Saccharomyces cerevisiae as a model system, we provide evidence that the middle domain of the FACT subunit Spt16 – the Spt16-M domain – is involved in functional interactions with histone H3. Our results show that the Spt16-M domain plays a role in the prevention of cryptic intragenic transcription during transcription elongation and also suggest that the Spt16-M domain has a function in regulating dissociation of Spt16 from chromatin at the end of the transcription process. We also provide evidence for a role for the extreme carboxy terminus of Spt16 in functional interactions with histone H3. Taken together, our studies point to previously undescribed roles for the Spt16 M-domain and extreme carboxy terminus in regulating interactions between Spt16 and chromatin during the process of transcription elongation.In eukaryotic cells, the highly conserved FACT (FAcilitates Chromatin Transcription) complex plays important roles in several chromatin-based processes including transcription initiation and elongation. During transcription elongation, the FACT complex interacts directly with nucleosomes to facilitate histone removal upon RNA polymerase II (Pol II) passage and assists in the reconstitution of nucleosomes following Pol II passage. Although the contribution of the FACT complex to the process of transcription elongation has been well established, the mechanisms that govern interactions between FACT and chromatin still remain to be fully elucidated. Using the budding yeast  In eukaryotic cells, genomic DNA associates with several proteins to form a protein-DNA structure referred to as chromatin. The fundamental unit of chromatin is the nucleosome, a structure composed of 147 base pairs of DNA associated with a protein complex known as the histone octamer Saccharomyces cerevisiae model system in vitroFAcilitates Chromatin Transcription) FAcilitates Chromatin Transactions to more accurately reflect the varied chromatin-based processes that depend on FACT, including transcription initiation and DNA replication An area of intense research in recent years has focused on elucidating the mechanisms that allow for productive Pol II passage over transcribed units despite the presence of nucleosomes across these regions. These studies have identified a large number of factors possessing a variety of biochemical activities that have been implicated in the process of transcription elongation in the context of chromatin The yeast FACT complex (yFACT) is composed of two subunits essential for viability, Spt16 and Pob3, which can interact with nucleosomes through the assistance of the HMG box-containing protein Nhp6 S. cerevisiae and S. pombe Spt16-NTDs show the presence of two potential peptide-binding modules – one within the N-terminal lobe of the Spt16-NTD and the other, structurally similar to bacterial aminopeptidases, in the more C-terminal lobe of the Spt16-NTD S. cerevisiae, the Spt16-NTD domain, in conjunction with the Pob3-M domain, is involved in functional interactions with the C-terminal extension of histone H2A S. pombe, the Spt16-NTD domain has been shown to interact directly with both the globular domains and N-terminal tails of histone H3 and H4 in vivoin vitroAdditional studies revealed the presence of four structural domains within the Spt16 subunit of yFACT – these domains are referred to, from the N-terminus to the C-terminus direction, as Spt16-NTD, Spt16-D, Spt16-M and Spt16-C The role of the Spt16-M domain within yFACT is still under investigation. Mutations in residues within the Spt16-M domain have been shown to result in defects in cell integrity S. cerevisiae as a model system, we described the isolation and characterization of a histone H3 mutant, H3-L61W, that confers several mutant phenotypes when expressed as the sole source of histone H3 in cells, including a cold sensitive (Cs−) phenotype and sensitivities to formamide, hydroxyurea and caffeine FLO8 gene FLO8 cryptic transcription phenotypes, although the Spt16-E790K mutation only suppressed the latter phenotype at the low temperature of 14°C and not at the permissive temperature of 30°C In previous work using SPT16 gene that suppress the Cs− phenotype of H3-L61W cells. For these experiments, we PCR-amplified either different fragments  or the entire length (screen D) of the SPT16 gene using the high-fidelity polymerase Pfu under standard PCR conditions  across the region from −773 to +3521 and are the focus of the studies presented here. We were surprised to find that in several cases the mutations we identified in these mutants were located outside of the regions that were targeted by the PCR-mediated mutagenesis , indicating that only specific mutations within the Spt16-M domain display genetic interactions with H3-L61W. The genetic interactions displayed between the Spt16-truncation mutants (Spt16-E989stop and Spt16-E1004stop) and the H3-L61W mutation , is expressed constitutively at high levels, and has been used extensively to study the transcription elongation process. As shown in PMA1 assayed in our experiments overlaps with the promoter of the LEU1 gene; however, several lines of evidence indicate that in both H3-WT and H3-L61W cells binding of Spt16 in this region is attributable to PMA1 transcription and, at least for the most part, does not reflect events related to LEU1 transcription. First, microarray experiments have shown that under the growth conditions used in our studies (rich medium) LEU1 expression is minimal in H3-WT cells and this expression is unaffected by the H3-L61W mutation PMA1 3′ region PMA1 3′ region is not recruited to facilitate LEU1 transcription initiation or elongation in either strain. Third, the patterns of Spt16 binding in H3-WT and H3-L61W cells at the PMA1 3′ region are similar to those seen at the 3′ regions of several other genes PMA1 in H3-L61W cells is caused by PMA1 transcription and not due to LEU1 transcription initiation. Taken together, these observations indicate that PMA1 serves as a good model gene with which to study the effects of H3-L61W on the distribution pattern of Spt16 across transcribed genes.The H3-L61W mutation causes a shift in distribution of Spt16 across transcribed genes, in which slightly lower levels of Spt16 are found at 5′ ends of genes and dramatically increased levels are seen at the 3′ ends of genes compared to what is seen in H3-WT cells PMA1 and lower occupancy levels at the 3′ region of the gene , suggesting that the Spt16 mutants cannot exert their suppressive effects in the context of robust transcription.Consistent with our previous findings showing that H3-L61W causes cryptic transcription within the To further elucidate the nature of our Spt16 mutants, we performed several experiments to determine their effects when expressed in cells carrying wild-type histones. In the first set of experiments, we carried out assays to ascertain the functional stability of the Spt16 mutants when exposed to different temperatures and to obtain insights into whether