Somatic nuclear transfer is a cloning technique that shows great promise in the application to regenerative medicine. Although cloned animals are genetically identical to their donor counterparts, abnormalities in phenotype and gene expression are frequently observed. One hypothesis is that the cause of these abnormalities is due to epigenetic aberration. In this report, we focused our analysis on the acetylation of histone H3 at lysine9 (H3K9Ac). Through the use of whole genome tiling arrays and quantitative PCR, we examined this epigenetic event and directly compared and assessed the differences between a cloned mouse (C1) and its parental nuclear donor (D1) counterpart. We identified 4720 regions of chromosomal DNA that showed notable differences in H3K9Ac and report here many genes identified in these hyper- and hypo-acetylated regions. Analysis of a second clone (C2) and its parental donor counterpart (D2) for H3K9Ac showed a high degree of similarity to the C1/D1 pair. This conservation of aberrant acetylation is suggestive of a reproducible epigenetic phenomenon that may lead to the frequent abnormalities observed in cloned mice, such as obesity. Furthermore, we demonstrated Crp which was identified as a hyper-acetylated gene in this study is related to the body mass, suggesting that Crp is a possible candidate of a cause for the abnormal obesity in cloned mice. In this, one of the first reports describing genome-wide epigenetic abberation between parental and nuclear transfer-cloned mammals, we propose that aberrant acetylation of histones (H3K9Ac) flanking promoter regions highly correlates with gene-expression and may itself be an epigenetic change that accounts for variable expression patterns observed in cloned animals. Furthermore, different uterine environment from the donor may contribute to the abnormalities as well. In many labs, general attempts to identify abnormalities at a molecular level revealed atypical profiles of gene expression compared to naturally conceived animals. These abnormal gene expression patterns were observed not only in early development, such as in embryos but also in placentas and normally birthed cloned mice et al. have reported that cloned mice offspring show an over-diversity of gene expression compared to normal mice, but common abnormalities are surprisingly few. They also showed that some of these genes map in a linked cluster and are commonly changed in the same manner. Taken together, this evidence suggest that although there are many abnormalities caused by random components, cloned mice indeed exhibit common abnormalities that result from non-random components.Because success in cloning techniques using somatic nuclear transfer (NT) has led to the generation of genetically identical animals, the technique is now being developed for regenerative medicine. But despite substantial improvements in mouse NT cloning during recent years DNA methylation and histone modifications are known to be important epigenetic events that control cellular morphology and function Acetylation of histone H3 at Lysine 9 (H3K9Ac), one of the most well known epigenetic markers, is enriched in the promoter region of activated genes Application to a regenerative medicine, a comparison of clones to their donor counterparts may provide more valuable insights than comparing clones and normal animals. However, nearly all studies of cloned mice so far, have focused on this latter comparison. In the gene expression analysis, individual cloned mice show diverse results, suggesting that combined data from multiple cloned mice will not accurately represent abnormalities or differences of significance ChIP on chip) using the Affymetrix mouse tiling 1.0 array, which spans the entire mouse genome at a 30 bp resolution with 47 million 25-mer probes (excluding repetitive sequence elements). Fixed chromatin preparations from cloned mouse (C1) and donor mouse (D1) livers were immunoprecipitated with an antibody directed against the H3K9Ac. The immunoprecipitated DNA (treatment sample) and the control DNA from a whole cell extract (WCE) were amplified by the Ligation-Mediated PCR (LM-PCR) method and hybridized to the tiling arrays as previously described −4 as a significant cut-off To identify the H3K9Ac enriched sites, we performed ChIP followed by tiling array analysis  as a validation tool and picked 43 genomic sites to test. No consideration was given to the P-values of the selected sites but rather they were chosen for their general dispersion across the genome . In the sites showing 2-fold enrichment and above (≥log21), increasing enrichment clearly correlates with an increasing significance level of P-value. Out of 18 sites, Seventeen (17) sites which were detected as P≤10−4 yielded an enrichment greater than 4-fold in ChIP-qPCR . Based on this, we conclude that the cut-off of P≤10−4 is reliable to identify the H3K9Ac enriched sites with little false positives and high certainty. Furthermore, the total Pearson correlation coefficients were 0.68 and 0.74 in C1 and D1 liver samples, respectively. In addition, from the data, we can interpret that amplification of ChIP DNA by LM-PCR prior to hybridization to the tiling array did not introduce a significant bias into the ChIP on chip results.Although e genome . Of thes on chip . Sites dChIP on chip tiling array P-values from over 16,000 promoter region data points between C1 and D1 livers samples showed a high degree of similarity, with a Spearman correlation coefficient of 0.898 . Cut-off values for this comparison were relaxed relative to the previous ChIP-qPCR analysis because the reference material was another ChIP sample (not WCE) and as a consequence, the results displayed more variability. In addition, since the difference of H3K9Ac was relatively insignificant between C1 and D1 (2 (C1/D1)]) of an H3K9Ac level in the C1 sample was detected on 26 hyper-acetylated sites (2 (C1/D1)]) was detected on 22 hypo-acetylated sites  and donor (D2) pair that was derived using the same cumulus cell NT technique. We repeated the ChIP-qPCR experiment using the same primer sets for 26 hyper- and 22 hypo-acetylated genes  on ChIP The abnormalities of cloned mice have been recognized not only by phenotype In higher eukaryotes, transcriptional regulation is complex. Many factors and elements influence and directly regulate transcription, such as transcriptional regulatory factors, DNA methylation, and Chromatin structure. Furthermore, these elements work together and clearly influence one other, establishing a concerted functional “network” et al. reported that Crp induces leptin resistance, inhibiting the binding of leptin to its receptor As a possible phenotype-related H3K9Ac aberration, we noted C-reactive protein (Crp) is a hyper-acetylated gene identified in this study . Crp is To our knowledge, this is the first report describing genome-wide epigenetic aberration between donor and cloned animals. We propose that epigenetic abnormalities, such as those in H3K9Ac lead to abnormal gene expression, and that abnormal gene expression results in abnormal phenotypes. Hence, controlling the epigenetic landscape of a cell or by selecting a proper donor cell type possessing epigenetic information suitable to cloning we may be able to improve cloning success rates and the health of cloned animals.2 in Ca2+-free CZB medium in the presence of 5 g/mL cytochalasin B, and cultured for 24 h in CZB medium. When cloned embryos were developed to the 2-cell stage, it was transferred to the oviduct of foster mothers (ICR strain) which had been mated with vasectomized ICR males 1 day previously. All recipient females were euthanized at 19.5 dpc (days post coitum) and pups raised by lactating ICR foster mothers. All animals  were maintained in accordance with the Animal Experiment Handbook at Riken Center of Developmental Biology.BDF1 (C57BL/6×DBA/2) oocytes and fresh isolated BD129F1 (BDF1×129/Sv) cumulus cells were collected and used for the production of cloned mice as previously described 3, 1% SDS). The WCE aliquot and the eluted immune complexes were reverse cross-linked at 65°C for 3.5h and treated with RNase and Proteinase K. The DNA from WCE and the ChIP DNA was extracted using phenol/chloroform method and ethanol precipitation.Livers extracted from cloned and donor mice at the age of 2.5 month-old were homogenized using the BioMasher™ extraction device (Nippi Corp.). ChIP experiments using an anti-acetylated K9 histone H3 antibody  were performed as described Taq™-Plus (Cosmo Bio Corp.). The amplified DNA was purified, fragmented with DNase I, and end-labeled with biotin-ddATP by using terminal deoxytransferase. The labeled fragments were hybridized to the GeneChip® Mouse Tiling 1.0 Array Set (Affymetrix Corp.). The arrays were washed, and scanned using the Affymetrix GeneChip® System. Each sample was hybridized and tested as duplicate samples.LM-PCR and array hybridization were performed as previously described http://www.affymetrix.com/support/developer/downloads/TilingArrayTools) for the P-value calculation. All tiling array data including raw signal intensity and calculated P-value are available in Center for Information Biology gene EXpression database (CIBEX) (http://cibex.nig.ac.jp/) accession number CBX38.The hybridization intensities  of the probes were measured in two technical replicates for each of the treatment and control samples, and were mapped to the genome using exact 25-mer matching to mouse genome (NCBI version 32). Raw array data were quantile normalized within treatment/control replicate groups and scaled to a median feature intensity of 500. A shift of the intensities in the treatment relative to control data in a 400 bp window centered at each probe was evaluated by a Wilcoxon Rank Sum test, which assigned a P-value to the probe position ftp://ftp.ncbi.nih.gov/refseq) aligned to genome as TSS, we attempted to measure the magnitude of H3K9Ac enrichment in the promoter region. The alignment was carried out by using BLATThe profile of H3K9Ac enrichment in the promoter region (±1 kb of TSS) of protein-coding genes was compared between the donor and cloned mice. Using the 5′ ends of mRNA sequences  was used as instructed in the manual. The PCR conditions were an initial step of 10 sec at 95°C, followed by 40 cycles of 3 sec at 95°C and 20 sec at 62.5°C. To generate a standard curve for each amplicon, Ct values of serially diluted WCE DNA which extracted in ChIP experiment  were determined and plotted against the amount of WCE DNA template. ChIPed DNA was measured by real-time PCR in duplicates and the median of the average Ct values of all the amplicons in each sample was scaled to 22. The amount of an amplicon was estimated from the standard curve. Enrichment, (fold change or relative enrichment) was determined as multiple of the amount for each amplicon.PCR primers for evaluating ChIP assays  were desed genes . PCR amp5′-TGTGTCCGTCGTGGATCTGA-3′ and 5′-CCTGCTTCACCACCTTCTTGA-3′) for data normalization. The PCR primers used for each gene in this analysis are given  ELISA (High-Sensitivity), Mouse kit  was used to measure the Crp levels in the serums in accordance with the manufacturer's protocol.Table S1ChIP-qPCR Primers used for validation of the tiling array data.(0.02 MB XLS)Click here for additional data file.Table S2Hyper- or hypo-acetylated sites (start and end position on the mouse genome NCBI version 32) and primers used for ChIP-qPCR.(0.05 MB XLS)Click here for additional data file.Table S3Hyper- or hypo-acetylated genes and corresponding coding region primers used for qRT-PCR.(0.02 MB XLS)Click here for additional data file.


