of medaka dmrt1 promoters with other vertebrates is restricted to the MHCL pseudogene regions.mVISTA plots of vertebrate (3.97 MB PDF)Click here for additional data file.Figure S2dmrt1bY promoter. KIAA0172p and MHCLbp regions are green shaded, regions IV–III and repeat 2 are grey shaded. The Izanagi element (repeat 1) is red shaded, its terminal inverted repeats are black shaded and the 8 bp target site duplication is underlined. The three identified putative THAP domain-encoding exons are pink shaded. The dmrt1bY exon 0 is blue shaded. Red lines mark border segments for the transcriptional regulation analysis . See also Annotation of the (0.02 MB PDF)Click here for additional data file.Figure S3Izanagi element. (A) Structure of the Izanagi element. Pink boxes indicate the three putative exons constituting the THAP domain are. The insertion of repeat 2 in the dmrt1bY promoter splitting repeat 1 into repeat 1b is indicated by the arrow. Note that the Izanagi element is shown here in reverse complement compared to the dmrt1bY promoter. (B) Alignment of the putative THAP protein domain from the Izanagi element consensus sequence with the THAP domain from the PFAM database. Identical essential residues are yellow shaded; other identical residues are blue shaded. + indicates similar residues.Putative THAP domain in the (1.01 MB PDF)Click here for additional data file.Figure S4dmrt1bY promoter. The three segments correspond to the regions used for transcriptional regulation analysis. Transcription factor binding sites conserved with the dmrt1a promoter are boxed. Of particular importance for transcriptional regulation of dmrt1bY might be the repeat 1b area (beige box) with multiple Sox5 and Pax2 binding sites, as well as a Dmrt1 binding site (red). Further upstream, two SF1 binding sites are located (red). For further characterization see Repeat elements and transcription factor binding sites in the (2.37 MB PDF)Click here for additional data file.Figure S5dmrt1bY promoter deletion constructs in different cell lines. (A–C) Various 5′-deletions mutants (3 Kb and 6 Kb) from pBSII-ISceI::9 Kb dmrt1bY prom::GLuc plasmid were transfected either into Mouse TM4 Sertoli, Xiphophorus embryonic epithelial A2 or medaka HN2 fibroblast like cells. Transfections were repeated three times; error bars represent the standard errors of the means.Activity of (0.62 MB PDF)Click here for additional data file.Figure S6dmrt1bY promoter. Linearized NJ trees for dmrt1 (A) and tyra (B) genes based on third codon positions using the Kimura-2-parameter model are shown. The split between fugu and Oryzias species was set to 95 million years ago (MYA) dmrt1 gene duplication has occurred dmrt1 duplication period (blue). The repeat 2 insertion is very recent. Other models of sequence evolution gave similar results.Timing of repeat insertions into the (2.49 MB PDF)Click here for additional data file.Figure S7Transient transfection analysis of the Dmrt1bY promoter nested Dmrt1 binding site. Transcriptional activity of the mutant 3 Kb proximal Dmrt1bY promoter (mutated Dmrt1 binding site) was not significantly impaired while overexpressing dmrt1bY or not.(1.78 MB PDF)Click here for additional data file.Table S1Location and adjacent genes of repeat 1 elements containing Dmrt1 binding sites in the medaka genome.(0.06 MB DOC)Click here for additional data file.Text S1Izanagi and Izanami: Creators of Japan.(6.88 MB PDF)Click here for additional data file.


The enzyme polyphosphate kinase 1 (PPK1) regulates this pathway by synthesizing polyphosphate required for the activation of MprB. The precise manner in which only a subpopulation of bacterial cells develops persistence, remains unknown. Rel is required for mycobacterial persistence. Here we show that the distribution of rel expression levels in a growing population of mycobacteria is bimodal with two distinct peaks corresponding to low (L) and high (H) expression states, and further establish that a positive feedback loop involving the mprAB operon along with stochastic gene expression are responsible for the phenotypic heterogeneity. Combining single cell analysis by flow cytometry with theoretical modeling, we observe that during growth, noise-driven transitions take a subpopulation of cells from the L to the H state within a “window of opportunity” in time preceding the stationary phase. It is these cells which adapt to nutrient depletion in the stationary phase via the stringent response. We find evidence of hysteresis in the expression of rel in response to changing concentrations of PPK1. Hysteresis promotes robustness in the maintenance of the induced state. Our results provide, for the first time, evidence that bistability and stochastic gene expression could be important for the development of “heterogeneity with an advantage” in mycobacteria and suggest strategies for tackling tuberculosis like targeting transitions from the low to the high rel expression state.Phenotypic heterogeneity in an isogenic, microbial population enables a subset of the population to persist under stress. In mycobacteria, stresses like nutrient and oxygen deprivation activate the stress response pathway involving the two-component system MprAB and the sigma factor, SigE. SigE in turn activates the expression of the stringent response regulator,  M. tuberculosis, the causative agent of the disease, is remarkably resilient against various physiological and environmental stresses including that induced by drugs. The “successful” containment of tubercular infection either through the host immune response or by the application of drugs is beset by the problem of persistence Tuberculosis, responsible for more than a million deaths every year, constitutes a major health concern worldwide. The effectiveness of the killer disease stems from the fact that the mycobacterial pathogen M. tuberculosis has been found to persist for years in the latent state characterized by the absence of active replication and metabolism rel initiates the stringent response which leads to persistence. The importance of Rel arises from the fact that it synthesizes the stringent response regulator ppGpp (guanosine tetraphosphate) M. tuberculosis under starvation On tubercular infection, granulomas form in the host tissues enclosing the infected cells. Mycobacteria encounter an altered physical environment in the restricted space of granulomas with a paucity of life-sustaining constituents like nutrients, oxygen and iron E. coli at the single cell level using microfluidic devices B. subtilis established the role of noise in driving the phenotypic transitions from the non-competent to the competent state. The network of regulatory interactions controlling competence development contains a core module, an autostimulatory positive feedback loop in which ComK proteins, synthesized from the comK gene, promote their own production. The non-competent and competent states correspond to low and high ComK levels. In the alternative scenario leading to positive feedback-based phenotypic heterogeneity, there is one stable expression state, mostly, the low expression state. The high expression state is unstable so that noise-driven activation to this state is followed by a relaxation to the low expression state. Competence development in B. subtilis has also been analysed in this alternative scenario Persistence is a general phenomenon observed in microbial populations subjected to stress, e.g., when kept in fluctuating environments mprA and mprB encode the histidine kinase