the mutants are defective for the process of DNA replication. For these studies, H3-WT cells expressing wild-type or mutant Spt16 proteins were assayed for growth on rich medium at 30°C, 37°C, or 16°C and on rich medium containing hydroxyurea, a drug that reduces intracellular levels of dNTPs and that is often used to score for DNA replication defects PMA1 gene in cells expressing H3-WT. As shown in PMA1 in an abnormal fashion. The most defective mutant, the M-domain mutant Spt16-G836S, showed essentially the same level of occupancy at all three PMA1 regions assayed, a pattern that is very different from that seen for wild-type Spt16 , suggesting that the Spt16 mutants do not cause cryptic transcription initiation defects in H3-WT cells in the absence of significant levels of transcription. Next, we wished to determine if the Spt16 mutants can confer Spt- phenotypes – which are associated with abnormal chromatin structure and defective transcription regulation lys2-128δ allele for growth on medium lacking lysine. As shown in the right two panels of - phenotype. Interestingly, we found a general, but not perfect, correlation in the abilities of the mutants to confer the Spt- and cryptic transcription initiation defects, suggesting that in some instances the two phenotypes can be related to each other. In past studies, we reported that the two original Spt16 suppressors of H3-L61W phenotypes, Spt16-E790K and Spt16-E857Q, confer an Spt- phenotype only in the context of low intracellular levels of histones H3 and H4 (hht1-hhf1)Δ background, we wished to assess if the Spt16 mutants that confer an Spt- phenotype in the (hht1-hhf1)Δ background can also do so in the context of wild-type levels of histones H3 and H4. As shown in the right two panels of - phenotype in HHT1-HHF1 cells. Similarly, only a subset of the Spt16 mutants that cause a cryptic transcription initiation defect at the FLO8-HIS3 reporter in (hht1-hhf1)Δ cells were found to also cause the cryptic transcription phenotype in cells expressing histones H3 and H4 at wild-type levels Δ mutation, likely as a result of a more relaxed chromatin structure in this genetic background. The Spt16 mutants showing the strongest cryptic initiation defects are those harboring mutations within the Spt16-M domain, thus highlighting the importance of the integrity of this domain in preventing cryptic transcription.In the third set of experiments, we asked if the Spt16 mutants confer phenotypes indicative of defects in transcription and chromatin structure. For these studies, we first assessed if the Spt16 mutants cause cryptic transcription initiation defects at the To determine if the Spt16 mutants we isolated cause the various effects described in this work by affecting Spt16 protein abundance, we performed western-blot analysis to measure Spt16 protein levels in the context of the different Spt16 mutations. As shown in PMA1 gene and all Spt16-M domain mutations suppress an intragenic cryptic transcription initiation phenotype seen in H3-L61W cells. The extreme C-terminus of Spt16 is also implicated in functional interactions with histone H3 since two small C-terminal truncations of Spt16 were also found to suppress H3-L61W growth and intragenic cryptic transcription initiation phenotypes. Finally, in experiments carried out in the context of wild-type histone H3, we have provided evidence in support for roles for the Spt16-M domain in ensuring proper distribution of Spt16 across transcribed genes and in preventing aberrant cryptic transcription initiation.In this work we have shown that the Spt16-M domain is an important player in functional interactions between Spt16 and histone H3. Mutations within the Spt16-M domain suppress several mutant growth phenotypes conferred by the histone H3 mutant H3-L61W, including growth defects at low temperatures and in the presence of several drugs. At the molecular level, many Spt16-M domain mutations suppress the Spt16 3′-accumulation phenotype at the PMA1 seen in H3-L61W cells points to a role for this domain in regulating the departure of Spt16 following transcription. However, in the context of H3-WT, the levels of Spt16 detected at the 3′ region of PMA1 were comparable between wild-type Spt16 cells and cells expressing any of the Spt16 mutants (see green bar-graphs in i.e. in the context of H3-L61W nucleosomes), but they would have no effect in situations where the signal is fully operational (i.e. in the context of H3-WT nucleosomes). Thus, our results point to a role for the Spt16-M domain in regulating Spt16 dissociation from chromatin following the transcription process and are consistent with a model in which the Spt16-M domain serves as a sensor for a putative Spt16-departure signal.The observation that the Spt16-M domain mutants suppress the Spt16 3′-accumulation phenotype at FLO8:HIS3 reporter gene when cells are grown in glucose-containing medium while causing a cryptic transcription initiation phenotype at the same gene in the context of H3-WT when cells are grown in galactose-containing medium. These results can be explained by a model of allele-specific Spt16-histone H3 interactions. In this model, the Spt16-M domain and histone H3 interact directly to promote nucleosome reassembly during transcription elongation. The H3-L61W mutation disrupts this interaction, leading to impaired nucleosome reassembly and cryptic intragenic transcription. The Spt16-M domain mutants restore the interaction with H3-L61W through compensatory structural changes leading to more efficient nucleosome reassembly and, as a result, suppression of cryptic transcription. These structural changes, however, decrease the affinity between the Spt16-M domain and H3-WT, resulting in defective nucleosome reassembly and cryptic transcription in H3-WT cells. The Spt16-M domain may interact with histone H3 through the histone H3 N-terminal tail since this tail has been shown to interact with S. cerevisiae yFACT in vitro through yFACT regions other than the Spt16-NTD domain FLO8-HIS3 only in conditions in which the reporter gene is expected to be largely inactive seems puzzling at first given the fact that cryptic intragenic transcription is thought to be dependent on active transcription originating from a bona fide upstream promoter. However, recent studies have suggested that even at very lowly transcribed genes and at genes expected to be inactive, transcription-dependent nucleosome disassembly and Spt16-mediated reassembly can still occur FLO8-HIS3 cryptic transcription phenotype by the Spt16 mutants is likely to be due to an increased ability of the Spt16 mutants to reassemble H3-L61W nucleosomes during the transcription elongation process. We do not rule out, however, alternative models that could account for cryptic transcription at FLO8-HIS3 in H3-L61W cells grown in glucose-containing medium and for the mechanism of suppression by the Spt16 mutants. For example, it is possible that an abnormally open chromatin structure permissive