The ultimate goal of synthetic biology is the conception and construction of genetic circuits that are reliable with respect to their designed function . This task remains still to be attained due to the inherent synergy of the biological building blocks and to an insufficient feedback between experiments and mathematical models. Nevertheless, the progress in these directions has been substantial.It has been emphasized in the literature that the architecture of a genetic oscillator must include positive (activating) and negative (inhibiting) genetic interactions in order to yield robust oscillations. Our results point out that the oscillatory capacity is not only affected by the interaction polarity but by how it is implemented at promoter level. For a chosen oscillator architecture, we show by means of numerical simulations that the existence or lack of competition between activator and inhibitor at promoter level affects the probability of producing oscillations and also leaves characteristic fingerprints on the associated period/amplitude features.In comparison with non-competitive binding at promoters, competition drastically reduces the region of the parameters space characterized by oscillatory solutions. Moreover, while competition leads to pulse-like oscillations with long-tail distribution in period and amplitude for various parameters or noisy conditions, the non-competitive scenario shows a characteristic frequency and confined amplitude values. Our study also situates the competition mechanism in the context of existing genetic oscillators, with emphasis on the Atkinson oscillator. In the relatively young field of synthetic biology ,2, thereIn the context of genetic designs, two- and three-element networks have been shown to be enough for implementing oscillatory behavior either of transient or sustained type ,6. SeverThe way towards understanding and reliably predicting the information encoded by combinations of cis-regulatory sites lies in the coupling of such mathematical models with the experimental synthesis of regulatory promoter libraries -17. ReceIn this paper, we show how the dynamics of a simple two-component activator-inhibitor oscillator is drastically affected by the architecture of the promoters and the nature of binding by the transcription factors. We show that competitive DNA-binding of several transcription factors acting at the level of a promoter leads to a more fragile oscillator than with non-competitive binding. The type of binding mechanism and the circuit architecture together determine the features of the resultant oscillations. The intention of the present work is to emphasize the importance of choosing the appropriate mathematical modeling in ensuring the experimental achievement of the desired biological function.We also argue that detailed studies of these DNA-binding mechanisms could provide new insights into old experimental and theoretical studies of genetic circuits. A clear example is the experimental implementation of the Atkinson oscillator  for whicBackground section continues with an extended introductory section on transcriptional regulation and genetic circuits that provides the framework of discussion of the existing models and experimental implementations. Results and discussion section details the main results on the competitive and non-competitive transcriptional binding, especially the mathematical characteristics of the route towards sustained oscillations. In Conclusions section we emphasize the implications of the DNA-binding mechanism on the reliability of the genetic circuit in terms of its desired function.This paper is organized as follows: Genetic circuits involve a certain number of genes that regulate one another's expression . This rein silico progenitors of today's genetic circuits. Moreover, the coupling of transcriptional regulation with metabolic flux [sign" of genetic interaction (positive or negative), but also the type of interaction  will affect the behavior of the circuit. These dynamic consequences have only begun to be explored and are far from being clarified.Among the reasons for constructing synthetic biological circuits, there is the goal of understanding the fundamental building blocks and regulation mechanisms of biology ,25, and lic flux  has openlic flux ,39. Thislic flux . Given aWhen referring to an interaction network, one considers two types of interactions or loosely called feedbacks: positive (protein A promotes the production of protein B) and negative (A inhibits the production of B). Beyond the details of the interaction itself, the theory of circuits associates an overall sign to the interaction network itself, with the circuit being positive or negative depending only on the parity of the number of negative interactions in the network. A network is positive if it contains either positive interactions alone, or an even number of negative ones, while a negative network has an odd number of negative feedbacks ,50. The The situation might be different in non-genetic feedback loops, for example enzymatic circuits. This is the case commented by Smolen et al.  regardinThe simplest genetic circuit capable of producing sustained oscillations is a circuit of one gene with delayed self-inhibiting regulation Figure . The theAs discussed above, a circuit of odd-number negative feedbacks is equivalent to a positive-and-negative feedback circuit, and thus oscillations are theoretically possible. For example, a three-gene synthetic oscillator based on (non-explicitly delayed) negative interactions alone has been already verified experimentally  required for sustained oscillations (i.e. non-damped). For a multimer binding to a single binding site, the Hill coefficient coincides with the multimer's degree . From the experimental point of view, one hopes to obtain a high Hill exponent by introducing several binding sites for the transcription factor (TF). In this way and depending on the binding affinities of the TF, binding cooperativity may occur, leading to a higher exponent than the one corresponding to the degree of the multimer employed. For example, a Hill coefficient of n = 3 as in the example above may be obtained with dimers binding to two sites if some degree of cooperativity exists [Sufficient conditions for the occurrence of sustained oscillations are still an open issue in nonlinear dynamics theory, and naturally they are continuing to be delineated in feedback-based gene networks . It is cy exists ,63.relaxation-based oscillator containing three interactions: two positive and one negative. When the expression of a TF, say protein A, is controlled by two TFs, say A and R, through a positive and a negative feedback, respectively, one can imagine several transcriptional scenarios. In Figure A). For the competition case shown in (B), the activator A (circle) and the repressor R (square) compete for the binding on the promoter in the activator's module. In this case, the transcription of the activator occurs from two possible states of the activator promoter: occupied with A or occupied with R. For the design in (C), the inhibition occurs through post-translational control: the degradation of the activator is catalyzed by the repressor. These two designs have been studied by Guantes&Poyatos (2006) [Figure s (2006)  and we fA results from several combinations of the A promoter: AX, XB, AB, with X implying free site. Compared to Design I, the mathematical form of Design III is closer to the model associated to the Atkinson oscillator introduced by Scott et al. [damped oscillations rather than the sustained ones expected from the model [We introduce in Figure t et al. . Guantest et al.  claim tht et al.  envisiont et al. . This exhe model . There isustained oscillations. The way these equilibria lose their stability, that is the underlying bifurcation type, determines the type of oscillators. More precisely, when a parameter of the system is varied, a bifurcation is said to occur at a certain parameter value (bifurcation point) for which a qualitative change in the behavior of the system appears, such as the birth of sustained oscillations [Below we shall assess the implications of binding competition by comparing Design I with Design III. Before detailing these results, we wish to establish a framework for their comparison in the light of two existing works: Guantes&Poyatos  and Conrllations ,66.We suggest that circuit architecture AND DNA-binding processes determine the type of bifurcations leading to oscillatory solutions, types that confer specific features to the oscillations. Previous results show that Design I induces oscillations with large activator amplitudes and arbitrarily small frequencies, and acts as an "integrator" of external stimuli, while Design II shows emergence of oscillations with finite, and less variable, frequencies and smaller amplitudes, and detects better frequency-encoded signals ("resonator"). This classification originates from neuronal oscillations where the type of neuron (type I or type II) determines entrainment properties, phase response curves and robustness to noise . In a nuIn terms of equations, the systems describing the implementations from Figure dx/dτ, and the rest of the variables and parameters being defined in Table α gives a measure of how much stronger the transcription from the activator-bound promoter is, compared to the basal transcription from the free one. The parameters β and γ contain the total non-dimensional strength of the transcription and translation for the activator and repressor, respectively. The σ parameter contains the DNA-binding properties of the repressor multimer scaled to the activator's. For Design II, the parameter σ' differs in expression from that of σ in the other models, justifying the different notation . For th = 1, as . A paramad hoc general rule cited in the literature for the case of more proteins affecting a gene is that the functions appear multiplied [In addition to the above discussion of the parameters and their meaning, let us comment briefly on the similarities and differences on the equations describing the three designs. All designs share the same rate law for the repressor, while the definitory features concern the rate law for the activator. All rate-law terms have a form of the type: production term minus degradation term. Even from the equations, one can notice that Design II is different from the other two by its particular degradation term, the post-transcriptional regulation. On the other hand, the production terms for Designs I and III differ in a subtle manner: while the transcription factors appear summed in the denominator of Design I, they appear multiplied for Design III. The ltiplied . By our ltiplied , the curβ (abscissa) and γ (ordinate), where the period value is represented by a color from the color code in the right-hand-side of the figure. The dark-blue color is associated to cases lacking sustained oscillations.We developed numerical algorithms to simulate the equations characterizing Design I and Design III from Figure α and σ, and varied β and γ, in order to compare to existing results [We have chosen fixed values for Δ,  results . By this results , directlβ and γ to vary, we may relate to the experimental context. More precisely, the binding constants β and γ to take different values within a certain interval. These parameters could also be varied by varying the total promoter concentration, β and γ, respectively  , allowin008 , allβ) for two fixed values of γ: γ = 0:01 (panels A and C) and γ = 0:05 (panels B and D). As already identified by the studies mentioned above, two main bifurcation types are observed: the Hopf bifurcation and the saddle-node on an invariant cycle, or SNIC, bifurcation [In addition to the existence of the sustained oscillations, we also identified the bifurcation types that allowed this behavior. We illustrate in Figure urcation . Below, supercritical (or soft) and the subcritical (or hard) (defined below). In both cases, the real part of the complex eigenvalues becomes positive, and the unique stable state of the system loses stability. For the supercritical type (referred to as Hopf(p) in the figures), the oscillations begin with low amplitude near the bifurcation, which increases with the change in the parameter. It is the case of Figure sustained oscillations start or end with high amplitude at the bifurcation point. It is a consequence of two limit cycles, a stable and an unstable one, colliding at the bifurcation point, leaving an unstable fixed point surrounded by a stable limit cycle , whereas systems near saddle-node bifurcations do not . One woβ for which damped oscillations exist. In these ranges, the eigenvalues are complex, but with negative real part. The existence of an imaginary part of the eigenvalues leads to the oscillatory behavior, but due to the real part being negative, these oscillations are damped.In Figure β >βc = 0.225 in Figure For example, damped oscillations exist for β ≈ 0.1 is characterized by a SNIC bifurcation as β is increased, as the region shows long periods . This bs Figure . Crossins Figure . On the β and γ. In Figure β, γ . This analysis directly compares with the previous studies [As discussed by Conrad et al. , the par studies ,19. As cC matches the behavior of Design II from [biochemical mechanism  allows these bifurcations to occur at all. For example, Design I cannot pass from an integrator to a resonator by changing one parameter as happens for Design II and Design III.Returning to Figure  II from  in spitAs a final remark and returning to the discussion on the Atkinson oscillator, it is interesting to notice that the experimental implementation showed damped oscillations. We have mentioned that damped oscillations occur prior to a Hopf bifurcation, and not to SNIC bifurcation. Judging from the discussion of the two designs Figure , it is rboth damped and sustained, for a larger fraction of the parameter space , further reinforcing Figure Apart from the computation of the parameters leading to oscillations, let us now present a dynamical perspective on the differences between Design I and III. For a better understanding of the oscillatory features of the systems, we have studied in more detail the stability properties of the fixed points associated to the three designs. More