sensor MprB and its partner the cytoplasmic response regulator MprA respectively. The protein pair responds to environmental stimuli by initiating adaptive transcriptional programs. Polyphosphate kinase 1 (PPK1) catalyses the synthesis of polyphosphate (poly P) which is a linear polymer composed of several orthophosphate residues. MprB autophosphorylates itself with poly P serving as the phosphate donor mprAB operon mprAB operon has a basal level of gene expression mprAB operon is activated, MprA-P regulates the transcription of the alternate sigma factor gene Sig E, which in turn controls the transcription of rel.Recent experiments provide knowledge of the stress signaling pathway in mycobacteria linking polyphosphate (poly P), the two-component response regulator MprAB, the alternate sigma factor SigE and Rel rel expression in mycobacteria grown up to stationary phase, a model where nutrient depletion serves as the source of stress. We combined mathematical modeling with experiments to establish that bistability along with gene expression noise give rise to phenotypic heterogeneity in terms of the expression of rel. Our results indicate that noise influences cell fate under stringency in mycobacteria. By regulated expression of PPK1 from a tetracycline-inducible promoter in a ppk1 knockout mutant, we further demonstrated hysteresis in rel expression.We hypothesized that positive feedback and stochastic gene expression likely play a role in the development of persistence in mycobacteria. To test this hypothesis, we studied the dynamics of mprAB  expression in M. smegmatis harboring the rel promoter fused to gfphsp60 promoter as a function of time. A single bright population was observed at different times of growth . In this strain, only a negligible fraction of the cell population was in the H state at any phase of growth  of M. smegmatisrel expression. We reasoned that regulated expression of PPK1 would serve to tune the positive feedback by virtue of the ability of the enzyme to synthesize poly P, the phosphate donor for phosphorylation of MprA. We therefore introduced the ppk1 gene under the control of the tet promoter rel-gfp plasmid, and confirmed tetracycline-inducible rel expression by fluorescence microscopy (ppk1 and rel-gfp in medium with increasing concentrations of tetracycline (inducer) and analyzed the distribution of cells expressing GFP by flow cytometry in the stationary phase (steady state)  in PPK-KO harboring a tetracycline-inducible kinase-dead mutant of ppk1. No induction of rel expression was observed when bacteria were grown in the presence of tetracycline (data not shown) confirming the role of the enzymatic activity of PPK1 in the induction of rel expression. The set of GFP distributions as a function of the inducer concentration are shown in In order to obtain further definitive evidence of bistability in croscopy . The gencroscopy  providedy state)  as well y state)  in the “rel expression in a growing population of mycobacteria with nutrient depletion serving as the source of stress. Summarising our present findings, we observe that rel expression is bistable and a combination of positive feedback along with gene expression noise select a subset of cells to be in the high rel expression state. Our results suggest that there is a temporal window during which fluctuations in the basal expression level drive the transitions from the low (L) to the high (H) expression state. Similar experimental observations have been made on competence development in B. subtilisThe phenomenon of persistence has been investigated in a host of microorganisms but the molecular mechanism which activates the switch to persistence in a cell is little understood in most cases. Persistence can develop in response to a variety of stresses some of which, like nutrient and oxygen deprivation, bring about the stringent response in bacterial systems. Rel is widely acknowledged as a key player enabling mycobacteria to persist under conditions of stress. In this paper, we identified the triggering mechanism of the switch to high B. subtilisrel expression in the stationary phase. We also find experimental evidence of another important feature of bistability, namely, hysteresis. Hysteresis is known to promote robustness in the maintenance of the induced state Recent studies have pinpointed the contributions of positive feedback and gene expression noise towards creating phenotypic heterogeneity in mprAB generates phenotypic heterogeneity, we observed that sigE-GFP expression is also bimodal ppGpp favor the accumulation of poly P in E. colirel expression at least under a certain set of conditions sigE and rel is now well established ppk1 expression is regulated, whether mprAB preferentially utilizes poly P and whether only ppk1 transcription is responsible for poly P accumulation rel in persistence, it would also be of great interest to analyze whether the high-rel population is antibiotic-tolerant and represent persistors. While the present study does not address these questions, our characterization of the high rel state and a systems level analysis of how mycobacteria attain this state offers important clues regarding possible drug targets. One aim of drug therapy could be the inhibition of the switching transition from the low to the high rel expression state thus reducing the chances of mycobacterial survival via the stringent response.As has been recently pointed out M. smegmatis mc2155 rel promoter  fused to gfp in pFPV27 has been described rel promoter were generated by overlap extension PCR. The primers used are depicted in Supplementary rel promoter was electroporated into wild-type M. smegmatis mc2155, or the isogenic strain inactivated in the ppk1 gene (PPK-KO) ppk1 gene under the control of a tetracycline-inducible promoter or in the sigE-inactivated (sigE KO) strain RH243, a gift from Prof. R.N. Husson hsp promoter was excised from pCKB115 gfp in the replicative promoter-less gfp vector pFPV27 The sigE promoterM. smegmatis using the sense and antisense primersThe GGTACCGCGGGATACAGTTCCCCATGC-3′5′-TAA [KpnI site in bold]GGATCCTTCAACATGCTAAACGCATGT-3′5′-TA [BamHI site in bold]M. smegmatis mc2155, the isogenic PPK-KO and the tet-ppk1-complemented strain.respectively and cloned into the promoter-less replicative gfp vector pFPV27 ppk1 under a tetracycline-inducible promoter was achieved by constructing an integrating vector containing a hygromycin-resistance cassette along with a positive-selection L5 integrase cassette. ppk1 was first amplified from genomic DNA using the sense and antisense primers CATATGATAAGCAATGATCGCAAG-3′5′-TAA [NdeI site in bold] and AAGCTTCAGGGGCTGCGGTGCCGTT-3′5′-TTA [HindIII site in bold] respectively and cloned between same sites of pMIND tet promoter. The ppk1 gene along with the tet promoter was excised by digesting with KpnI and HindIII and cloned between the same sites in pUC19 to generate pTC-PPK. A 3.7 kb Hyg-integrase cassette from pUC-HY-INT ppk1. The kinase-dead ppk1 carrying mutations of two histidine residues (required for autophosphorylation) to alanine ppk1-KD] was amplified by PCR from a suitable construct essentially as described by Sureka et al. M. smegmatis mc2155 as described above.Expression of M. smegmatis cells were grown for different periods of time, harvested, washed twice and resuspended in PBS. Fluorescent bacteria were analyzed on a FACS Caliber (BD Biosciences) flow cytometer. All data modes were set to log, and the forward scatter detector level was set to E-1. The