to intragenic cryptic transcription is generated during DNA replication in the context of H3-L61W and that the Spt16 mutants suppress cryptic transcription by promoting a more compact chromatin structure on the newly replicated DNA molecules. Alternatively, expression of HIS3 in the context of the FLO8:HIS3 reporter in H3-L61W cells may be a result of changes in chromatin structure over the cryptic promoter element of FLO8 that allows for transcription initiation to occur in the absence of any transcription originating from the GAL1 promoter. In this scenario, the Spt16 mutants would suppress cryptic transcription by reestablishing a closed chromatin structure over the cryptic promoter element through activities related to its role in transcription initiation. Our results lay the foundations for future experiments, such as in vitro biochemical assays, that will further explore the significance of the interactions between the Spt16-M domain and histone H3 in regulating chromatin dynamics.Our studies show that the Spt16-M domain mutants can suppress the H3-L61W cryptic transcription initiation phenotype at the We have entertained the possibility that the abilities of Spt16 to reassemble nucleosomes during transcription elongation and to depart 3′ ends of genes following transcription are functionally related to each other. For example, it could be envisioned that in order to properly depart chromatin following transcription, Spt16 needs to efficiently reassemble the last nucleosome at the 3′ end of a transcribed gene. However, this hypothesis is not supported by our data. For example, in the context of H3-L61W nucleosomes, Spt16-E735G is among the strongest suppressors of the Spt16 3′-accumulation phenotype  but is oFLO8 (see PMA1 (see (hht1-hhf1)Δ background  and the SPT16 gene (marked with the KanMX4 gene) and express either H3-WT or H3-L61W from the HHT2 or hht2-11 alleles, respectively, located at the endogenous HHT2 locus (see hht2-11 strains). Strains yADP51-yADP66 express H3-WT from both the HHT1-HHF1 and HHT2-HHF2 loci. In addition, most strains express wild-type or mutant versions of Spt16 from a centromeric, LEU2-marked plasmid. The wild-type version of this plasmid  has been described elsewhere All is study  are GAL2PT16 see . The Spt- phenotype conferred by the H3-L61W mutations  and OAD356 (5′ GTTAGCACTTTGAGAATCTTTCAG) were used to amplify the region from –773 to +1230 from plasmid pAO01. The PCR amplifications were done using Pfu polymerase (Stratagene) using standard PCR conditions. Two separate pools of PCR products were generated. Plasmid pAO01 was linearized with restriction enzymes KpnI and EagI (both from New England Biolabs). Strain yADP50 was co-transformed with each of the PCR pools and the linearized plasmid and cells were plated for growth on SC medium lacking leucine to select for gap-repair events generating circular plasmids. Transformants were then replica-plated to medium containing 5-FOA to select for loss of the URA3-marked centromeric plasmid carrying the wild-type SPT16 gene. The resulting colonies were then replica-plated to YPD medium and incubated at 14°C to screen for suppression of the H3-L61W Cs− phenotype. Candidate suppressing plasmids were then recovered from the yeast cells and retested for suppression in independent experiments.Four screens were performed to identify Spt16 mutants able to suppress the CsGGACCTATTCTCCAATCATTCAGTCC) and OAD358 (5′ GATCCAGTCTTTTCGTTCTTCCAG), which amplify the region from +764 to +2044 (three independent pools of PCR products were obtained for this screen); screen C, primers OAD359 (5′ GAACAAAGTTACGTGGCGAAGCCCG) and OAD341 (5′ TGATTCTGTGGATAACCGTA), which amplify the region from +1430 to +3521 (three independent pools of PCR products were obtained for this screen); screen D, primers OAD342 and OAD341, which amplify the region from –773 to +3521 (four independent pools of PCR products were obtained for this screen). The enzymes used to linearize pAO01  were as follows: screen B, EagI and BsaAI; screen C, SalI; screen D, KpnI and SalI.Screens B, C, and D were carried out in a fashion similar to that described for screen A, except that the primers used were as follows: screen B, primers OAD357  and OAD395 (5′ CCGTAAGATGGGTCAGTTTGTAAAT); internal region of PMA1: OAD416 (5′ TGATGTTGCTACTTTGGCTATTGC) and OAD417 (5′ TTCCATTTAACGGGCTTTGG); 3′ region of PMA1 OAD383 (5′ CGTAAGCGAGACTTCCAAATGG) and OAD384 (5′ TCCTGCCCAGCTCTTCTATAATACTT); NO ORF region: OAD377 (5′ TGGTGATAGGCGTTGAGTATGTG) and OAD378 (5′ GTGCGCAGTACTTGTGAAAACC). Control experiments in which the Spt16 antibody was omitted from the immunoprecipitation reactions were carried for each sample and compared with immunoprecipitation reactions in which the Spt16 antibody had been included to ensure specificity of the antibody.The % immunoprecipitation (%IP) for each of the four regions indicated in 2, 1 mM EDTA, 5% glycerol, 0.3 M ammonium sulfate, 1 mM DTT, 1 µg /ml leupeptin, 1 µg /ml pepstatin A, and 1 mM PMSF). 50 µg (Cells were grown in liquid YPD medium to logarithmic phase and were harvested by centrifugation. Protein samples were obtained by disrupting cells using 425-600 microns acid-washed glass beads (Sigma) and strong agitation using a mini-beadbeater (Biospec Products) in protein lysis buffer . 50 µg  or 75 µg). 50 µg  of totalFigure S1Growth phenotypes of H3-WT cells expressing wild-type or mutant Spt16 proteins assayed under various conditions. Strains yADP18, yADP19, and yADP35-49 were patched onto a YPD plate in the order shown in the table above the pictures, incubated at 30°C and then replicate plated to the plates indicated. The approximate incubation times were as follows: YPD 30°C, 1 day; YPD 37°C, 1 day; YPD 16°C, 6 days; YPD +150 mM hydroxyurea (HU), 4 days. The growth pattern of the H3-L61W Spt16-WT strain on the same plates is provided as a reference since it displays significant cold sensitivity and sensitivity to hydroxyurea.(TIF)Click here for additional data file.Table S1Saccharomyces cerevisiae strains.(DOC)Click here for additional data file.