specifically, we have calculated the eigenvalues for the fixed points associated to the three designs for various parameters spaces. The Additional File σx2y2 at the denominator. Both Design II and III show an interesting cut-off in activator's amplitude limiting it at low values. From these features can be seen that the biochemical mechanism leaves a characteristic fingerprint on the nature of the resultant oscillations. Having observed these features in amplitude and period, we have inquired on the dynamical causes responsible for these limitations. As already illustrated by [yx and yy represent the function y(x) obtained from the conditions yx and yy, and the form of these functions also gives clues on the general dynamics of the system beyond the steady states. The definition of the nullclines and the extended results of this analysis are included in Additional File yx for Design II and III are tightly related, with the first being the square of the other. This "similarity" however does not imply necessarily a similarity in dynamic behavior. The analysis of the nullclines as detailed in Additional File β does not imply a change in the shape of the nullclines, as the shape is mainly controlled by α. For this reason, these two designs present a characteristic frequency and a limitation in x amplitude. Moreover, from the equations can be seen that, starting from an oscillation-producing parameter case, an increase in β implies upwards displacement of yx , and an increase in Δ or γ implies upwards displacement of yy. For this reason, by increasing both β and γ simultaneously, their crossing is maintained and thus the oscillations too. This does not occur for Design I for which a change in β implies not only a displacement, but a change in shape, leading to a three-crossing case: no oscillations. Besides explaining the loss of oscillation capability for this design beyond a certain threshold value in β, the shape of the nullclines, as discussed in the additional file, is responsible for the high amplitudes in both x and y, as can be see in Figure S8.5.It is interesting to mention that, while the repressor's amplitude for the three models is similar, the distribution of activator's amplitude is specific for each design. It ranges from the queue of high amplitudes for the spiky oscillations of Design I, to an almost uniform and narrow distribution for Design III . The difference in this aspect between Design I and III is somehow expected, as the amplitude of the latter is further restrained by the term rated by  in theirThere has been increasing acknowledgment of the fact that internal noise at cellular level - low number of molecules -70 - canδA ≫δR. As commented also by Atkinson et al. [δA and/or decreasing δR. However, decreasing δR is not a good strategy as we have seen that the resulting oscillation period depends on δR. The shorter δR, the longer the period. For example, a value of δR = 0.02 h-1 typical value of δA = 1 h-1 would result in a good Δ for the occurrence of oscillations, but in a period of about 100 hours for Design III. Such a long period, much longer than the cell-cycle period, could be altered by the intrinsic cellular division period. On the other hand, Elowitz&Leibler [δA, that is synthetically design proteins that have a short life time (of the order of minutes). This strategy appears to be widespread nowadays.Once more, we wish to emphasize an important ingredient that makes oscillations possible in the above models: the time scale difference between the degradation rates of the two TFs given by Δ (the quotient of degradation rates). We have seen that for all these models, no oscillations are possible unless a substantial difference between the two degradation rates exists (Δ ≫ 1). The requirement thus says that n et al. , strateg&Leibler  follow tgeneral we refer to a non-competitive design similar to Design III, but having several bindings sites for activators and repressors. In this case thus, an approximate model would employ the general form of Hill function:Another strategy to facilitate oscillatory conditions is a more pronounced nonlinearity of the regulatory function, that is consider a Hill function of higher exponent. The mathematical model for Design III considered dimers as active TFs and one binding site for each feedback. For example, let us assume the general case for Design III. By the term β and γ depend on the degree of multimerization of the TFs. If n ≠ 2 and m ≠ 2, the parameters β and γ differ in expression from those employed in the previous section, and thus a direct comparison with the previous results is difficult. Nevertheless, the interesting remark to make here is that oscillations are possible even for Δ = 1 (no difference in degradation rates) when n = 3, as seen in Figure n, the nonlinearity of the positive feedback, is responsible for the existence of the oscillations, while m relates to the extension of the parameter space leading to these oscillations: the higher the m (degree of repressor's multimerization), the wider the oscillatory region.However, keep in mind that the parameters n = m = 4:Again concerning the nonlinearity of the feedback, let us return to the experimental implementation of the Atkinson oscillator. The experimental design  appears β and γ have the same expression as in Design I and thus are directly comparable. Notice in Figure and where Δ, The model from eqs. (1-2) is a simplified version of the Atkinson model as described by Scott et al. . There, Still in two-gene systems, another strategy to facilitate oscillations is introducing another positive feedback for Design I, more precisely an autocatalysis on the repressor. This eliminates the necessity of large Δ, while maintaining dimers and one binding site. Of course, this leads to another architecture than the one shown in Figure Notice in Figure In the present work, we have analyzed two DNA-binding mechanisms in composed promoter sites and how they affect the existence of oscillations in genetic circuits. There is general consensus that both positive and negative genetic interactions are needed to obtain a robust oscillator . Given aFrom our results, we have emphasized the fact that, when these interactions, either positive or negative, are accomplished through transcriptional regulation, the interactions between various binding sites on the promoter need to be taken into consideration. We showed the non-competition scenario to present robustness to parameter changes defined by the existence of oscillatory solutions for a much wider parameters range than for the competition one. Moreover, the two scenarios imply different routes toward the occurrence of oscillations, that is different bifurcation types. While the competition model is mainly associated to the SNIC bifurcation, the non-competition one relies more on the Hopf bifurcation. While the former bifurcation type leads to more spiking-like oscillations, the latter shows more sinusoidal ones. In addition, a characteristic of the Hopf bifurcation is that sustained oscillations are preceded by damping oscillations. This may prove to be an important feature to consider when interpreting experimental data. Many other interesting properties arise as a consequence of the type of the oscillation-generating bifurcation, an issue that has been extensively analyzed in previous works ,19,67 anThe Atkinson oscillator introduced and experimentally implemented by  has beenHere we have used simplified models of genetic circuits. In this simplification, the modeling of genetic circuits includes only the temporal evolution of the protein concentration ,74. ThisFor the numerical simulations, we developed C++ codes employing routines from the free-software GNU Scientific Library (Additional File All authors contributed to the design and coordination of the study. AM performed the computational implementations and prepared the original draft, which was revised by RVS, and subsequently by MC and MI. All authors read and approved the final manuscript.Transcriptional regulation and model description. A PDF file containing a brief introduction to transcriptional regulation and the terminology employed in the main text. The details necessary for obtaining the equations employed in this study are also included here.Click here for fileThe C++ codes developed for the numerical simulations, as well as the instructions of their use.Click here for fileOscillatory region for an extended parameter space for Design III.Click here for fileComparison between Design I and Design III for Δ = 20.Click here for fileThe behavior of Design II from Guantes&Poyatos[s&Poyatos.Click here for fileExample of a subcritical Hopf bifurcation.Click here for fileThe trajectory behavior prior to a Hopf and to a SNIC bifurcation.Click here for fileRobustness to parameter changes.Click here for fileDynamical differences between the designs: discussion of the nullclines.Click here for fileThe influence of internal noise.Click here for file


The spindle checkpoint delays anaphase onset until all chromosomes have attached in a bi-polar manner to the mitotic spindle. Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors. Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1. Here, fission yeast FRAP experiments confirm that Bub1 stably binds kinetochores, and by tethering Bub1 to telomeres we demonstrate that it is sufficient to recruit anaphase inhibitors in a kinase-independent manner. We propose that the major checkpoint role for Bub1 is as a signalling scaffold. Cells employ many mechanisms to ensure that their genomes are replicated and segregated with high fidelity every cell cycle in vitroThe molecular mechanism of action of the spindle checkpoint remains unclear, although several important findings have been made. First, a single unattached kinetochore is sufficient to activate the checkpoint in vitro, using Mad1/Mad2 “scaffolds” coupled to beads in vitro work has been reported for BubR1/Mad3, Bub3 or Bub1 dynamics.Here we focus on the mechanism of recruitment of checkpoint proteins to kinetochores, and their exchange dynamics once recruited. Several fluorescence recovery after photo-bleaching (FRAP) studies have described the dynamics of spindle checkpoint proteins and Cdc20 in vertebrate cells In fission yeast, Bub1p is necessary for the efficient recruitment of Bub3p and Mad3p to kinetochores, and their targeting is independent of Mad1p and Mad2p 1/2 of ∼30 seconds bub1+ gene has been C-terminally tagged with GFP, such that it is expressed from its own promoter at the endogenous locus, and a range of checkpoint and chromosome segregation assays demonstrate that it is fully functional nda3). These cells arrest early in mitosis, with no microtubules, unattached kinetochores and hyper-condensed chromosomes nda3 cells with anti-microtubule drugs (25 µg/ml carbendazim) to ensure the arrest was maintained. We carried out FRAP experiments and a representative example is shown  and arrested it in mitosis at 18°C for 6 hours. Here we observed 82 (+/−9) % recovery of Mad3-GFP, with a recovery half-time of 17 (+/−4) seconds  and the Bub1-Tel (GFP)  and S4. Here we have tested critical aspects of the scaffolding model for Bub1 checkpoint function. Bub1p is relatively stably associated with kinetochores, and is sufficient to recruit other checkpoint proteins, and therefore displays the two key characteristics of a signalling scaffold. Note, our scaffold lacks the C-terminal kinase domain, confirming that this is not necessary for the recruitment of Bub3p and Mad3p. This ectopic targeting of Bub1p, Bub3p and Mad3p to telomeres had no consequence on cell cycle progression. Unfortunately the levels of Bub3p and Mad3p recruited to the telomeres were not sufficient to carry out detailed FRAP studies. These are important experiments for the future, and we will extend this approach by co-targeting of a Mad1p scaffold, and test whether the concerted action of Mad1p and Bub1p is sufficient for generation of “wait anaphase” signals.It is not clear why all the checkpoint proteins are recruited to kinetochores. We speculate that certain checkpoint proteins, and perhaps the Cdc20 effector, not only undergo structural conformational change See 1–586-GFP-Taz1Myb was constructed as follows. The bub1 promoter (500bp upstream of the bub1 ATG) plus a 1758 bp fragment of bub1+ encoding the first 586 residues was amplified from S. pombe genomic DNA with a NotI added at the 5′ end and SmaI at the 3′ end of the fragment. This was inserted into the MCS of the plasmid pJK148 PstI added at the 5′ end and SalI at the 3′ end of the fragment and ligated into the same unique sites of the plasmid, in-frame to the 3′ end of the Bub11–586 fragment. Primers used were: GFPTaz1PstIFW–CGATCTGCAGATGAGTAAAGGAGAAGAAC and GFPTaz1SalIRV–GCCGTCGACTTAAGATTGATAATTAACAAG. The resulting plasmid was integrated into the chromosome at the leu1 gene locus in cells disrupted for the bub1+ gene.Bub1The mCherry and tdTomato fusion constructs with Bub3 and Mad3 respectively were made using the PCR-based gene targeting method Preparation of yeast cell extracts, SDS-PAGE and immunoblotting were carried out as previously described Live-cell imaging was typically performed in minimal media, on a pad of 1% agarose. Some GFP, mCherry and tdTomato imaging  was alsoThis system was also used for FRAP: cells were mounted on a pad of 1% agarose and PMG media with 25 µg/ml carbendazim (CBZ) and sealed with VALAP (Vaseline/Lanolin/Paraffin). Photobleaching was carried out using a 514nm Nitrogen dye laser system (Photonic Instruments). Images were captured and analysed using Slidebook software . To calculate fluorescence recovery, images were captured with 200ms exposure at 2x2 binning at various time intervals post bleaching. The kinetics of FRAP were analysed as described Table S1Analysis of co-localisation between Bub1-TEL, Mad3, and kinetochores (Ndc80).(0.05 MB PDF)Click here for additional data file.Table S2Analysis of co-localisation between Bub1-Tel, Bub3 and kinetochores (Ndc80).(0.05 MB PDF)Click here for additional data file.Table S3Analysis of co-localisation between Bub1-Tel, Mad3 and telomeres (Pot1).(0.04 MB PDF)Click here for additional data file.Table S4Analysis of co-localisation between Bub1-Tel, Bub3 and telomeres (Pot1).(0.05 MB PDF)Click here for additional data file.