fluorescence intensity of 20,000 ungated events was measured in FL-1 at a detector sensitivity of 780. A side scatter threshold was set to gate out inappropriate heterogeneities. It is difficult to analyze single mycobacteria by FACS because of their tendency to clump. Careful gating procedures were adopted to minimize the effect of clumping. The data files were analyzed using CellQuest Pro (BD Biosciences) and WINMIDI. The flow cytometry data is represented in histogram plots where the x-axis is a measure of fluorescence intensity and the y-axis represents the number of events.Cells were fixed by incubation for 15 minutes at room temperature followed by 45 minutes on ice in 2.5% (v/v) paraformaldehyde, 0.04% (v/v) glutaraldehyde, 30 mM sodium phosphate (pH 7.5). The cells were transferred to slides, washed with PBS, air-dried, dipped in methanol (−20°C) for 5 min and then in acetone (−20°C) for 30 s and allowed to dry. Brightfield and fluorescence images were taken using a 100× oil immersion objective on a Zeiss Axioimager A1 microscope equipped with a cooled CCD camera.Figure S1s (Eq. 2) (A) and the parameter α (Eq. 19) (B). A generic stimulus-response curve in the steady state. The points 1u and 2u denote the upper and lower bifurcation points respectively at which transitions occur from one branch to the other.Bistability and hysteresis in the deterministic model. Steady state concentrations of phosphorylated MprA versus basal production rate (6.62 MB TIF)Click here for additional data file.Figure S2M. smegmatis harboring rel-GFP was grown as described in Fluorescence microscopy of rel-GFP expression. (5.64 MB TIF)Click here for additional data file.Figure S3M. smegmatis mc2155. Cells were grown for different periods of time as indicated and the growth was monitored by recording the absorbance at 600 nm spectrophotometrically.Growth kinetics of (2.94 MB TIF)Click here for additional data file.Figure S4M. smegmatis harboring hsp-GFP was grown for different periods of time and the expression of GFP was monitored by flow cytometry as described in Time course of hsp-GFP expression. (6.13 MB TIF)Click here for additional data file.Figure S5ppk1 and the construct of rel-gfp was grown upto stationary phase in the presence of different concentrations of tetracycline (inducer). Lysates were analyzed by SDS-PAGE and Western blotting using anti-PPK1. Blots were reprobed with anti-PBP3 to ensure equal loading. B. Densitometric analysis of Western blots (panel A) of PPK1. C. Cells were grown as described under panel A, and poly P levels were measured as described in Ref. 1.A. Western blots of tetracycline-inducible PPK1 in PPK-KO. PPK-KO harboring the integrated tet-inducible (5.90 MB TIF)Click here for additional data file.Figure S6GFP distributions obtained through stochastic simulation via the Gillespie algorithm. 5, 18, 25 and 50 represent time in h.(3.84 MB TIF)Click here for additional data file.Figure S7Rate of transition from the L to the H state and the CV of the low expression state versus time obtained through stochastic simulation based on the Gillespie algorithm.(3.44 MB TIF)Click here for additional data file.Figure S8sigE-GFP expression. M. smegmatis expressing the sigE-GFP construct was grown for different periods of time  as indicated and the expression of GFP was monitored by flow cytometry. The figure provides evidence of bimodal sigE expression.Time course of (3.40 MB TIF)Click here for additional data file.Text S1Mathematical models(0.14 MB DOC)Click here for additional data file.Table S1rel promoterPrimers used for mutation of the (0.02 MB DOC)Click here for additional data file.


One means by which patterns of gene expression are achieved is through the differential packaging of DNA into distinct types of chromatin. While chromatin state exerts a major influence on gene expression, the extent to which cis-acting DNA sequences contribute to the specification of chromatin state remains incompletely understood. To address this, we have used a fission yeast sequence element (L5), known to be sufficient to nucleate heterochromatin, to establish de novo heterochromatin domains in the Schizosaccharomyces pombe genome. The resulting heterochromatin domains were queried for the presence of H3K9 di-methylation and Swi6p, both hallmarks of heterochromatin, and for levels of gene expression. We describe a major effect of genomic sequences in determining the size and extent of such de novo heterochromatin domains. Heterochromatin spreading is antagonized by the presence of genes, in a manner that can occur independent of strength of transcription. Increasing the dosage of Swi6p results in increased heterochromatin proximal to the L5 element, but does not result in an expansion of the heterochromatin domain, suggesting that in this context genomic effects are dominant over trans effects. Finally, we show that the ratio of Swi6p to H3K9 di-methylation is sequence-dependent and correlates with the extent of gene repression. Taken together, these data demonstrate that the sequence content of a genomic region plays a significant role in shaping its response to encroaching heterochromatin and suggest a role of DNA sequence in specifying chromatin state.Characterizing how genomic sequence interacts with  Epigenetic packaging of DNA sequence into chromatin is a major force in shaping the function of complex genomes. Different types of chromatin have distinct effects on gene expression, and thus chromatin state imparts distinct features on the associated genomic DNA. Our study focuses on the transition between two opposing chromatin states: euchromatin, which generally correlates with gene expression, and heterochromatin, which is typically refractive to gene expression. While heterochromatin is capable of spreading into euchromatic domains, the parameters that influence such spreading are unknown. We established heterochromatin at ectopic sites in the genome and evaluated whether specific DNA sequences affected the extent of heterochromatin spreading and the transition between heterochromatin and euchromatin. We found that the nature of the genomic DNA neighboring the heterochromatic sequence dramatically affected the extent of heterochromatin spreading. In particular, the presence of genes antagonized the spread of heterochromatin, whereas neutral sequence elements were incorporated into the domain. This study demonstrates that genome sequence and chromatin identity are inextricably linked; features of both interact to determine the structural and functional fate of underlying DNA sequences. How these interactions are coordinated and to what extent genomic sequence serves as a blueprint, directing these interactions towards normal growth and development, remain major questions in genome biology.Correct patterns of gene expression are established by orchestrated interactions among Chromatin has classically been divided into two functionally distinct types: heterochromatin and euchromatin. Genes inserted within, or proximal to, major heterochromatin domains can exhibit either variegated or complete silencing Several studies have pointed towards the importance of genome sequence in shaping epigenetic states. For example, insulators are specific DNA sequences that protect genes from the regulatory effects of neighboring domains, thus enforcing domain boundaries de novo nucleation site in the fission yeast, Schizosaccharomyces pombe. The unique advantage of this system, in addition to its genetic tractability, is the presence of well-defined