In contrast, the Model Description Language (MDL) adopted by the modeling tool ProMoT is highly modular and it enables a faithful representation of biological parts and pools.The modular design of synthetic gene circuits via composable parts (DNA segments) and pools of signal carriers (molecules such as RNA polymerases and ribosomes) has been successfully applied to bacterial systems. However, eukaryotic cells are becoming a preferential host for new synthetic biology applications. Therefore, an accurate description of the intricate network of reactions that take place inside eukaryotic parts and pools is necessary. Rule-based modeling approaches are increasingly used to obtain compact representations of reaction networks in biological systems. However, this approach is intrinsically non-modular and not suitable We developed a computational framework for the design of complex (eukaryotic) gene circuits by generating dynamic models of parts and pools via the joint usage of the BioNetGen rule-based modeling approach and MDL. The framework converts the specification of a part (or pool) structure into rules that serve as inputs for BioNetGen to calculate the part’s species and reactions. The BioNetGen output is translated into an MDL file that gives a complete description of all the reactions that take place inside the part (or pool) together with a proper interface to connect it to other modules in the circuit. In proof-of-principle applications to eukaryotic Boolean circuits with more than ten genes and more than one thousand reactions, our framework yielded proper representations of the circuits’ truth tables.For the model-based design of increasingly complex gene circuits, it is critical to achieve exact and systematic representations of the biological processes with minimal effort. Our computational framework provides such a detailed and intuitive way to design new and complex synthetic gene circuits. Originally, RNA polymerases and ribosomes were chosen as the only common signal carriers [Signal carriers are molecules that play the role of b͡io-electrons: they are exchanged between parts in such a way that their fluxes ; between chemicals and transcription factors. Analogously, the accuracy with which both small RNAs and chemicals modify the mRNA secondary structure influences translation strength. Moreover, transcription factors and chemicals can bind DNA (the former) and mRNA (the latter) cooperatively.in silico most of the synthetic circuits realized in E. coli during the first half of the last decade [eukaryotic gene parts is necessary to properly model and simulate in silico synthetic constructs for yeast and mammalian cells. Moreover, cellular compartments of various volumes, such as nucleus and cytoplasm, have to be taken explicitly into account. Since they host reactions without counterparts in prokaryotes, new pools have to be introduced.In our bacterial part model, we considered only promoters and RBSs regulated by no more than two regulatory factors ,8. This t decade . Howevert decade -13. Therper se is no longer necessary and the binding site for the ribosomes is embedded in the protein coding region. Furthermore, the nucleus has a spliceosome pool, and the cytoplasm contains as many mRNA pools as there are coding regions in the circuit. While eukaryotic riboswitches/ribozymes-mediated translation regulation does not show any particular difference to bacteria, RNA interference (RNAi) includes more steps than the sole antisense RNA base-pair binding. Indeed, siRNAs  are processed in the nucleus and exported to the cytoplasm where they bind the so called RISC (RNA Induced Silencing Complex). In this configuration, they bind and cleave their targets on mRNA, after which the mRNA is degraded rapidly [Several other features distinguish eukaryotic from prokaryotic transcription and translation, and corresponding modular mathematical descriptions are missing to date. Once transcribed, mRNA undergoes splicing and maturation in a eukaryotic cell’s nucleus before being transported into the cytoplasm where it is translated. Moreover, eukaryotic mRNA does not have a unequivocal sequence  recognized by the ribosomes. Therefore, the RBS as a part  rapidly . We thercombinatorial explosion problem.Recent advances in transcription factors engineering,16 and RRule-based modeling approaches tackle this combinatorial explosion of species and reactions. A biological system is specified by: a) a general description of the species types and their possible states (in our example: a transcription factor that can be bound or unbound and a promoter with six operators that can be either free or bound); b) a list of seed species, i.e., the ones present before any interaction takes place (an unbound transcription factor and a promoter with six free operators); c) a set of rules that describe the interactions between the species (unbound transcription factors bind free operators). According to these input specifications, software such as BioNetGen [ioNetGen  and the ioNetGen  compute ioNetGen .However, rule-based languages cannot be used to generate directly models for single composable parts (and pools) that interact via the exchange of fluxes of molecules such as signal carriers, as in our model.In contrast, the Model Description Language (MDL)  adopted combinatorial composable biological parts for both complex prokaryotic and eukaryotic systems via the joint usage of BNGL and MDL. Figure In this work, we present an extension of our modeling tool  to buildThis modeling framework represents a novelty in the field of computational synthetic biology. Several other computational tools for the modular design and analysis of synthetic gene circuits are available -33. TheyRNAi-based logic evaluator[The paper is structured as follows: In the next section, we provide a detailed description of our novel models for eukaryotic parts and pools (see Methods for details). As an example application, we subsequently show how to build within this framework an evaluator and a poWith the joint usage of MDL and BNGL, we propose models for eukaryotic parts and pools that arise, when possible, from the corresponding bacterial modules ,8. We aiA more exhaustive description of eukaryotic systems might take into account mechanisms that have been neglected here. For instance, cell metabolic reactions can be described by a network of pools that either store free molecules (e.g. kinases and phosphatases) or that represent enzymatic reactions (e.g. phosphorylation and dephosphorylation). Furthermore, part models presented below might be enriched by considering also operator positional effects and transcription squelching, for instance. Such a precise picture might be useful for the analysis of specific cellular phenomena–and the evaluation of the corresponding kinetic parameter values–on rather simple gene circuits.orthogonal systems that is broadly exploited in synthetic biology [To model promoters, operator position is not explicitly taken into account, but activator binding sites are supposed to be placed upstream of the TATA box whereas repressors bind the DNA between the TATA box and the TSS (Transcription Starting Site). We take a prokaryotic repression model based on competition between repressors and RNA polymerases, where DNA-bound repressors prevent RNA polymerases from reaching their binding sites and initiating transcription. The use of bacterial transcription factors in eukaryotic cells is a way of combining  biology ,40.N operators, in principle. Transcription factors of the same species can bind cooperatively. Here, as in our previous work [Different transcription factors can bind a promoter, each on ous work , we assuous work .RNA polymerase binds the DNA in the absence of any repressors. If it is recruited by activators, two scenarios are possible: a) if the activators do not bind cooperatively, only one of their operators has to