Toxoplasma gondii. Promoters of T. gondii ribosomal proteins (RPs) were analyzed by EMSAs and ChIP. One RP promoter domain, known to bind an Apetela 2, bound to nuclear extract proteins. Promoter domains appeared to associate with histone acetyl transferases. To study effects of a RP gene's regulation in T. gondii, mutant parasites (Δrps13) were engineered with integration of tetracycline repressor (TetR) response elements in a critical location in the rps13 promoter and transfection of a yellow fluorescent-tetracycline repressor (YFP-TetR). This permitted conditional knockdown of rps13 expression in a tightly regulated manner. Δrps13 parasites were studied in the presence (+ATc) or absence of anhydrotetracycline (-ATc) in culture. -ATc, transcription of the rps13 gene and expression of RPS13 protein were markedly diminished, with concomitant cessation of parasite replication. Study of Δrps13 expressing Myc-tagged RPL22, -ATc, showed RPL22 diminished but at a slower rate. Quantitation of RNA showed diminution of 18S RNA. Depletion of RPS13 caused arrest of parasites in the G1 cell cycle phase, thereby stopping parasite proliferation. Transcriptional differences ±ATc implicate molecules likely to function in regulation of these processes. In vitro, -ATc, Δrps13 persists for months and the proliferation phenotype can be rescued with ATc. In vivo, however, Δrps13 could only be rescued when ATc was given simultaneously and not at any time after 1 week, even when L-NAME and ATc were administered. Immunization with Δrps13 parasites protects mice completely against subsequent challenge with wildtype clonal Type 1 parasites, and robustly protects mice against wildtype clonal Type 2 parasites. Our results demonstrate that G1 arrest by ribosomal protein depletion is associated with persistence of T. gondii in a model system in vitro and immunization with Δrps13 protects mice against subsequent challenge with wildtype parasites.Molecular pathways regulating rapid proliferation and persistence are fundamental for pathogens but are not elucidated fully in  Toxoplasma gondii could play a role in control of this parasite's cell cycle and consequent phenotype in a model system in vitro. Recent studies of ribosomes and/or cell cycle Saccharomyces cerevisiae the absence of RPs cause cell cycle arrest in G1 ArabidopsisDrosophila melanogaster “minutes” T. gondii ribosomal proteins (RP) T. gondii contain highly conserved T. gondii RP (TRPs) promoter sequences TCGGCTTATATTCGG) and TRP2 ([T/C]GCATGC[G/A]), are located close to transcriptional start sites of RPs T. gondii genome. For example, a small RP (rps13’s) promoter has two TRP2s, in addition to a motif found to regulate bradyzoite gene expression, TGTGTGIt was of interest to determine whether transcriptional regulation of a ribosomal protein (RP) in T. gondii also has orthologous molecules to those in other organisms that interact with transcription factors regulating RPs. Similar to other eukaryotes, activation of gene expression in Toxoplasma involves acetylation of histone proteins S. cerevisiae, MYST family histone acetyltransferase (HAT), Esa1, is recruited to RP gene promoters to regulate transcription Plasmodia, T. gondii and plants Plasmodium, a global yeast two hybrid analysis suggested that ApiAP2 proteins interact with chromatin remodeling factors and each other P. falciparum ApiAP2 protein PfSIP2 is key in chromosome end biology involving heterochromatin formation and epigenetic factors Arabidopsis, especially in stress conditions, a HAT interacts with an AP2, CBFI T. gondii. Consistent with this hypothesis, the TRP2 promoter element had been reported to contain bases that bind a Plasmodium AP2 T. gondii's ribosomal components are essential for ribosomes to function properly. Thus, a new system using reagents described earlier rps13) had been selected earlier T. gondii profoundly.Little was known about which of T. gondii genes T. gondii had been described earlier although regulation of a functional endogenous gene product was not accomplished T. gondii that would stably modify gene expression, independent of promoter strength. In studies, foundational for the present work, tetracycline repressor response element (TetO)-modified promoters for T. gondii rps13 had been created The use of a transactivator system had successfully created conditional knockdowns of T. gondii ribosomal protein, (and hence ribosomes) including its regulation and production, was investigated using an inducible knockdown system T. gondii proliferation and persistence in an in vitro model system was obtained. Conditional knockdown of rps13 created a G1 arrested parasite that persisted in this tissue culture system for months. However, this mutant parasite did not appear to persist in vivo and protected mice against challenge with wildtype parasites.Herein, our studies provide new empirical information concerning ribosomal protein (RPs) promoters and promoter associated proteins. A role of a T. gondii RPS13 was produced using the phyre modeling server. This model used the solved Thermophilus ribosomal protein RPS15 structure as a starting model. Analysis of the sequence alignment between various RPS13 proteins and the Thermophilus RPS15, protein from which the T. gondii model is based, shows a significant amount of sequence similarity between not only the RPS13 family, but the RPS13 and RPS15 proteins . RNA samples were taken from clone 3 grown for 4 and 24 hours. Amount of measured rps13 was normalized to total RNA. Primers at the C terminus outside the region of the construct for homologous recombinants were used. The effects of culturing the conditional knockdown parasites -ATc on abrogating rps13 mRNA were apparent early, at the 4-hour time point. Reduction of mRNA was robust by 24 hours. We found a 3-fold diminution of mRNA at 4 hours and a 10-fold diminution at 24 hours   . No multrps13 on ribosomes.To further characterize the effect of knockdown of RPS13 on expression and localization of large ribosomal proteins and assembly of large subunits of ribosomes, stable transfectant parasites expressing RPL22-Myc were created as described in vitro culture, the mutant parasites that had been continuously cultured for over 40 passages in the presence of ATc were then cultured in the presence or absence of ATc. In the presence of ATc, the mutant parasites maintained a proliferative phenotype. In the absence of ATc, proliferating parasites were not noted in the cultures examined using an inverted microscope to monitor tissue cultures. The phenotype persisted in parasites that had been passaged many more than 40 times in the presence of ATc. These results indicate that the mutant parasites are stable in vitro even over prolonged continuous culture conditions and do not revert to the wildtype genotype.To determine the stability of the RPS13 conditional knockdown system in the mutant parasites under prolonged rps13 was lethal, mutant parasites (clone 3) with conditional knockdown of rps13 (-ATc) were cultured in multiple experiments for times up to 75 days during which time no lytic plaques or obviously replicating parasites could be observed microscopically. After the varying periods of culture -ATc, the infected fibroblasts were scraped off and aliquots of the cell suspension split into two equal parts and used to infect fresh fibroblasts ±ATc. In the fibroblast cultures +ATc, rapidly proliferating parasites could be observed as early by approximately 5 days for parasites previously maintained for 27 and 75 days -ATc. These parasites went on to destroy fibroblast monolayers in the presence of ATc. In contrast, parasites in -ATc, still did not proliferate and did not destroy the monolayers. There were two replicate trials performed to 2.5 months. By 5 days after addition of ATc there were replicating parasites in these cultures (Data not shown).To determine whether conditional knockdown of rps13 might initiate a stress response. Thus, real-time PCR analysis of the transcript levels of stage specific markers for tachyzoites (SAG1) and bradyzoites (BAG1) in clone 3 cultured ±ATc for 4 hours, 24 hours and 5 days was performed. At 4 hours of culture, there was no change in SAG1 expression while there was a notable increase in BAG1 transcript levels. By 24 hours of culture BAG1 transcripts had increased substantially while that of SAG1 had decreased. The dynamics of both SAG1 and BAG1 transcript levels suggested a coordinated effect of perturbation of gene expression. The ratio of BAG1 to SAG1 was elevated in the absence of ATc suggesting an early “stress” response. These findings were reproducible in two replicate experiments  is markedly diminished in RPS13 conditional mutants, but large subunit RNA is not (28S) . RibosomToxoplasma microarray data was analyzed to determine whether there was an association with a G1 transcript profile. Four hundred and seventy-five genes with significant gene expression differences  between ±ATc samples were identified. These gene expression differences are represented in the heat map in T. gondii . Over 60% of the genes that have diminished expression -ATc overlap with genes that are diminished at G1, and over 40% of genes with increased expression -ATc overlap with genes that are up-regulated at G1. A hypergeometric distribution of this association suggests that the probability of this happening by chance is essentially zero. Thus, by both FACS and microarray analysis, parasites -ATc display a G1 profile. Affymetrix in vivo or would be eliminated by a competent host immune response was of interest. Thus, 100 tachyzoites of the conditional mutant clone 3 were administered intraperitoneally to female Swiss Webster mice, half of which were given 0.2 mg/mL ATc to drink in their water and half of which were given water -ATc. A third group was left uninfected but given ATc to drink in their water. By 10 days, all infected mice given ATc had died, whereas all other mice were alive and remained alive for up to a year , and persist at least for a short period of time while they elicited an immune response. Thus, in two replicate experiments, groups of mice were given 100,000 RPS13 conditional mutant parasites and all mice survived. They then received booster doses of 100,000 RPS13 conditional mutant parasites 2 weeks and 4 weeks later without ill effect. Two weeks later they were challenged with 2,000 RH tachyzoites and all unimmunized control mice died while all immunized mice survived  gene DNA