DNA sequences, referred to here as heterochromatin-nucleating sequences, that are sufficient to induce heterochromatin formation de novode novo heterochromatin domain at a euchromatic locus permits a simplified view of this process, in contrast to native domains of heterochromatin that result from the complex interplay of multiple sites of nucleation and heterochromatin barriers de novo heterochromatin domains clearly implicates primary DNA sequence in defining both the magnitude and extent of the heterochromatin domain. The conceptual framework that emerges from this study provides a basis for exploring the nature of complex genomes and the impact of genome sequence on the establishment and maintenance of chromatin domains, in organisms ranging from yeast to mammals.Elucidating the role of genome sequence in shaping chromatin domains requires an experimental system in which heterochromatin nucleation can be initiated in a controlled manner. To this end, we have examined heterochromatin spreading from a S. pombe have demonstrated that a fragment of pericentromeric DNA, called L5, is capable of nucleating heterochromatin, marked by di-methylation at H3K9 (H3K9me2) and the presence of the HP1 homologue, Swi6p, at an ectopic site through an RNAi-dependent mechanism Previous studies in +ade6 reporter gene. This construct was then integrated at the euchromatic ura4+ locus in order to create ura4::L5-ade6+ strains. In addition to the L5-containing construct, a control construct bearing only the +ade6 gene was also integrated at the +ura4 locus (ura4::+ade6). The effect of L5-integration on the chromatin environment of sequences within the ura4 locus was characterized by quantifying H3K9me2 and Swi6p levels throughout the region using chromatin immunoprecipitation (ChIP).To address this question, we created a construct containing the 1.6 kb L5 element upstream of an de novo heterochromatin domain that spans approximately 15 kb. Throughout, we will describe the properties of a heterochromatin domain by its extent, the distance over which heterochromatin is enriched, and its magnitude, the level of heterochromatin enrichment at a given location.In the presence of the L5 element, H3K9me2 was enriched ∼2- to >10-fold over both the reporter gene and the surrounding genomic neighborhood , extendiura4 changes upon insertion of L5, we sought to determine whether gene expression at the ectopic locus was also altered. Quantitative RT- PCR (qRT–PCR) was used to quantify the levels of mRNA in the presence of L5 relative to control strains lacking L5. As expected from earlier studies +ade6 expression; however, the reduction in expression was moderate (34±5%), indicating that silencing is incomplete in these strains . Comparison of H3K9me2 and Swi6p enrichment at L5 and +ade6 between the two sites of integration reveals similar patterns of enrichment, suggesting that the nucleation of heterochromatin and local spreading are not sensitive to the changes in genomic location from ura4 to spbc2f12.03  was positioned between L5 and +ade6. This region was selected because it is one of the larger regions in the S. pombe genome in which known protein-coding genes are absent and because it normally lacks heterochromatic modifications ura4 locus in strains lacking L5  at its promoter  +his3, consistent with the levels observed in uninterrupted spacer strains . While the local magnitude of H3K9me2 proximal to L5 was increased in these strains . The stability of the silenced state was determined by the proportion of progeny that exhibited silencing after a period of overnight growth. We examined the phenotypic stability of +ade6 and −ade6 controls, and as expected, the progeny maintained the appropriate phenotype  occurs less often when +ade6 is distal to 7 kb of spacer DNA and less enriched in Swi6p, despite comparable levels of the epigenetic mark H3K9me2.In addition to the total level of gene silencing, another manner in which the reduced levels of Swi6p in spacer strains could affect gene expression is by altering the stability of gene repression. When transgenes are placed within the centromere, or at locations throughout the mating type locus, their phenotypic stability (silenced or expressed) can vary with location and Swi6p dosage henotype . Howevered state , suggestura4::L5-+ade6 strains, ∼18% of the progeny exhibited phenotypes indicative of complete silencing and 90% exhibited at least partial silencing. In contrast, silenced colonies from ura4::L5-7kb-+ade6 strains were less likely to give rise to progeny that exhibited complete or partial silencing , suggesting that maintenance of silencing is also less frequent when +ade6 is separated from L5 by spacer DNA and reduced in Swi6p localization  into subclasses that include attenuators, neutral elements and protosilencers/boosters. While this is helpful for purposes of discussion, we do not wish to impose strict definitions, especially in light of data from this study, suggesting that particular sequences may be placed at multiple points along the continuum, depending on their context.de novo heterochromatin domain encompassing multiple endogenous genes and altering gene expression  and non-coding RNAs  de novo heterochromatin domains warrants further genome-wide studies.In addition to protein-coding genes, the ing RNAs . The uraAla within the S. pombe centromere 1 prevents the spread of heterochromatin into the abutting domain of centromeric chromatin. In contrast, the tDNAGly gene is not coincident with the domain boundary of the ectopic heterochromatin domain formed at +ura4; however it is deficient in H3K9me2 enrichment, due to the absence of a nucleosome(s)  . Nucleos+swi6 resulted in increased levels of H3K9me2 over sequences adjacent to L5, as well as enhanced repression of +ade6, consistent with an increase in local heterochromatin  and used for further analysis. All transformants were then crossed into a +swi6 strain and the ura4::L5-+ade6 allele was selected for on the basis of FOA resistance. To create swi6+333 strains, ura4::L5-+ade6 strains were crossed into SPG1232  ura4::L5-7kb::(Pnmt1-+his3)-+ade6 the Pnmt1-+his3 construct was transformed into Kfy589, colonies were selected for on the basis of growth on media lacking histidine. After integration within lambda was confirmed by Southern analysis, these strains were crossed into a +swi6 strain.The genotypes for strains used in this study are as listed . Fissionade6+ was amplified from S. pombe genomic DNA using primers BWP34F and BWP34Rb  with BamHI and purifying the 7.2 kb fragment, which was then ligated into the BglII sites of BW5 and BW7, respectively. To create BW20 an additional copy of L5, as a BamHI–BglII fragment, was inserted into the BglII site of BW7.Plasmid BW7 was constructed through digestion of YL317 with SpeI and ClaI and subsequent purification of the L5-containing fragment +his3 within the plasmids pFA6a-kanMX6-P3nmt1-GFP and pFA6a-kanMX6-P41nmt1-GFP, respectively (A gift from Jian-Qiu Wu) +his3 containing fragment was then inserted within the PstI site in the lambda fragment.Plasmids BWP40 and BWP41 were created by replacing the GFP ORF with ura4 locus, we selected transformants on the basis of growth on PMG–adenine and death on FOA. These strains were then confirmed via Southern blot to have a single +ade6 insertion and the site of integration was mapped using an inverse PCR protocol modified from To identify random integrants that did not disrupt the Strains were grown overnight with shaking in YES media at 32°C and diluted to a concentration of 1e6 cells/mL. Cultures were diluted serially (1∶9) and plated on YES media lacking adenine.To assess the stability of silencing, colonies that were scored as either completely white or completely red were identified using a Leica MZ7.5 microscope and grown for 24 hours in YES media before plating on YE plates lacking adenine.For both protocols, plates were grown for three nights at 32°C and shifted to 4°C for 24 hours before photographing or counting.