be occupied in order to let RNA polymerase bind; b) if the activators bind cooperatively, all their operators must be bound to get transcription started. Chemicals can bind and inactivate transcription factors anchored to their operators. Depending on the presence or absence of cooperativity, the binding of a co-repressor  to an acPromoter leakage is proportional to all the configurations where at least one repressor is bound or, in the absence of repressors, where there is no activator whatsoever on the DNA (without cooperativity) or all the right-most operators (with cooperativity) are free.As already mentioned above, eukaryotic cells do not have an RBS. Therefore, translation regulation–together with gene expression–concerns the coding region. Here, in contrast to our bacterial framework, each coding region has a corresponding mRNA pool in the cytoplasm. mRNA pools are connected to the ribosome pool and, potentially, to chemical and siRNA pools as well. In the nucleus, mRNA is transcribed and spliced, and it becomes mature inside the coding region part. Free molecules of the spliceosome have their own pool and they interact with the immature mRNA by following a Michaelis-Menten (enzyme-like) scheme. All the other steps of mRNA maturation and transport into the cytoplasm are lumped into a single reaction to minimize the model’s number of kinetic parameters.Translation regulation occurs either via riboswitch activation/deactivation or RNA interference. As in the promoter case, position along the mRNA is not explicitly taken into account. However, riboswitches are normally placed on the 5’-UTR (Untranslated Region)  whereas on) and inactive (off). Only the active state allow ribosome binding to the mRNA. Riboswitches change their state upon chemical binding to their aptamers. Only when all the riboswitches’ aptamers are on, ribosomes are allowed to bind the mRNA and to start translation. As an improvement of our previous representation [N different riboswitches can be placed along the 5’-UTR.Riboswitches are, essentially, RNA hairpin loops that can prevent ribosome binding. In our framework, they assume two different states: active , ∧ for AND, and ∨ for OR. As inputs, we considered four external chemicals that interact with promoters and RBSs. However, since our composable parts accommodated at most two binding sites on DNA or mRNA, we could not predict a circuit design that only employs transcription or translation control, respectively. With the new set of eukaryotic parts and pools, we are now able to reconstruct an RNAi-based logic evaluator that is close to the original one, and to design an alternative circuit that performs the same Boolean function via transcription regulation alone.As a benchmark for our eukaryotic part and pool model, we chose the RNAi logic evaluator by Rinaudo et al.. In thiset al., we preset al. version of the circuit, each Boolean variable corresponds to an endogenous signal. When a signal is present, its corresponding siRNA is “inactivated” that is, no longer produced. Moreover, signal a also activates the siRNA associated with the a,-b,-c, and -d under the control of an activator that is inhibited when bound by the corresponding input chemical, whereas the transcription of siRNA-a  has a total of 6 binding sites, whereas 4 lie on AND2AND1 (AND2) gate has 17 (13) species, 45 (33) reactions, and 5 (4) exchange fluxes. Overall, the circuit contains 197 species and 474 reactions. We assume that parts of the same type–such as promoters regulated by an activator or siRNA coding regions–are identical. This means that we have to specify only a limited amount of parameter values with respect to the number of circuit reactions  is regulated by three repressors and it contains a total of 6 operators; AND2’s promoter (pand2) is controlled by two repressors and hosts 4 operators. These promoter configurations show a high degree of complexity: pand1 hosts 65 species, exchanges 12 fluxes, and contains 834 reactions; pand2 hosts 17 species, exchanges 7 fluxes, and contains 130 reactions. The promoter that leads to the synthesis of the repressor associated with pand1 and pand2 have the same number of binding sites as the mRNAs for AND1 and AND2 in the RNAi-based circuit version, the promoters’ species are much more numerous than the corresponding mRNAs’, because mRNA is degraded as soon as one siRNA binds (states where more than one siRNA is bound are forbidden). Overall, the transcription-based version of the circuit is made of 187 species and 1165 reactions. Hence, our modular, rule-based modeling approach turns out to be extremely useful in designing systems with complex promoters.In the transcriptional version of this circuit, siRNAs are replaced by repressors  in order to simplify some interaction schemes and to reduce the number of reactions and unknown parameters in the system.A possible tool extension might exploit a new, recently developed ProMoT feature: the Process Interaction Model  (PIM) coFinally, our framework is highly abstract and still misses a connection with real DNA sequences: for this reason, a future link to the Synthetic Biology Open Language (SBOL) ,52 is unseed species , and reaction rules are written to an BNGL file (part.bngl). BioNetGen then reads the part.bngl file, calculates all the species and reactions involved in the part, and writes them to the part.net file. At this point, our program takes part.net as an input and converts its content into an MDL file (part.mdl).As stated above, only promoters, bacterial RBSs, and eukaryotic coding regions  require a rule-based modeling approach. Part structure is specified into an input file. Here, the binding site number, the kind of regulatory factors acting on the part, their mutual interactions (i.e. cooperative or not), and their activation or inhibition via chemicals have to be specified. Moreover, a set of kinetics parameters is required too. Our program converts this information into rules that represent generic descriptions of the reactions that take place in the part. Kinetic parameters, molecules, storage-intras), and adapter-fluxes, i.e., entities that handle the exchange of fluxes of molecules with external modules and permit to build the part interface. Indeed, adapter-fluxes have two terminals: one is connected to either an internal storage-intra or a reaction, the other to a part terminal. The export function from part.net to the MDL format converts species into storage-intras–if they belong to the part–or into adapter-fluxes–if they belong to a pool, such as RNA polymerase and ribosomes, or they are fictitious  via the python script “compartment_parser.py”. Finally, by running another python script, “link_compartment.py”, the two compartments are connected to form a cell that, in ProMoT, is an instance of the class sbml-model. All the circuit components  are written in a single MDL file that can be loaded into ProMoT to visualize, modify if necessary, and finally export the system to SBML or Matlab