in any of 8 mice (data not shown). RH tachyzoite DNA (this PCR method was sensitive in detection of 4 parasites) was the positive control for this quantitative PCR experiment (r2 = 0.99).To determine whether any parasite DNA could be found to persist in brains of these immunized and challenged mice, PCR with primers to detect the multi-copy B1 gene and quantitative PCR to detect the 300 repeat copy 529 base pair gene was performed with brain from mice infected for prolonged periods of time after immunization with P<0.01). In these experiments, qPCR of brain using the 300 copy 529 bp gene was performed following challenge of control and immunized mice. This demonstrated marked diminution in parasite burden in brain following immunization, most dramatically for those immunized two times compared to the unimmunized group  and 5 mice immunized two times (2×) mice in each trial and thus a total of 10 immunized mice for each type of immunization . They were challenged with 50 Me49 cysts i.p. and at 30 days after challenge, cyst numbers were found to decrease markedly in immunized mice ; P<0.01.ed group P≤0.001.ed group .T. gondii, although there is precedent for this fundamental biological process in other eukaryotes such as yeast. Our findings provide an in vitro model which suggests that T. gondii's sensing of intra-parasite stresses could modulate regulation of transcription of its rp genes. RPs influence biogenesis of ribosomes, and subsequently, the ability of the parasite to either proliferate or arrest in G1. Analysis of promoters of RPs led Schaap and van Poppel to postulate that two conserved rp gene promoter cis elements, TRP1 and TRP2, regulate expression of rp genes, and thereby, biogenesis of ribosomes rp transcription T. gondii AP2, X1-3 . Movement of AP2s to the nucleus occurs in waves associated with the cell cycle and transcription of genes rpl9 enhances transcription of a reporter gene T. gondii, as van Poppel and Schaap had postulated. These latter studies occurred while we found that these six bases that are part of the TRP2 promoter element were necessary, but not sufficient, for binding the nucleoproteins in the complex in our EMSAs. We found that additional C terminal bases also were critical for binding/associating with nuclear extract proteins. The loss of different bands with differing mutations in the full TRP2 promoter element suggests that additional bases also contribute to a complex or complexes of associated nuclear extract proteins. For mutants 7 and 8 all associations with all nuclear extract proteins are abrogated. One explanation for this might be that an association with some of the other proteins with bases absent in mutants 7 and 8 could be critically dependent for or on protein(s) associated with TGCATG. Otherwise, it might be expected that only some of the bands would disappear because the TGCATG core site is intact in mutants 7 and 8. Recent work of others rps13 promoter using a luciferase reporter may be useful in clarifying the importance of these bases in both of its TRP2 elements in enhancing transcription. Their relative importance and interaction with the TGTGTG promoter element, previously shown to be important in bradyzoite gene transcription, in the rps13 promoter also will be of interest in future studies.Our finding that abrogation of a ribosomal protein causes a specific G1 arrest confirms the strength of the refined Tet model for conditional gene knockdown. This influence of ribosomes on proliferation and persistence has not been demonstrated previously in Toxoplasmarp promoters. These results are similar to the association of HATS with AP2 transcription factors that regulate genes in ArabidopsisPlasmodiaPlasmodia demonstrated an AP2 interaction with GCN5 histone acetyl transferases and Swi2/Snf2 ATPases Arabidopsisin vitroT. gondii. Further, future studies with knockdown and tagging these genes, ChIP, and immunolocalization will be useful in definitely proving there is truly an association and if so in characterizing the association.In addition, we found that αMYST A and B caused shifts in our EMSAs with both TRP1 and TRP2. Myst B (GCN5) was identified by mass spectrometry of nuclear factors binding TRPs 1 and 2. Also, in ChIPs, antibody to flag tagged MYST B suggested interactions with TRP1 and 2. Attempts to perform this ChIP study for TgMYST A were complicated by the fact that an additional copy of TgMYST A is lethal to T. gondii and a TOR-like protein appears to be transported into the nucleus. Putative homologues of kinases, transcription factors, cyclins and a ceramide activated protein phosphatase that is involved in sensing cellular stress and mediates G1 arrest via TORC2 in yeast have been identified in T. gondiirps13 and TRP1 in the promoters of other RPs in T. gondii. This further suggests that TOR and PKA pathways may participate in regulation of proliferation and persistence in T. gondii. Roles for long chain fatty acids and ceramide synthases producing ceramides and signaling through TORC2 T. gondii. Sensing of cellular stress in plants is mediated by PYR/PYL/RCAR T. gondiiWe note, relevant to our model , as suggT. gondii and whether it is operative in vivo remains to be determined. Plasmodia monitor nutrient support for translation of proteins before committing to DNA synthesis. The cell cycle mechanisms sensing ribosomes or translation factors Further, growth arrest in yeast and animals can be mediated by depletion of ribosomal proteins. In yeast, ribosome biogenesis is linked to G1 of the cell cycle T. gondii in vitro, a new conditional knockdown system was used with a TetR knockdown system using constructs discussed earlier rps13 using a reporter gene in T. gondiiT. gondii endogenous RPS13 gene. Separately we have found this system is effective in regulating a novel dense granule protein and parasite enzymes .To study effects of ribosomal protein S13 on proliferation and persistence in Thermophilus RPS15 as a basis because this latter structure had been solved. This confirmed that this T. gondii molecule has all the features to be a RPS13. It has residues likely to interact with ribosomal RNA  suggests that most ribosomal proteins, including RPS13, do not have altered expression across strain or developmental stages. There are a few exceptions, however. For example, RPL4 transcript levels are increased in type III CTG. RPL4 (42.m05824) was recently found to be a part of the channel in ribosomes for assembling proteins The forced proliferation arrest observed herein shares the G1 arrest feature associated with the Δrps13 mutant parasites can persist in vitro in this proliferation arrested state for prolonged periods of time (months) and yet be revived by adding ATc. This is reminiscent of how plants can “go to sleep for a long, long time” through the PYR abscisic acid calcineurin stress response pathway T. gondii has this pathway. The recent work of Sibley et al demonstrates that this ABA system is operative in T. gondii.Our work provides novel insights into significance of this protein for this parasite's cell cycle, proliferation, and persistence in tissue culture after removal of ATc. Our Δrps13 can maintain functions vital for persistence while neither replicating nor differentiating to encysted bradyzoites fully. Lack of ribosomes themselves are a model leading to arrest in G1 in other organisms T. gondii  that allows escape from the immune response, an imbalance of ribosomal proteins and diminution of ribosomes might allow a variety of microorganisms that can stop replicating and persist in a basal state to persist in vivo for a long time.Some of the genes that are expressed in fully differentiated bradyzoites were not transcriptionally upregulated -ATc . This su. gondii . The mecin vivo. They appear to be incapable of persistence in the brain in the presence of a competent immune response. They cannot be demonstrated to be revived in vivo by subsequent administration of ATc administered alone or with L-NAME. Nonetheless, they protect mice against challenge with wildtype clonal Type 1 parasites completely, or clonal Type 2 parasites robustly. They are proving useful as internal “gold standard of protection” controls in ongoing studies to develop biomarkers and to produce a component vaccine to protect humans against toxoplasmosis .Our conditional mutant parasites do not appear able to evade the immune response in vivo, like Δrps13 in vitro, also are arrested in G1 in vivo and could contribute to the slowed replication in the full encysted, bradyzoite phenotype. However, although it may be a component part of development of a bradyzoite phenotype and even necessary, the studies herein demonstrate that it is not sufficient for the full phenotype. At a minimum, other genes which allow for the sequestration from the host's immune response and for other essential bradyzoite genes to be expressed must also be necessary for bradyzoites to form and encyst since Δrps13 does not persist in brain in vivo in the presence of a competent immune response.Since bradyzoites in vitro persistent, G1 arrested phenotype in a “stressed” parasite is common to other parasites that are attenuated and proving to be immunogenic and protective. Examples of such parasites are those attenuated by knockout of genes encoding components of nutrient pathways and other unknown means such as in temperature sensitive mutant 4. The robustness of protection conferred by Δrps13 combined with its lack of persistence in vivo, and inability to induce growth of the parasite with ATc or L-NAME after the first week, may provide insights into establishing protection by other means. These results suggest that a similar persistent exposure to immunogenic epitopes for several days might be useful in development of component vaccines as well. This conditional knockdown parasite appears to be sufficient to stimulate the immune system without establishing a chronic infection  and conducted according to AAALAC and USDA guidelines .T. gondii and assays that measure uracil uptake Methods are presented in detail in Figure S1(A) TRP1 and TRP2 EMSA with radio labeled TRP1 and TRP2 probes with (+) and without (−) cold competitor TRP1 or TRP2 with tachyzoite nuclear extract. Note mobility-shift which indicates that there is protein in the nuclear extract that binds to TRP2, which is competed by cold TRP2. TRP2 arrow indicates level in the gel where this occurs. (B) EMSAs with radio labeled TRP1 [left lanes] and TRP2 [right lanes] with nuclear extracts from tachyzoites competed by cold probe second lane, and with antibody to MYST A [third lane] and MYST B [fourth lane]. Some of the differences are highlighted by inclusion in green rectangles. Bands are marked by letters a to g so they can be identified in each of the corresponding lanes. In S1A * indicates a nonspecific band that is not competed by cold probe. S1A shows part of the EMSA beginning with band A. In S1A and S1B, bands of similar mobility for TRP1 and TRP2 are not necessarily binding the same nuclear extract proteins. Bands are indicated with letters to indicate corresponding bands between (2.62 MB TIF)Click here for additional data file.Figure S2in vitro +ATc. (A) Creation of parasites. These parasites were created as described previously for parasites with one TetO in the rps13 promoter Δrps13 conditional knockdown, genomic integration of four TetO elements in the rps13 locus was accomplished with a hit-and-run mutagenesis strategy TCCCCGACAACACCTTCTAC) and native T. gondii genomic DNA resulting in different pseudodiploid conformations rps13 promoter (prps13) as shown in the schematic diagram top, right. An alternate pseudodiploid that did not occur could have formed with a crossover within the rps13 intron. PCR using primers 3 (GTCGAGTCCTGTAGGTTCATC) and 10 (GGAGATCTCTATCACTGATAGGGA) on DNA isolated from mycophenolic acid-xanthine-resistant clones showed that only one clone, B28, had the replacement construct integrated at the rps13 locus . PCR with primers 3 and 10 yielded a product of the correct size to include the rps13 gene promoter and four TetOs. Primers 9 (TCCCTATCAGTATAGAGATCTCC) and 4 did not amplify products (data not shown). PCR with DNA from 6-thioxanthine-resistant clones using both HXGPRT primers  as well as primers 3 and 4 (CTCCGAAGGAGTCTCTCAGTG)  show that pseudodiploidy has been lost for clones 1, 6, 12, 15, and 18. PCR using primers 3 and 10  show that clones 1, 6, 12, 15, and 18 retained TetO elements in the rps13 promoter. Parasites, e.g., chloramphenicol-resistant clones 3 and 9, express YFP (data not shown). (B) Comparison of uracil uptake in Δrps13 mutant parasites and parental strains +ATc. Uracil uptake in the mutant parasite compared to wildtype or parental strain was similar. There was no diminution of uptake in Δrps+ATc. Parental and conditional mutant knockdown parasites are shown.Creation of conditional mutant parasite and replication (0.61 MB TIF)Click here for additional data file.Figure S3Expression of RPS13 and RPL22. (A) Western blot of cultures ±ATc at 4 and 48 hours probed with αRPS13 and αSAG1. (B) IFA at 24 hours and 4 days probed of cultures ±ATc with αMyc to detect Myc-tagged RPL22.(2.22 MB TIF)Click here for additional data file.Table S1Mass Spectrometry.(0.07 MB XLS)Click here for additional data file.Table S2±ATc Transcriptome.(0.29 MB XLS)Click here for additional data file.Text S1(0.22 MB DOC)Click here for additional data file.Movie S1RPS13.(3.65 MB MOV)Click here for additional data file.


Plasmodium falciparum. While recent studies assigned important roles for reversible histone modifications, silent information regulator 2 and heterochromatin protein 1 (PfHP1) in epigenetic control of variegated expression, factors involved in the recruitment and organization of subtelomeric heterochromatin remain unknown. Here, we describe the purification and characterization of PfSIP2, a member of the ApiAP2 family of putative transcription factors, as the unknown nuclear factor interacting specifically with cis-acting SPE2 motif arrays in subtelomeric domains. Interestingly, SPE2 is not bound by the full-length protein but rather by a 60kDa N-terminal domain, PfSIP2-N, which is released during schizogony. Our experimental re-definition of the SPE2/PfSIP2-N interaction highlights the strict requirement of both adjacent AP2 domains and a conserved bipartite SPE2 consensus motif for high-affinity binding. Genome-wide in silico mapping identified 777 putative binding sites, 94% of which cluster in heterochromatic domains upstream of subtelomeric var genes and in telomere-associated repeat elements. Immunofluorescence and chromatin immunoprecipitation (ChIP) assays revealed co-localization of PfSIP2-N with PfHP1 at chromosome ends. Genome-wide ChIP demonstrated the exclusive binding of PfSIP2-N to subtelomeric SPE2 landmarks in vivo but not to single chromosome-internal sites. Consistent with this specialized distribution pattern, PfSIP2-N over-expression has no effect on global gene transcription. Hence, contrary to the previously proposed role for this factor in gene activation, our results provide strong evidence for the first time for the involvement of an ApiAP2 factor in heterochromatin formation and genome integrity. These findings are highly relevant for our understanding of chromosome end biology and variegated expression in P. falciparum and other eukaryotes, and for the future analysis of the role of ApiAP2-DNA interactions in parasite biology.The heterochromatic environment and physical clustering of chromosome ends at the nuclear periphery provide a functional and structural framework for antigenic variation and evolution of subtelomeric virulence gene families in the malaria parasite  Plasmodium falciparum, like many other unicellular pathogens, uses antigenic variation of surface proteins as a prime mechanism for immune evasion. Subtelomeric gene families, including the major virulence family var, encode these antigens, and recent studies highlighted the important contribution of the specific biology of chromosome ends in regulation of their expression. P. falciparum chromosome ends are enriched in epigenetic marks of eukaryotic heterochromatin; however, it remains unknown which proteins and regulatory DNA motifs are involved in the recruitment and organization of heterochromatin, and how they contribute to the role of subtelomere biology in this important pathogen. Here, we present the experimental identification and genome-wide characterization of PfSIP2, a member of the ApiAP2 family of transcription factors that are specific to Plasmodium and related apicomplexan parasites. PfSIP2 binds to a conserved recognition sequence exclusively in heterochromatic domains upstream of subtelomeric var genes and within telomere-associated repeat elements. Our results suggest important roles for this protein in several aspects of chromosome end biology, including var gene silencing. They are furthermore highly relevant for future efforts to dissect the specific biology of P. falciparum chromosome ends, and for our understanding of the role of ApiAP2 factors in parasite biology. Throughout the eukaryotic kingdom, the overall structure of chromosome ends is conserved and characterized by the telomeric tract, composed of short G-rich repeats, and an extensive subtelomeric region consisting of various types and lengths of repeats, also known as telomere-associated sequences (TAS) Plasmodium falciparum causes the most severe form of malaria in humans with up to two million deaths annually P. falciparum erythrocyte membrane protein 1 (PfEMP1) to a variety of endothelial receptors var gene family comprising approx. 60 mostly subtelomeric members var genes, only one PfEMP1 variant is exposed per parasite at any time and switches in var gene expression result in antigenic variation of PfEMP1 var genes and additional subtelomeric gene families coding for proteins involved in host-parasite interactions The apicomplexan parasite P. falciparum chromosome ends consist of a stretch of telomeric GGGTT(T/C)A repeats with an average size of 1.2 kb, followed by an extensive 20 to 40 kb TAS domain var, rif, stevor and pfmc-2tmP. falciparum chromosome ends associate into clusters that are anchored to the nuclear periphery var genes on heterologous chromosomes P. falciparum subtelomeric gene families is clonally variant and restricted to only one member (or a few) in each family var genes are reversibly silenced in a manner reminiscent to telomere-postion effect in other eukaryotes S. pombe and higher eukaryotes var gene transcription and switching have been studied in some detail. Active var loci are enriched in acetylated H3K9 and H3K4me2/me3 var loci lack these activation marks and are enriched in H3K9me3 and PfHP1 instead var and a subset of rif genes is dependent on the two P. falciparum orthologs of silent information regulator 2 (PfSIR2) Expression of P. falciparum. However, it remains completely unknown which proteins and cis-acting sequences are involved in the recruitment and organization of P. falciparum heterochromatin, and how they contribute to the important role of chromosome end biology in this pathogen. Our understanding of sequence-specific DNA-protein interactions in P. falciparum is negligible and mostly limited to the description of upstream sequence elements and their interaction with unknown nuclear proteins, and to in silico mapping of over-represented motifs in candidate promoter sequences in vivocis-acting elements upstream of P. falciparum genes in vitro has recently been demonstrated P. berghei ookinetes Overall, these results show that conserved epigenetic mechanisms that are also in place in other eukaryotes dictate heterochromatic silencing in var genes at the border of the non-coding and coding parts of P. falciparum chromosomes in vivo to release a 60kDa functional N-terminal domain, PfSIP2-N. We show that both AP2 domains are strictly required for binding to the bipartite SPE2 motif. In vivo, PfSIP2-N is associated with over 700 SPE2 consensus sites that cluster upstream of subtelomeric var genes and in TARE2/3, but not to single chromosome internal sites. Consistent with this striking and exclusive binding of PfSIP2-N to heterochromatic regions, we found no effect of PfSIP2-N over-expression on global gene transcription. Instead, our results imply major roles for PfSIP2-N in var gene silencing and chromosome end biology.Here, we identified a member of the ApiAP2 family as the unknown protein binding to SPE2 arrays upstream of subtelomeric P. falciparum annotated protein database using TurboSequest software P. falciparum proteins represented by two or more unique peptides were identified in the SPE2- and the SPE2M-bound fractions, respectively  produced a shift of similar size and specificity that was supershifted in presence of anti-6×HIS antibodies. Consistent with the smaller size of the SIP2-N-HIS_B protein, we observed a faster