+ade6 (BWP85F/R). Levels of mRNA from +ade6, and other genes queried, were expressed relative to +act1(BWP74F/R). The standard curve was generated using genomic DNA isolated from strain Kfy1. In order to be included in this study a PCR experiment had to have a PCR efficiency between 90–110% and a correlation coefficient >0.99.Total nucleic acid was isolated from logarithmically growing cells in YES media at 32°C, and was then subjected to DNAse treatment and RT–PCR using oligodT as a primer. Expression was analyzed by quantitative PCR using SYBR Green on a Bio-rad myCycler, using primers specific to the wild type copy of The H3K9me2 ChIP protocol was adapted from H3 ChIPs were preformed as above using an antibody to H3 (abcam 1791).Swi6p ChIPs were performed using the above protocol modified from +act1 in IP reactions relative to a no-antibody control.Quantitative PCR was used to assay levels of query/Figure S1ura4 genomic region is not enriched in H3K9me2 in the absence of L5, and exhibits varible nucleosome occupancy. (A) The ura4 locus is depicted, and assayed for the presence of H3K9me2 (black) and Swi6p (grey), in the absence of the L5 element. (B) Nucleosome occupancy was characterized using an antibody to the c-terminus of histone H3. The data are expressed relative to the nucleosome occupancy at the act1+ locus.The (3.00 MB TIF)Click here for additional data file.Figure S2S. pombe intergenic and lambda spacer fragments do not recruit H3K9me2 in the absence of L5. H3K9me2 enrichment over S. pombe intergenic (A) and lambda (B) sequences in the absence of L5. The intergenic spacer DNA is in duplicate copies in the genome (at the ura4 locus as well as its endogenous locus) thus no enrichment is represented by a value of 2.(1.88 MB TIF)Click here for additional data file.Figure S3ura4 locus is unaffected by the presence of lambda spacer DNA. Analysis of H3K9me2 in the ura4 locus in +ura4::L5-7kb-ade6 strains (black) and +ura4::L5-ade6 strains. To facilitate comparison between the two strains H3K9me2 enrichment for the +ura4::L5-ade6 is depicted with a gap over the lambda insert.Heterochromatin spreading in the (1.93 MB TIF)Click here for additional data file.Figure S4+swi6 copy number results in decreased +ade6 expression but does not alter expression of other genes within the de novo heterochromatin domain. Steady state mRNA levels are depicted relative to +ura4::ade6 strains for wild type strains (grey) and strains bearing 3 copies of +swi6 (black).Increased (1.59 MB TIF)Click here for additional data file.Figure S5+act1. +his3 mRNA was isolated from highly transcribed  and weakly transcribed conditions  in swi6- ura4::L5-7kb::(Pnmt1-+his3)-+ade6 strains. For comparison the level of +ade6 and +spcc330.03 mRNA are shown for +ura4::ade6 strains. (B) Relative H3K9me2 enrichment for strongly and weakly transcribed conditions.Insertion of a gene within lambda attenuates heterochromatin independent of the level of transcription. (A) Levels of steady state mRNA relative to (2.72 MB TIF)Click here for additional data file.Figure S6de novo heterochromatin domains.The ratio of Swi6p/H3K9me2 is reduced over spacer DNA. Scatter plot of the levels of Swi6p/H3K9me2 for heterochromatic regions genomewide (1.47 MB TIF)Click here for additional data file.Table S1Strains used in this study.(0.04 MB PDF)Click here for additional data file.Table S2Primers used in this study.(0.08 MB PDF)Click here for additional data file.


Drosophila Pumilio (Pum) protein is a translational regulator involved in embryonic patterning and germline development. Recent findings demonstrate that Pum also plays an important role in the nervous system, both at the neuromuscular junction (NMJ) and in long-term memory formation. In neurons, Pum appears to play a role in homeostatic control of excitability via down regulation of para, a voltage gated sodium channel, and may more generally modulate local protein synthesis in neurons via translational repression of eIF-4E. Aside from these, the biologically relevant targets of Pum in the nervous system remain largely unknown. We hypothesized that Pum might play a role in regulating the local translation underlying synapse-specific modifications during memory formation. To identify relevant translational targets, we used an informatics approach to predict Pum targets among mRNAs whose products have synaptic localization. We then used both in vitro binding and two in vivo assays to functionally confirm the fidelity of this informatics screening method. We find that Pum strongly and specifically binds to RNA sequences in the 3′UTR of four of the predicted target genes, demonstrating the validity of our method. We then demonstrate that one of these predicted target sequences, in the 3′UTR of discs large (dlg1), the Drosophila PSD95 ortholog, can functionally substitute for a canonical NRE (Nanos response element) in vivo in a heterologous functional assay. Finally, we show that the endogenous dlg1 mRNA can be regulated by Pumilio in a neuronal context, the adult mushroom bodies (MB), which is an anatomical site of memory storage. Drosophila Pumilio (Pum) protein was originally identified as a translational control factor for embryo patterning. Subsequent studies have identified Pum's role in multiple biological processes, including the maintenance of germline stem cell, the proliferation and migration of primordial germ cells, olfactory leaning and memory, and synaptic plasticity. Pum is highly conserved across phyla, i.e., from worm to human; however, the mRNA targets of Pum within each tissue and organism are largely unknown. On the other hand, the prediction of RNA binding sites remains a hard question in the computational field. We were interested in finding Pum targets in the nervous system using fruit flies as a model organism. To accomplish this, we used the few Pum binding sequences that had previously been shown in vivo as “training sequences” to construct bioinformatic models of the Pum binding site. We then predicted a few Pum mRNA targets among the genes known to function in neuronal synapses. We then used a combination of “golden standards” to verify these predictions: a biochemical assay called gel shifts, and in vivo functional assays both in embryo and neurons. With these approaches, we successfully confirmed one of the targets as Dlg, which is the Drosophila ortholog of human PSD95. Therefore, we present a complete story from computational study to real biological functions.The  Drosophila melanogaster Pumilio (Pum) protein is one of the founding members of the PUF RNA-binding protein family. Its function in the posterior body patterning of Drosophila embryos is relatively well studied. Wharton and Struhl hunchback (hb) mRNA, named Nanos Response Elements (NREs), which are essential for normal abdominal segmentation. It was later shown that Pum binds these elements, recruits Nanos (Nos) and Brain Tumor (Brat), and represses the translation of maternal hb mRNA Drosophila bicoid (bcd) mRNA, which plays a key role in