format. All the simulations presented in this paper have been performed with COPASI [Our software is a set of Perl and Python scripts. Scripts for promoters, bacterial RBSs, and eukaryotic coding regions call BioNetGen to compute all the part or pool reactions and species. Eukaryotic coding region and siRNA scripts generate two MDL files: one for the nuclear part and the other for the corresponding mRNA/siRNA pool in the cytoplasm. Once written, part and pool MDL files can be loaded into ProMoT and the cellular nucleus and cytoplasm designed, separately, in a drag-and-drop way. Both the nucleus and the cytoplasm have to be saved as h COPASI . The sofThe authors declare that they do not have competing interests.Main project’s idea: MAM, JS. Modeling: MAM, MC, EW. Simulations and data analysis: MAM. Paper writing: MAM, MC, EW, JS. All authors read and approved the final manuscript.Supplementary material. Supplementary Material contains the list of parameter values we used for the simulations of the two logic evaluators and the simpler circuit where a reporter protein is regulated by an activator and an siRNA. This small circuit is described in details: for each of its parts and pools we give all the reactions, the corresponding BNGL rules, and the parameter values that have to be specified as inputs. Results from the simulations of both kinds of circuits are reported. Moreover, figures that elucidate some interactions (at DNA and mRNA level) considered in our framework have been inserted.Click here for file


Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes. Plasmodium falciparum is a protist parasite that causes malaria and kills more than 800,000 people per year. The parasite escapes from the human immune response by antigenic variation through switching between expression of different var genes. These encode different variant antigens that are expressed on the surface of the infected erythrocyte and mediate pathogenic adhesion of the infected erythrocytes to host receptors. Understanding how this process is regulated may lead to the identification of factors that are essential for immune evasion and that could represent novel drug targets. Here, we have identified the parasite's histone variant PfH2A.Z as a novel contributor to the transcriptional regulation of antigenic variation. PfH2A.Z is enriched in the promoter of many genes, but enrichment correlates with gene expression only in var genes. Furthermore we show that PfH2A.Z enrichment in var promoters is antagonised by the var gene silencing factor PfSir2A. These findings further extend our knowledge of the complex mechanisms regulating gene expression in P. falciparum. Plasmodium falciparum causes the majority of malaria-induced morbidity and mortality, resulting in approximately 860,000 deaths each year Plasmodium parasites have a complicated life cycle, during which they differentiate into several morphologically distinct asexual and sexual stages in the human host and the mosquito vector.P. falciparum erythrocyte membrane protein 1 (PfEMP1) variant antigens, which are expressed on the IE surface and are encoded by approximately 60 genes of the var multigene family var gene is expressed at a time P. falciparum and chronic infection Disease occurs during the repeated cycles of invasion and asexual replication of the parasite inside human erythrocytes. A central mechanism of malaria pathogenesis is the ability of the infected erythrocytes (IE) to sequester at vascular sites by cytoadherence to host receptors. Through this process the parasite avoids clearance by splenic macrophages and contributes to severe malaria complications such as cerebral and placental malaria P. falciparum employs stringent regulatory mechanisms to achieve stage-specific gene expression P. falciparum, emphasizing the significance of epigenetic control in the parasite During its asexual, intaerythrocytic life cycle, Plasmodium. The Plasmodium genome encodes a broad set of common chromatin remodelling and modifying factors, including many that are novel and that may be employed in unique epigenetic mechanisms Epigenetic gene regulation confers a heritable state of gene expression and is typically mediated by changes in chromatin structure without a change in DNA sequence. The fundamental units of chromatin are nucleosomes and are formed by ∼146 bp of DNA wrapping around an octamer of histones. The canonical nucleosome components are two H2A/H2B dimers and an H3/H4 tetramer. In most eukaruyotes an additional subunit, H1, links nucleosomes, but this subunit is missing in Plasmodium species. Chromatin can exist as compact, silent heterochromatin and as open, transcriptionally competent euchromatin. These different physical states are essentially determined by the composition and distribution of nucleosomes, post-translational modification of histones, presence of chromatin associated trans-factors and by the covalent modification of DNA, although this latter mechanism has not yet been shown in P. falciparum chromatin has unique features that differ markedly from other eukaryotes. P. falciparum intergenic regions, including promoters, display a global nucleosome depletion P. falciparum promoters does not correlate inversely with steady-state mRNA levels P. falciparum exists in an unusually euchromatic state conferred by the euchromatin marks trimethylated lysine 4 of histone 3 (H3K4me3) and acetylated lysine 9 of histone 3 (H3K9ac) P. falciparum heterochromatin, defined by the histone post-translational modification H3K9me3 and its cognate trans-factor heterochromatin protein 1 (HP1), is restricted to subtelomeric and several chromosome internal domains in P. falciparum that contain gene families including the majority of var genes The organisation of var genes present both in subtelomeric and central chromosomal positions form clusters at the nuclear periphery var gene appears to require it to leave the heterochromatic cluster and relocate to a specific perinuclear expression site var genes is controlled by epigenetic mechanisms var gene must acquire the histone marks H3K4me3 and H3K9ac in its promoter var promoter is the only cis element required for monoallelic exclusive transcription var gene intron is important for var gene silencing cis sequence elements contribute to the rate of var gene switching var gene regulation was proven by the upregulation of numerous var genes and the loss of monoallelic var gene expression that occurred when either of two Sir2 histone deacetylase genes were disrupted The P. falciparum gene regulation is apparent, the role of alternative histones has not yet been investigated. H2A.Z is an H2A variant, which is essential for viability in most organisms apart from yeast and has been highly conserved through evolution Toxoplasma gondii, H2A.Z enrichment at promoters (and enhancers in humans) correlates with gene activity Alternative histones can replace canonical histones through ATP-dependent deposition to create structurally and