migrating complex of identical specificity. For completeness, we also expressed a 150kDa protein in P. falciparum containing all three AP2 domains of the second ApiAP2 protein PF10_0075 but were unable to detect binding to SPE2 (data not shown). Together, these results unambiguously identified PFF0200c as the P. falciparum SPE2-binding protein, which we termed PfSIP2 (SPE2-interacting protein).PFF0200c encodes a large 230kDa protein containing two N-terminal AP2 domains as the only annotated features . However E. coli . Nuclear E. coli , and antin vitro. To test this, we performed SPE2 pull-down experiments and confirmed that only N-terminal PfSIP2-N-Ty bound to SPE2 beads, but not full-length PfSIP2-Ty nor the C-terminal fragment PfSIP2-C-Ty  . WesternIP2-C-Ty . Togethevar genes we expected PfSIP2-N to mark chromosome end clusters. Consistent with this assumption, indirect immunofluorescence (IFA) microscopy identified discrete PfSIP2-N-HA foci at the nuclear periphery with increasing numbers of foci in replicating stages (in vivo by targeted chromatin immunoprecipitation (ChIP-qPCR). We observed specific enrichment of PfSIP2-N-HA at the SPE2 array upstream of the upsB var gene PFL0005w while three regions further downstream showed no association  to compare the activities of an episomal upsB promoter and a truncated version lacking a 500bp region containing the entire SPE2 array in transfected parasites lines 3D7/upsBR var promoter activity.We recently demonstrated that bona fide SPE2 motif and to a DNA sequence of identical length carrying the GTGCA motif only. E. coli and used gel shift assays to confirm that indeed both adjacent AP2 domains are required for binding . Only three motifs competed efficiently  and two competed moderately . These motifs are most closely related to the original SPE2 sequence. Furthermore, in two instances a 5bp spacer was tolerated. Interestingly, the rap2 (non-competing) and rap3 (competing) sequences are identical except for the fifth base in the spacer, indicating that these positions can also contribute to specificity. The competition EMSAs were repeated several times with independent batches of competitors and input protein (both nuclear extracts and E. coli lysates) and yielded identical results  occur in sense-oriented tandem arrays approx. 2.2kb upstream of 23 subtelomeric upsB loci with an average of eleven motifs spaced by 12bp per locus, and the only chromosome-internal upsB var gene contains two upstream SPE2 sites. 66 sites define a second highly conserved cluster of upsB-associated SPE2 sites approx. 2.7 kb upstream of every subtelomeric locus. Interestingly, 393 sites (51.7%) are located in TAS concentrated in the TARE2/3 region on every chromosome end, with conserved position and orientation and an average of 18 motifs per chromosome end, most of which are spaced by 120bp. P. falciparum chromosomes.Next, we used gel shift competition assays to pinpoint as accurately as possible the minimal sequence requirements for a functional SPE2 consensus motif  and S3.  results . Genome-e genome  and S4. in silico prediction and to identify potential additional PfSIP2-N target sites we performed genome-wide ChIP (ChIP-on-chip) on a high-density whole genome tiling array (NimbleGen Systems Inc.) in silico prediction revealed a high degree of overlap . Over-expression of PfSIP2-N-HA was evident by up to eight-fold higher levels of  control . The ovevar genes. Our comprehensive analysis reveals important novel insights into the nature and specificity of the PfSIP2/SPE2 interaction and provides compelling evidence for a major role of PfSIP2 in chromosome end biology.Sequence-specific DNA-protein interactions serve to target specific activities to specific sites in the genome and are instrumental in genome organization and gene regulation. Here, we identified PfSIP2, a member of the ApiAP2 family of putative transcription factors, as the unknown nuclear protein binding to SPE2 tandem arrays upstream of subtelomeric in vitro indicates that the release of PfSIP2-N does not occur accidentally during extraction but reflects a true proteolytic cleavage event required to activate the DNA-binding activity. Our thorough re-definition of the specificity of the PfSIP2-N/SPE2 interaction in gel shift competition assays allowed us to determine a highly specific SPE2 consensus motif. Both 6bp half sites of the bipartite SPE2 element are strictly required for binding, which is in agreement with our earlier findings PlasmodiumArabidopsis tandem AP2-domain protein AINTEGUMENTA to a 16bp sequence, which also requires both AP2 domains for binding in vitro nor in vivo and, for that matter, consider PfSIP2-N unlikely to act as transcriptional activator of GTGCA-associated genes. We explain these conflicting results by fact that the protein binding microarray technology used in the former study was limited to the screening of random 10mers only, which precluded identification of the 16bp SPE2 element as high affinity binding motif. Our results are highly relevant for our understanding of the role of PfSIP2 in parasite biology, and provide important information for the future investigation of specific ApiAP2-DNA interactions.Surprisingly, we found that the SPE2-binding activity is not exerted by the full-length protein but rather by a processed N-terminal fragment, PfSIP2-N. The inability of full-length PfSIP2 to interact with SPE2 in silico prediction identified 777 PfSIP2-N target sites, 94% of which are located in two distinct landmark regions within subtelomeric heterochromatin on all chromosomes. One cluster corresponds to the previously described tandem arrays upstream of subtelomeric var genes in silico prediction of subtelomeric target sites, showing that both subtelomeric landmark regions were bound by PfSIP2-N in vivo. In contrast, PfSIP2-N was absent at internal sites, except for the only internal upsB locus with two upstream SPE2 motifs. Therefore, binding of PfSIP2-N is restricted to heterochromatic regions including the entire subset of upsB var genes. The co-localization of PfSIP2-N with PfHP1 at perinuclear chromosome end clusters and upstream of upsB var genes corroborates this exclusive association. The observed increase in default upsB promoter activity upon deletion of the SPE2 array suggests a role for PfSIP2-N in var gene silencing, possibly through direct or indirect recruitment of effector proteins. However, the overall distribution pattern of PfSIP2-N implies important roles in structural and functional organization and maintenance of P. falciparum chromosome ends that go beyond regulating var gene expression.Our genome-wide S. cerevisiae. One such factor is ABF1, which is involved in silencing, initiation of DNA replication, alteration of chromatin structure and nucleotide excision repair P. falciparum. First, multiple attempts to generate a PfSIP2 knockout line failed due to refractoriness of the pfsip2 locus to disruption (data not shown). Since pfsip2 was readily accessible for 3′ replacement, we believe PfSIP2 is essential for parasite survival. Second, given the close connection between DNA replication and heterochromatin formation, the specific co-purification of several DNA replication/repair and chromatin remodeling factors  with PfSIP2-N . Hence, if SIP2 has the same binding specificity in other species (which is likely due to the remarkable sequence identity in their DNA-binding domains) then SIP2 must have a function other than being involved in subtelomere biology. It will be interesting to test if processing of PfSIP2 reflects a specific gain-of-function process during evolution of the P. falciparum lineage in order to cope with the massive expansion and control of subtelomeric virulence gene families.ApiAP2 factors have been proposed to act as regulators of stage-specific expression P. falciparum. To the best of our knowledge, PfSIP2-N/SPE2 represents a novel type of sequence-specific interaction at chromosome ends that has not been reported in any other eukaryote. Our results are highly relevant in the dissection of the specific biology of P. falciparum chromosome ends, which is key to the evolution and variable expression of subtelomeric virulence gene families, and will help to understand similar processes in other systems. Efforts to analyze a loss-of-function phenotype and to identify PfSIP2 interaction partners will be important future steps into this direction.In conclusion, we have identified the first sequence-specific component of subtelomeric regions in P. falciparum 3D7 parasites were cultured as described previously E. coli lysates were diluted to avoid excess input of recombinant protein. Competition EMSAs were performed in presence of non-specific competitor DNA  and a 50- to 100-fold molar excess of specific ds competitors.High salt nuclear extracts and EMSAs were prepared and carried out as described 32P-labeled SPE2 probe in presence or absence of a 25-fold molar excess of SPE2 or SPE2M competitors. DNA-protein interactions were UV-crosslinked for 60min (107 Joule) in a Stratalinker 1800 (Stratagene) and separated by SDS-PAGE. Gels were directly exposed to X-ray film.Protein samples were incubated for 20min in EMSA buffer with 20fmol P. falciparum/human annotated protein database.The complete protocol is explained in detail in BamHI and HindIII and analysed by Southern blot. The blot was probed with a 32P-dATP-labeled 702bp fragment derived from the 3′ end of PFF0200c.To verify successful 3′ replacement at the PFF0200c locus, gDNA from 3D7 wild-type parasites and drug-cycled 3D7/SIP2-Ty parasites was digested with E. coli Tuner (DE3) cells (Novagen) replicating pMICO Plasmids are described in Primary antibody dilutions were: anti-HA 3F10 (Roche Diagnostics) 1∶2,000; anti-Ty BB2 (kind gift of K. Gull) 1∶10,000; anti-6×HIS (R&D Systems) 1∶5,000. High salt nuclear extracts from 3D7/SIP2-Ty and 3D7/SIP2-N-Ty schizonts were incubated for 1hr with streptavidin agarose beads carrying immobilized SPE2 or mutated SPE2M motifs in EMSA buffer supplemented with non-specific competitor DNA. After centrifugation at 2000rpm the supernatant was saved and beads washed three times in binding buffer. Bound proteins were eluted with 2M KCl.Methanol-fixed cells were analysed using rat anti-HA 3F10 (1∶100) or mouse anti-Ty BB2  antibodies. Alexa-Fluor® 568-conjugated anti-rat IgG (Molecular Probes) 1∶500; FITC-conjugated anti-mouse IgG (Kirkegaard Perry Laboratories) 1∶300; TexasRed-conjugated