anterior development Drosophila embryos, and Cyclin B (CycB) mRNA appears to be a target of translational repression by this complex Drosophila abdominal segmentation + current homeostasis in the CNS pum was found with both approaches: it is transcriptionally upregulated during memory formation after spaced training (which results in long-term memory) relative to massed training (which results only in shorter forms of memory), and two independent transposon insertions into pum yielded mutants with defective one-day memory after spaced training. In addition to pum, six other components of a pathway putatively involved in local translational control were identified: staufen, orb (CPEB), moesin and eIF-2G were transcriptionally regulated during memory formation, whereas transposon-mediated lesions were found in or near oskar (norka mutant) and eIF-5C (krasavietz mutant).More recently, Pum has been found to play a role in the nervous system at the neuromuscular junction Drosophila peripheral neurons, and that Nos colocalizes with RNA granules in dendrites. The role of Pum-dependent regulation in neurons also may be conserved Local mRNA translation within dendrites of neurons has been proposed to be a mechanism for activity-dependent synaptic plasticity  in CycBbcd of some fly species is one base longer than melanogaster, suggesting that the distance between the two boxes may be flexible. CycB TCE also contains short sequence segments like Box A and Box B and the distance between them is 23 bp. Therefore, we arbitrarily set the distance between 3 to 45 bases, to reduce the chance of missing some possible functional sites.We constructed three alternative models for Pum-binding sites, based on different assumptions. The first model is a simple consensus pattern, and the other two are based on positional weight matrices (PWM) hb and bcd NREs . Conservation is unknown for two genes, AP-1 gamma and mam, because corresponding D. pseudoobscura 3′UTR sequences were not available at the time we initiated this study. Non-conserved predictions on CaMKII and EP2237 could be false positives. However, it is also possible that the 3′UTR sequences of those two genes are incomplete or inaccurate for D. pseudoobscura (at the time we initiate this study), resulting in the failure to find conserved sites.The predicted Pum targets among the 151 synaptic genes with the above three NRE models are listed in dlg1 gene is predicted to be a candidate Pum target all three NRE models. In addition, the target dlg1 has also been previously suggested as a potential Pum target based on its presence in a collection of 1434 Drosophila genes containing the motif UGUAHAUA With NRE_M8, 31 transcripts (28 genes) are predicted to be candidate Pum targets. Here, we require that a 3′UTR sequence must contain at least two high-score sites to be considered as a candidate Pum target. Among them, 10 transcripts (8 genes) have at least two predicted sites that are conserved between the two fly species. With NRE_M10, 28 transcripts (25 genes) are predicted to be candidate Pum targets, among which 11 transcripts (9 genes) have at least one conserved site. Notably, dlg1 gene has predicted Pum binding sites in the 3′UTR of two non-overlapping transcript isoforms  fused with an N-terminal GST tag and has been shown to maintain the full binding activity of the wild-type Pum protein hb served as a positive control for Pum binding, whereas a random control RNA sequence, CRS that does not resemble an NRE-like element served as a negative control. Under the experimental conditions used, GST-Pum bound to hb NRE2 with high affinity, but did not bind to the control RNA sequence CRS, as shown in hb NRE was formed, ruling out the possibility that the complex between GST-Pum and hb NRE was generated by non-specific binding of GST to RNA. We also note that, under our experimental conditions, only one complex was formed between GST-Pum and hb NRE as we increased the concentration of GST-Pum, consistent with the presence of a single Pum binding site in a single hb NRE  using purified GST-Pum, which bears the RNA-binding domain of e hb NRE .dlg1 isoform C, which was not bound by Pum at all, even at a high molar ratio of protein to RNA. However, dlg1 transcript isoform C shares a different 3′UTR sequence from dlg1 transcript isoforms A and D. For the RNAs from hb NRE, dlg1 isoforms A and D, and AP-1 gamma, only one complex was formed upon binding of Pum. For the remaining RNAs, two or more complexes were formed, suggesting the existence of more than one Pum-binding site.Next, we determined the binding specificity of the predicted NRE-like elements by EMSA. As shown in hb NRE was bound by Pum. Under the same experimental condition, greater than 50% of the transcripts from dlg1 (isoforms A and D), shn, Csp, and mam were bound by Pum, suggesting that NRE-like elements in these transcripts have strong Pum-binding activities comparable to hb NRE. On the other hand, at the same 5∶1 molar ratio of protein to RNA, transcripts from Ace, AP-1gamma, EP2237, and Gad1 were largely unbound by Pum and showed weaker but substantial Pum-binding activities. We also tested Pum-binding activity of a 142-nt RNA fragment consisting of CycB TCE and flanking sequences (nts 400–541 of 3′UTR of CycB mRNA) and found that CycB TCE was able to bind to Pum with a much lower affinity compared to hb NRE  whereas the correlation for NRE_M8 is weaker and not statistically significant . While we do not understand why transcriptional responses for Pum's targets are in the same direction as that of Pum, this may reflect global transcriptional increases versus local translational repression  is transcriptionally induced by spaced training sion see . These thb mRNA be repressed by Pum/Nos in posterior regions of the early embryo. We started with a canonical genomic hb rescuing transgene in which the endogenous NRE motifs were deleted. In the absence of functional NRE elements, this construct causes a dominant sterility in transgenic females due to ectopic hb translation in the posterior half of the embryos produced. Such embryos are unable to form abdominal segments. Insertion of a functional NRE motif into this canonical construct restores Pum-mediated repression in the posterior, allowing production of viable progeny. Using this strategy, we tested the functional capacity of the predicted NRE motifs from Ace and dlg1. We chose these two putative targets because they showed relatively strong in vitro binding and also because both transcripts are induced by spaced training.To validate our target prediction method in vivo, we chose to use a Pum response assay described previously hb-transgene constructs (hb NREs had either been deleted entirely (hbΔ), replaced with a single hb NRE, NRE2 (hb2), had both hb NRE elements re-inserted , replaced with putative NRE elements from Ace or dlg1 genes (Ace or dlg1), or replaced with an anti-sense version of the predicted dlg1 NRE (dlg1-anti). We found that the predicted NRE from dlg1 is sufficient to partially restore abdominal patterning when compared with hb1,2 (dlg1 NRE is superior to that observed with a single copy of the hb NRE (hb NRE (hb2) yields partial rescue. In contrast, control transgenic lines in which no functional NRE was provided, or in which the dlg1 NRE was inserted in opposite orientation (dlg1-anti) generate progeny nearly devoid of abdominal segments (hb-transgene construct (dlg1-full), in which the two endogenous hb NREs are replaced by a longer version of the transcript, a 