functionally specialized chromatin domains P. falciparum. Using immunofluoresecence analysis (IFA), co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) in conjunction with quantitative real time PCR (qPCR), we show that PfH2A.Z is enriched in the promoter of a set of developmentally regulated genes in the euchromatin compartment. In these genes promoter occupancy of PfH2A.Z does not correlate with transcription levels, suggesting that presence of PfH2A.Z is important for providing a transcriptionally competent chromatin structure at the promoter but does not directly relate to promoter activity. In var genes by contrast, PfH2A.Z promoter occupancy is strongly associated with transcriptional activity, as PfH2AZ is periodically enriched in the active var gene and is depleted in silent var genes, which is consistent with our IFA evidence that H2A.Z is absent from the subtelomeric heterochromatin compartment. This balance is distorted in cells in which the histone deacetylase Sir2A has been disrupted. Thus, these data suggest that var gene silencing requires expulsion of PfH2A.Z from the var promoter and that this involves histone deacetylation.In this study, we provide the first characterization of the alternative histone H2A.Z in P. falciparum H2A.Z variant (PfH2A.Z) is encoded by the gene PFC0920w S. cerevisiae H2A.Z variant Htz1 and 67.3% identity with the human and mouse H2A.Z proteins  followed by quantitative PCR in ring stage parasites, trophozoites and schizonts. H2A.Z is enriched in nucleosomes surrounding the transcription start site (TSS) in other organisms, therefore at least two quantitative (q) PCR reactions were performed for each gene to amplify regions near the predicted TSS upstream of the start codon (ups) as well as in the open reading frame (orf).Our results show that PfH2A.Z is enriched in all three stages in the promoter regions of candidate genes that are differentially regulated throughout the life cycle , S3A. InTo determine whether the level of PfH2A.Z promoter occupancy in the examined genes correlated with gene expression, mRNA levels were quantified by q-RT-PCR . The upsTo further investigate the relationship between PfH2A.Z and euchromatic and heterochromatic histone marks, ChIP was performed in parallel for PfH2A.Z as well as H3K4me3, H3K9ac and H3K9me3. PfH2A.Z and the two euchromatic marks, H3K4me3, H3K9ac all showed significant enrichment in the ups region when compared to the orf in ring and schizont stage parasites, in contrast the levels of the heterochromatic mark H3K9me3 were the same in ups and orf . This isTogether, these results demonstrate that PfH2A.Z is enriched near the TSS in genes, independently of their transcriptional activity, and that PfH2A.Z enrichment near the TSS correlates with enrichment of the euchromatin marks H3K4me3 and H3K9ac.P. falciparum histone deacetylases Sir2A and Sir2B silence subtelomeric and central var genes S. cerevisiae H2A.Z antagonises subtelomeric gene silencing by Sir2 var genes. We harvested chromatin and RNA from a parasite culture that had been selected for the expression of a single var gene encoding VAR2CSA by panning on chondroitin sulphate A (CSA). To map the position of PfH2A.Z along the var2csa gene, we designed seven qPCR reactions spanning this var locus. The TSS of var2csa has previously been mapped to −1475 bp upstream of the start codon in the FCR3 parasite line The var2csa transcription peaks , and var2csa expressing parasites (var2csa ON) at ring stage. Transcription levels were verified by q-RT-PCR , but not the TSS of inactive var2csa (var2csa OFF) nor the TSS of the other two silent var genes PF08_0141 (var41) and PFL0020w (var20). In contrast to the upstream region, all three var genes were moderately enriched in PfH2A.Z in the introns  var genes in ring stages of both 3D7Δsir2 parasite lines  and p = 0.0152 (Δsir2B))  (var gene expression is down-regulated in mature 3D7Δsir2 parasites (data not shown) Although ups/orf ratios were generally quite low in ring stages, PfH2A.Z enrichment at Δsir2B)) . In schiΔsir2B)) . This re 0.0159) , althougvar genes in 3D7Δsir2A schizonts we analysed by ChIP the relationship between enrichment of PfH2A.Z and H3K9me3 or H3K4me3, respectively , orthologues of which mediate the post-replicative H2A.Z incorporation into nucleosomes in yeast and humans T. brucei and T. gondiiP. falciparum conforms to this pattern, with an enrichment of PfH2A.Z, but not H2A, in the upstream regions of genes , as described previously 5′-GGGATCCGGATGGAAGTTCCAGGAAAAGT and PfH2A.ZEcoRIRev: 5′-GAATTCTTATTGAGCTGTTGGGGGAAGTG. The PCR product was cloned into pGEX-5X-3 . PfH2A.ZGST fusion protein was expressed in BL21 cells in Luria Broth and induced with 1mM IPTG. Bacteria pellets were resuspended in Bugsbuster reagent and benzonase (Novagen) and the soluble proteins were purified on GST-Bind Resin (Novagen). Antibodies were generated in rabbits by the Walter and Eliza Hall Institute Monoclonal Antibody Facility .The full length PfH2A.Z (PFC0920w) sequence was amplified from 3D7 cDNA using oligonucleotides PfH2A.ZBamHIFor: Abcam Ab1791), rabbit anti-H2A (Millipore 07-146), rabbit anti-H2B (Abcam Ab1790), non-immune rabbit IgG (Abcam Ab 46540) and pre-immune rabbit serum. Primary antibodies used for IFA co-localization and WB were rabbit anti-H3K9me3 (Abcam Ab8898), rabbit anti-H3K4me3 (Millipore 04-745) and rabbit anti-H3K9ac (Millipore 06-942). Secondary antibodies for IFA were goat anti-rabbit AlexaFluor488 or chicken anti-rabbit AlexaFluor594 (Molecular Probes). Secondary antibodies for WB were goat anti-rabbit HRP (Invitrogen).Primary antibodies employed in ChIP assays in this study were rabbit anti-PfH2A.Z, rabbit anti-H3 (7 IE were separated by SDS-PAGE on 10% Bis-Tris gels (Invitrogen) and analysed by Western Blotting as described previously Parasites were harvested in 8 hour intervals at 5–10% parasitemia. Lysates were generated by saponin lysis of cultures and extraction of the resulting parasite pellets with 2 x SDS PAGE loading buffer. Equivalents of 5×10Molecular Probes) as a secondary antibody. PfH2A.Z was subsequently detected with affinity purified rabbit anti-PfH2A.Z (10 ng/µl) directly labelled using the Zenon Rabbit IgG labelling kit (Invitrogen) according to the manufacturers instructions. IE were mounted onto slides using ProLong antifade (Invitrogen), left overnight to cure and analysed with an Olympus FV1000 Confocal Laser Scanning Microscope and the FluoView software.IFA was performed on paraformaldehyde/glutaraldehyde-fixed cultures as described previously Electron Microscopy Sciences, Fort Washington, PA). Ultrathin (90–100 nm) sections were cut using a Leica Ultracut R microtome, labeled with rabbit anti-PfH2A.Z and goat-anti-rabbit IgG conjugated to 12 nm colloidal gold (Jackson ImmunoResearch Laboratories). Sections were poststained with uranyl acetate and lead citrate and observed using a Philips CM120 BioTwin Transmission Electron Microscope.Parasites were fixed in 1% glutaraldehyde for 1 h at 4°C, dehydrated in increasing ethanol concentrations, then embedded in LR Gold resin (Roche)) followed by dounce homogenization (Pestle B). 