anti-mouse IgG (Molecular Probes) 1∶500. Images were taken on a Leica DM 5000B microscope with a Leica DFC 300 FX camera and acquired via the Leica IM 1000 software and processed and overlayed using Adobe Photoshop CS2.P. falciparum, P. vivax and P. knowlesi, genome sequences available at PlasmoDB (version5.5) were searched using the PfSIP2-N-binding consensus sequence determined in competition gel shift assays (To identify the full complement of SPE2 consensus elements in t assays . A regul3). After incubation at 65°C for 10min to inactivate the BB2 antibody, the eluate was diluted 6× in incubation buffer lacking SDS (resulting in SDS concentration comparable to that of the first ChIP). Re-ChIP reactions were carried out in the presence of 0.2mg/ml Ty peptide to avoid immuno-precipitation by the BB2 antibody carried over from the first ChIP. Recovery values were defined in the percentage of the input material of the first ChIP. For genome-wide analysis, immunoprecipitated DNA was amplified by a modified T7 linear amplification method and analyzed on a tiling array  ChIP and ChIP-on-chip using formaldehyde-crosslinked chromatin was performed as described in detail elsewhere qPCR was performed on reverse transcribed total RNA and gDNA isolated from synchronous parasite cultures at six timepoints across the IDC. A detailed protocol, relative transcript calculation and primer sequences are provided in P. falciparum microarray as previously described Growth of 3D7/SIP2-N-HA parasites was tightly synchronized in parallel three times by sorbitol treatment to achieve a ten-hour growth window. Total RNA was isolated at four timepoints across the IDC at early ring stages (4–14 hours post-invasion (hpi)), late ring stages (14–24 hpi), trophozoites (24–34 hpi) and schizonts (32–42 hpi) by lysis of pelleted RBCs in TriReagent (Sigma). Transcript levels in 3D7/SIP2-N-HA were compared to those of the control line 3D7/camHG www.plasmoDB.org) accession numbers for genes and proteins discussed in this publication are: PfSIP2 (PFF0200c); PfHP1 (PFL1005c); upsB var genes ; rhoph1/clag2 (PFB0935w); rhoph1/clag3.1 (PFC0120w); rhoph1/clag9 (PFI1730w); rhoph2 (PFI1445w); rhoph3 (PFI0265c); rap1 (PF14_0102); rap2 (PFE0080c); rap3 (PFE0075c); rama (MAL7P1.208); rnp3 (PFL2505c); imp, putative (PFF0645c/MAL6P1.292); gap45 (PFL1090w); asp (PFD0295c); msp1 (PFI1475w); sera4 (PFB0345c); conserved Plasmodium proteins ; RFC, subunit 2 (PFB0840w); PfSNF2L (PF11_0053); DNA pol ε, subunit A (PFF1470c); transcription factor with AP2 domains (PF10_0075).PlasmoDB  Southern blot of BamHI/HindIII-digested genomic DNA from 3D7 and 3D7/SIP2-Ty parasites demonstrates the successful 3′ end replacement of the endogenous PFF0200c locus. Lane 1; pSIP2-2xTy_3′RP plasmid control; lane 2: 3D7 wild-type gDNA; lane 3: 3D7/SIP2-Ty gDNA after 2 cycles off and on WR99210 selection pressure; lane 4: 3D7/SIP2-Ty gDNA after three cycles off and on drug selection pressure. The membrane was probed with a 702bp encoding the 3′end of PFF0200c.C-terminal tagging of endogenous PfSIP2. (A) Schematic display of the genomic context of PFF0200c encoding PfSIP2, the C-terminal tagging plasmid pSIP2-2xTy_3′RP, and the integration event. 702bp of the very 3′ end of PFF0200c fused in frame to a sequence coding for the 2xTy tag was used for homologous recombination into the endogenous locus. (0.01 MB PDF)Click here for additional data file.Figure S2var gene silencing. Reporter constructs testing the effect of a 500bp deletion including the entire SPE2 array on upsB promoter activity are depicted on top. Stage-specific promoter activity was determined by qRT-PCR in absence of WR selection  and after WR selection . X-axis: synchronized cultures were harvested at six timepoints across the IDC. hpi: hours post-invasion. Y-axis: values represent relative transcript levels. Absolute hdhfr, msp8 and cam transcript numbers were divided by those obtained for the constitutively expressed gene PF13_0170 (www.plasmoDB.org). hdhfr transcript levels were additionally normalized for differences in plasmid copy number as determined by qPCR  and calmodulin  show comparable stage composition for each parasite line in each timepoint.Evidence for an involvement of the PfSIP2/SPE2 interaction in  by qPCR . Transcr(0.02 MB PDF)Click here for additional data file.Figure S3P. falciparum invasion genes. Altered nucleotides in the left or right half site of the original SPE2 sequence are highlighted in red. Additional nucleotides in the 4bp spacer are indicated in green. The arrow on top indicates the direction of electrophoretic separation. Red squares identify competitors unable to interact with PfSIP2-N_HIS_A, green squares highlight competitors that were able to compete with the SPE2/PfSIP2-N-HIS_A interaction. The experimentally determined SPE2 consensus site is shown at the bottom.Competition EMSA to determine a functional SPE2 consensus site. Competition EMSA using recombinant PfSIP2-N-HIS_A to determine the minimal sequence requirements for binding of PfSIP2-N to a SPE2 consensus site. The gel was rotated by 90° clockwise for simpler display. Lane 1: radiolabeled 28bp SPE2 probe only; lane 2: SPE2/PfSIP2-N-HIS_A interaction in absence of competitor; lanes 3–30: SPE2/PfSIP2-N-HIS_A interaction in presence of a 100-fold molar excess of specific competitors. The names of all competitors and the original SPE2 sequence and those of all competitors are indicated to the left. The dashed lines group competitors into artificially mutated SPE2 motifs and in naturally occurring SPE2-like elements upstream of (0.03 MB PDF)Click here for additional data file.Figure S4ChIP-re-ChIP shows that PfSIP2-N-HA and PfHP1-Ty co-localise in subtelomeric heterochromatin. ChIP-re-ChIP was performed on 3D7/SIP2-N-HA/HP1-Ty schizont stage parasites. Top panel: Recovery of PfSIP2-N-HA- and PfHP1-Ty-associated chromatin after the first ChIP using anti-HA and anti-Ty antibodies, respectively, compared to the negative control (rabbit IgG). Second panel: Chromatin fragments immuno-precipitated with anti-Ty antibodies in the first ChIP were used as input and were re-ChIPped using anti-HA, anti-Ty and rabbit IgG antibodies in presence of competing Ty peptide. Efficient and specific peptide competition is evident from the failure to precipitate chromatin with anti-Ty antibodies in presence, but not in absence, of Ty peptide. Re-ChIP using anti-HA antibodies shows that PfSIP2-N-HA is present on PfHP1-Ty-enriched chromatin but only at loci associated with SPE2 . Bottom panel: Results from second panel displayed as fold enrichment values for PfSIP2-N-HA on PfHP1-Ty-enriched chromatin fragments. qPCR primers were directed against upstream regions . This experiment was repeated using independently isolated chromatin and yielded similar results.(0.02 MB PDF)Click here for additional data file.Table S1SEQUEST search results of peptide tandem mass spectra for proteins eluted with SPE2.(0.08 MB PDF)Click here for additional data file.Table S2SEQUEST search results of peptide tandem mass spectra for proteins eluted with SPE2M.(0.08 MB PDF)Click here for additional data file.Table S3www.plasmodb.org).Genome-wide prediction of SPE2 consensus motifs. The position of 777 SPE2 consensus motifs experimentally determined by competition EMSA are listed. Column 1: Chromosome ID. Column 2: nucleotide position with respect to the left telomere end of the first bp of the SPE2 motif. Column 3: nucleotide position of the last bp of the SPE2 motif with respect to the left telomere on each chromosome. Column 4: “+” denominates positive association of the corresponding SPE2 motif in the ChIP-on-chip experiment . Column 5: SPE2 orientation on sense (“1”) or antisense (“−1”) strand. Column 6: “tel” indicates SPE2 location in TAREs, “var” indicates SPE2 location upstream of upsB var genes. SPE2 elements associated with genes that do not fall into the first to classes are identified by PlasmoDB annotation of the corresponding gene. Column 7: distance in bp to previous SPE2 motif. Column 8: blank cells represent canonical 4bp spacing between the two half sites of the bipartite SPE2 motif. “5” represents 5bp spacing. Column 9: PlasmoDB accession numbers of SPE2-associated genes. Column 10: SPE2 positions other than in TARE or upstream of coding sequences are indicated. Column 11: orientation of SPE2-associated genes on the sense (“1”) or antisense (“−1”) strand. Column 12: nucleotide position of the ATG start codon of SPE2-associated genes with respect to the left telomere on each chromosome. Values/information in individual columns was retrieved from PlasmoDB version 5.5 ((0.03 MB PDF)Click here for additional data file.Table S4var genes, other genes, TARE). Column 2: The groups classified in column 1 are further subgrouped according to various criteria Click here for additional data file.Table S5www.plasmodb.org).Single SPE2 sites upstream of internal genes. Column 1: Chromosome ID. Column 2: nucleotide position of the first bp of the SPE2 motif with respect to the left telomere end. Column 3: nucleotide position of the last bp of the SPE2 motif with respect to the left telomere on each chromosome. Column 4: SPE2 orientation on the sense (“1”) or antisense (“−1”) strand. Column 5: PlasmoDB gene annotation of associated genes. Column 6: PlasmoDB accession number of associated genes. Column 7: Location of SPE2 element with respect to the coding sequence. Column 8: blank cells represent canonical 4bp spacing between the two half sites of the bipartite SPE2 motif. “5” represents 5bp spacing. Column 9: orientation of SPE2-associated genes on the sense (“1”) or antisense (“−1”) strand. Column 10: nucleotide position of the ATG start codon of SPE2-associated genes with respect to the left telomere on each chromosome. Column 11: distance of SPE2 element in bp upstream of associated genes. Column 12: Peak transcription. Values/information in individual columns was retrieved from PlasmoDB version 5.5 ((0.02 MB PDF)Click here for additional data file.Protocol S1Affinity purification and mass spectrometry-based identification of the SPE2-interacting protein PfSIP2.(0.03 MB DOC)Click here for additional data file.Protocol S2Plasmid constructs.(0.03 MB DOC)Click here for additional data file.Protocol S3Quantitative reverse transcriptase PCR.(0.03 MB DOC)Click here for additional data file.Protocol S4Oligonucleotide sequences used in this study.(0.04 MB DOC)Click here for additional data file.