1.2-kb sequence including the predicted NRE element from the 2.8-kb sequence of dlg1 3′UTR , which is an anatomical site of memory storage The above data support the conclusion that the Our imaging studies support two conclusions. First, we found that transgenic expression of Pum in MB Kenyon cells results in a dramatic reduction of Dlg expression levels. Importantly Dlg expression in AL appears unaffected . In addiThe above observation suggests the interesting possibility that Pum normally plays a key developmental role in elaboration of MB structure. While we cannot rule out neo-morphic effects of Pum over-expression, these findings nevertheless are consistent with the previous observations of Pum's role in dendrite morphology hb and bcd genes; (3) the availability of a robust in vivo functional assay dlg1 and Ace, both of which are also induced during memory consolidation. In the case of dlg1, the identified NRE appears capable of functioning both in a heterologous in vivo context of the early embryo and an endogenous one in the adult brain .Our results also suggest that the binding specificity of Pum is conserved between dlg1, our in vivo evidence indicates that the predicted NRE can function, not only in context of the hb 3′UTR, but also in CNS while Pum is over-expressed.The fact that prediction scores of NRE_M10 and MmSelex_M8 are well correlated with in vitro binding data demonstrates the validity of these two models for Pum binding site prediction. The predicted hits by these two models in the synaptic gene set are significantly higher than random , furtherComparing our synaptic gene set with the pulled-down targets from Gerber et al. Studies in diverse organisms strongly indicate that sequences around BoxB play a major role in binding to Puf proteins CycB TCE with a lower affinity than hb NRE under our experimental conditions. CycB TCE was initially proposed due to its resemblance to bcd and hb NRE, and was required for translational repression control CycB TCE has a lower score according to our matrix. A new element downstream of TCE has recently been proposed and been shown to bind to Pum in gel mobility shift experiments and, when substituted for the native hb NRE in a chimeric hb mRNA, was able to mediate CycB-like regulation on hb mRNA ATTGTGCAAA, nts 561–570 of 3′UTR of CycB mRNA) in the RNA fragment used in these experiments. Our predicted site is close to the NRE element proposed by Kadyrova and colleagues, but not the same. Further work needs to be done to address this discrepancy. It is also worth mentioning that there are several significant differences between regulation of CycB mRNA and hb/bcd mRNAs bcd and hb, for example, regulation of CycB is Brat-independent. Kadyrova et al. CycB, Pum binding seems important only to recruit Nanos, because artificially tethering Nanos to the 3′UTR bypasses the requirement for Pum binding. This is in contrast to Pum's regulation of hb. Thus it seems that there are significant differences between the Pum-binding sites in CycB mRNA and those in hb and bcd mRNAs, as proposed previously hb and bcd. Discovery of additional Pum targets from a variety of cell types and biological contexts may uncover the relationship between NRE sequence and regulatory mechanism.Notably, Pum binds to a 142-nt RNA harboring Ace and dlg1, also appear to be transcriptionally induced after spaced training relative to massed training, suggesting that these are relevant targets for memory formation. We do not know why both a translational repressor and its putative targets are transcriptionally induced. It may be that transcripts are increased on a cell-wide level, while translation is spatially regulated within neurons. In the case of dlg1, our in vivo evidence supports the conclusion that the predicted NRE can mediate Pum-dependent repression both when it is in the context of the hb 3′UTR and in the endogenous dlg1 transcript in the CNS. Thus, our findings directly predict that dlg1 is a synaptic target of Pum.To our knowledge, this is the first study to characterize and predict Pum-binding sites with a PWM approach, which is typically more sensitive and more precise than consensus methods Drosophila member of a family of membrane-associated guanylate kinases (MAGUKs) that in mammals have been shown to play a key role in assembling the post-synaptic density in glutamatergic synapses. In Drosophila, Dlg expression is both pre- and post-synaptic at Type I boutons at the NMJ, and mutants exhibit post-synaptic structural defects as well as increased transmitter release + channels throughout the CNS Dlg is the sole para Na+ channels para may be direct, or may depend upon Pum's putative role in regulating translation of eIF-4E parapara is in our list of synaptic genes, yet our models did not predict any Pum binding sites in its 3′UTR. That is not surprising since Mee et al. para by Pum.Like Dlg, Pum also appears to have both pre- and post-synaptic effects at the NMJ and is co-localized with Dlg at Type I boutons dlg1 expression, thereby antagonizing the effects of Dlg on neuronal structure and/or function. We do not yet know whether other classic factors (Nanos and Brat) that cooperate with Pum in early embryos are also required in the translational control of Dlg in neurons. Further investigation also will be required to separate the roles of Pum in neuronal development and memory formation. Ultimate confirmation that Pum-dependent repression of dlg1 and the other predicted NRE-containing genes underlies Pum's role in neuronal structure, function and memory will also require additional examination.Our findings suggest that an additional role of Pum is direct regulation of Drosophila Genome Project  Release 3.1 annotation and keyword search in the FlyBase Vocabulary Report (http://flybase.org/) of gene expression. GO terms involved in neurotransmitter metabolism were not considered to relate directly to synaptic functions, and were thus excluded. 68 genes were obtained from the GO annotation and 132 genes were obtained from FlyBase search using the keyword “synapse.” Among those, a total of 151 genes were mapped to Release 3.1 Drosophila genome (with CG ID) and were used for further analysis (Synaptic genes were collected based on GeneOntology (GO) terms in the Berkeley analysis .hb and bcd from different fly species were retrieved from GenBank. The GenBank accessions are listed in Sequences of mRNA or genomic DNA that contain complete 3′UTRs of D. melanogaster were retrieved from BDGP Release 3.1 annotation. Putative D. pseudoobscura 3′UTR sequences were obtained based on whole-genome alignment between D. melanogaster and D. pseudoobscura produced by the BDGP at Lawrence Berkeley National Laboratory (http://pipeline.lbl.gov/). Distinct 3′UTR sequences of the mapped 151 synaptic genes are included in The 3′UTR sequences of all annotated genes for http://bayesweb.wadsworth.org/gibbs/gibbs.html) obtained from C. E. Lawrence's group was used to perform local multiple sequence alignment to identify the motif model. The base-frequency matrix output from the program was converted into the log-odds PWM with a background nucleotide frequency derived from all 3′UTRs in the genome of D. melanogaster, i.e.,b,jw is the matrix weight for base b at position j, b,jf is the frequency of base b at position j and bp is the background frequency of base b. b = A, C, G or T, j = 1 … n for a PWM of length n.The Gibbs Sampler program (hb and bcd of D. melanogaster and their corresponding sequence segments in other fly species (CycB (melanogaster and pseudoobscura) and eIF-4E (melanogaster only) were also included.Input sequences to Gibbs Sampler included known NREs in n, the score of a target sequence segment 1b2 … bnt = b, is:j is the position in the PWM, jb is the jth base of the target sequence.Pattern search was implemented with a Perl script as a regular expression match. Weight matrix scan on sequences was performed with an R script. For a PWM of length D. melanogaster. The matrix score cutoff was selected so that most of the known NREs scored above the threshold. Corresponding putative 3′UTR sequences of D. pseudoobscura were also searched when available. We define a predicted site in D. melanogaster as conserved if this site is aligned or overlaps with a predicted site in D. pseudoobscura in the LAGAN alignment provided by BDGP.We searched in the 3′UTR sequences of all 151 synaptic genes, including their distinct splicing variants in Drosoplila Pum as a fusion with GST E. coli and purified by affinity chromatography on glutathione-Sepharose (Amersham Biosciences) by standard procedures.The plasmid R6646 that encodes amino acids 1091–1533 of D. melanogaster genomic DNA. A T7 promoter was added at the 5′ terminus of the template by PCR. PCR products were purified and used as templates for in vitro transcription, which was done as described Transcription templates for the predicted NRE-like elements were obtained by PCR from hunchback NRE2 sequence used was AUUAUUUUGUUGUCGAAAAUUGUACAUAAGCC. The random control RNA sequence (CRS) is GGUAGUGCAUACAACUUCCUU. Binding reactions were carried out by mixing 10 fmol radiolabeled RNA with variable amounts of purified GST-Pum in a 10 μl binding buffer containing 0.1 mg/ml BSA, 10 mM Hepes/KOH pH 7.4, 50 mM KCl, 3 mM MgCl2, 1 mM EDTA, 0.1 mg/ml yeast tRNA, 2 mM dithiothreitol, 0.01% (w/v) Tween-20, 0.2 U rRNAsin (Promega), and 10% (v/v) glycerol. The protein-RNA complexes were allowed to form for 20 min at room temperature, followed by electrophoresis on a 5% non-denaturing acrylamide gel in 1× TBE buffer. The gel was dried, followed by autoradiography at −70°C or quantification on a phosphorimager (Fuji).EMSA was done as described RNA isolations were performed with Trizol (Invitrogen) as described before 5′-GCACTACCCAAGACAAATTTTATCGAAA-3′ and 5′-GCCCCGTACTACGCTTACAA-3′; probe: 5′-CACATTTTCGATCGATTCTT-3′Ace: primers: 5′-ATCCGCATAATAATGTAAACTACGACAGAA-3′ and 5′-ACTCATTATATAGGTTTAAATCAACGCGAfCAA-3′; probe: 5′-CAAATTCAATTTCTCCTTTTTTCC-3′dlg1: primers: 5′-GCATCATCCAAAAGCTCGGTTT-3′ and 5′-GAGCCGACCATGTTTTGAATCTTAA-3′; probe: 5′-CCCTGCAAAGTTCC-3′TBP: primers: Prior to QPCR quantification of Pum targets, all primers and probes underwent the linearity test using 1, 2 and 4 µg RNA for RT reaction. Expression levels were normalized to Drosophila TBP transcript levels. TBP was confirmed as an unchanged control by comparing in excess of 100 RNA extractions each after spaced and massed training (data not shown). All reactions were done in parallel by using at least eight independent RNA isolations for each group, with each RNA isolate being assayed once. Normalized threshold values (Ct) were subjected to parametric t-tests, with significance levels set at alpha = 0.05.hunchback genomic rescuing construct with a deletion of NRE elements. An Asp718I-BamHI fragment containing each modified hunchback gene was cut out from the resulting plasmid and inserted into the P-element transformation vector CaSpeR4 digested with the same restriction enzymes. The resulting constructs were injected separately into w1118(isoCJ1) hunchback gene, four independent transformant lines were analyzed for the effects on segmentation pattern in embryos. NRE function in each line was tested by collecting embryos from heterozygous females. Cuticle preparations were analyzed according to Wharton and Struhl The in vivo function of the predicted NRE-like elements was tested as described by Wharton and Struhl 1118(isoCJ1) or UAS-Pumilio (on chromosome II) homozygotes flies.A stock  which is homozygous for both MB-specific Gal4 driver OK107 (on chromosome IV) and UAS-mCD8::GFP (on chromosome II) was crossed with wild type wAdult brains were dissected in 1× PBS, fixed in 1× PBS containing 4% formaldehyde for 30 minutes, and blocked in penetration/blocking buffer consisting of 1× PBS, 2% Triton and 10% normal goat serum  for 2 hours at 4°C. Then dissected brains were placed in primary antibody  for overnight at 4°C. After washing by Washing Buffer  for 4×10 minutes in room temperature, dissected brains were placed in secondary antibody (1:200 dilution in Dilution Buffer) for overnight at 4°C. The following antibodies were used: monoclonal anti-discs large-s antibody 4F3  as primary antibody for Dlg staining, monoclonal anti-Fasciclin II-s antibody 1D4  as primary antibody for FasII staining, Cy3 conjugated AffniPure Goat Anti-Mouse IgG (H+L)  as secondary antibody. Finally, the brains were washed by washing buffer for 4×10 minutes at room temperature, treated with FocusClear  for 10 minutes and mounted onto slides with MountClear .z-axis and 1024×1024 resolution in x- and y-axes. The Cy3 signal is captured by HeNe1 543nm laser and GFP signal is captured by Argon/2 488nm laser. All brains were scanned from the anterior to the posterior to ensure good resolution of MB. The raw data were processed by LSM Image Browser Rel.4.2 (ZEISS) and further arranged into figures by Adobe Photoshop CS2.Confocal stacks of brains were acquired using a ZEISS LSM 510 confocal microscope. Following confocal settings were used: 40× water immersion lens, 1 µm spacing in the Text S1Supplementary Material(0.05 MB DOC)Click here for additional data file.Figure S1Correlation Between Matrix Prediction Scores and Pum-Binding Affinities. The abscissa is the measured percentage binding of Pum to the mRNA target. The ordinate is the prediction score, which is the maximum matrix score of all the sites in a sequence. The 12 data points represent 12 mRNA sequences (nine test sequences in (0.16 MB TIF)Click here for additional data file.Figure S2MmSelex_M8 Matrix and the Correlation of Its Prediction Scores to Pum-Binding Affinities. (A) Base-frequency matrices obtained using Gibbs Sampler with mouse SELEX sequence data from White et al. (0.19 MB TIF)Click here for additional data file.Figure S3Estimation of False Positives with Random Shuffle Tests on the 151 Synaptic Genes. Shuffling times n = 500. (A) Matrix NRE_M10. (B) Matrix MmSelex_M8. The gray bars represent the hits with the original matrix. The black bars represent the average hits with randomly shuffled matrices. The error bar is the standard deviation across the 500 shuffling tests.(0.21 MB TIF)Click here for additional data file.Figure S4dlg1 Gene Structure as Shown in the FlyBase Genome Browser. Transcript dlg1-RA and dlg1-RC are located on non-overlapping regions on the fly genome.(0.06 MB TIF)Click here for additional data file.Figure S5Comparison of the Overlap of Our Pum Target Predictions with the Adult Specific Targets from Gerber et al. (0.09 MB TIF)Click here for additional data file.Table S1NRE_PAT Predictions(0.02 MB XLS)Click here for additional data file.Table S2NRE_M8 Predictions(0.03 MB XLS)Click here for additional data file.Table S3NRE_M10 Predictions(0.02 MB XLS)Click here for additional data file.Table S4Segmentation Pattern in Embryos of Modified Hunchback Gene Transformant Lines(0.02 MB XLS)Click here for additional data file.Table S5Synaptic Gene List(0.04 MB XLS)Click here for additional data file.