0.25% NP40 was added to the parasite suspension prior to homogenization. Nuclei were pelleted by centrifugation at 21,000×g for 10 min at 4°C and resuspended in SDS lysis buffer . Chromatin was sheared into 200–1000 bp fragments by sonication for 2×8 min at 30 sec intervals using a Bioruptor UCD-200 (Diagenode) and diluted 1∶10 in ChIP dilution buffer .Chromatin was isolated at three time points during the intra-erythrocytic developmental cycle (IDC) from early ring (6–14 hpi), trophozoite (24–32 hpi) and schizont (36–44 hpi) stage parasites. Parasite cultures were cross-linked with 1% paraformaldehyde for 10 min at 37°C and the reaction subsequently quenched with 125 mM glycine. After one wash with PBS parasites were released from IE by saponin lysis, and nuclei were isolated by incubation for 30 min on ice in lysis buffer . For each IP, 1×109 ring stage parasites, 2.5×108 trophozoites or 1×108 schizonts were used. Optimal antibody dilutions were determined empirically and ranged between 1∶50 and 1∶200.Immunoprecipitation was performed with the EZ ChIP Kit (Applied Biosystems) (Applied Biosystems 7900HT) with SYBR PCR master mix (Applied Biosystems). Optimal PCR conditions were determined for each primer pair using serial dilutions of gDNA. PCR was performed in duplicates and melting curves were analysed after each run to confirm the specificity of the amplification. ChIP recoveries were normalized for input signals (ΔCt) and corrected for values obtained with non-immune control antibodies (ΔΔCt). The site specific enrichment of histones was calculated as the 2−ΔΔCt value. To correct for differences in nucleosome density, enrichment of PfH2A.Z and H2A was expressed as a ratio over H3. As in the course of the study different H3 antibody lots were used to precipitate biological replicates, the H3 recoveries and therefore ratios varied considerably between experiments. However, the pattern of histone variant enrichment across genes was reproducible between experiments (Primers amplifying upstream regions near the TSS (ups) and in the open reading frame (orf) were designed using the Primer Express software (systems) . For generiments , 4 & S4.For statistical analysis of PfH2A.Z enrichment in 3D7ΔSir2A and 3D7 parasites, the ratio of PfH2A.Z enrichment near the TSS and in the open reading frame (ups/orf ratio) was calculated. Paired t-test was performed using the GraphPad Prism software (Version 4).9 IE and extraction with salt-free buffers 8 IE were incubated with 10 µl of antiserum overnight at 4°C and precipitated with 30 µl protein G agarose (Millipore). After extensive washing proteins were eluted with 2 x SDS PAGE loading buffer and analysed by SDS-PAGE and Western Blotting.Mononucleosomes were prepared from freshly isolated nuclei by MNase (NEB) digestion with 20 KU MNase per 1×10Invitrogen). RNA was purified as described previously Invitrogen). Quantitative RT-PCR was performed as described previously −ΔΔCt analysis (Applied Biosystems user bulletin 2).Total RNA was harvested in parallel to the chromatin preparation by lysis of pelleted IEs in 20 pellet volumes of TRIzol  as well as loop 1 for H2A.Z/H2A.Z self interaction and loop 2 for nucleosome/DNA interaction (green). The region underlined in yellow encompasses the C-terminal docking domain which mediates interaction with the H3/H4 dimer. Lysine residues printed in red in PfH2A.Z were shown to be acetylated (0.98 MB TIF)Click here for additional data file.Figure S2Nuclear localization of PfH2A.ZGFP. Confocal microscopy of PfH2A.ZGFP parasites at the (A) merozoite, (B) ring, (C) trophozoite and (D) schizont stage. Blue: DNA stained with DAPI. Green: PfH2A.ZGFP. Overlays in the lower panels in each picture demonstrate the nuclear localization of PfH2A.ZGFP.(1.27 MB TIF)Click here for additional data file.Figure S3var genes. (B) Enrichment of H2A.Z/H3 in var genes var2csa, var20 and var41. Amplified regions are depicted in the gene models under each graph. Error bars represent standard deviation from two technical replicates.Biological replicate of ChIP analysis of PfH2A.Z occupancy in genes. ChIP was performed in ring , trophozoite (mid gray bars) and schizont stage (black bars) parasites with antibodies against PfH2A.Z and H3 and non-immune control antibodies. Real time qPCR was performed targeting sequences near the TSS and in the open reading frame of genes with different expression profiles . Enrichment was calculated and the data are presented as ratio over H3 to correct for differences in nucleosome density in the inter- and intra-genic regions. The left scale corresponds to ring data, the right scale to trophozoite and schizont data. (A) Enrichment of PfH2A.Z/H3 in non-(0.40 MB TIF)Click here for additional data file.Figure S4−ΔΔCt analysis. (A) Stage specific expression of ring, schizont, and constitutively expressed genes was verified. Sporozoite specific genes were not expressed in any stage. (B) Var2csa was expressed at high levels in ring stages but not in trophozoites and schizonts. Transcripts of two other var genes (var20 and var41) were undetectable.Verification of stage specific transcription profiles. Quantitative real time PCR was performed on cDNA prepared from ring (light grey), trophozoite (mid grey) and schizont (dark grey) stage parasites. The levels of each sequence in cDNA was determined relative to its levels in a constant quantity of 3D7 strain gDNA and the amount of cDNA normalised using the housekeeping gene arginyl-tRNA synthetase by 2(0.26 MB TIF)Click here for additional data file.Figure S5ChIP analysis of PfH2A.Z and histone modifications in upstream regions and open reading frames in (A) ring stage and (B) schizont stage 3D7 parasites. ChIP enrichment (over pre-immune serum) in the upstream region (ups) and open reading frame (orf) is shown for each antibody. PfH2A.Z (light grey), H3K4me3 (dark grey) and H3K9ac (white) concomitantly show a significant increase in the ups region in comparison to the orf. In contrast, H3K9me3 (black) is not significantly enriched. Shown is the median boxed with 25th and 75th percentile and minimum/maximum values as whiskers. A non-parametric Mann-Whitney test was performed and significant differences are indicated. 12 genes were analysed (N = 12) in two technical replicates of one experiment.(0.24 MB TIF)Click here for additional data file.Figure S6var genes. The var genes cluster together in a group with low PfH2A.Z enrichment and low euchromatic histone marks (H3K9ac and H3K4me3) but high levels of the heterochromatin mark H3K9me3. Data are compiled from two technical replicates of one ChIP experiment.Correlation between PfH2A.Z and histone modifications in the upstream region of genes in (A) ring and (B) schizont stage 3D7 parasites. Enrichment of PfH2A.Z (X-axis) positively correlates with H3K4me3 (p<0.0001) and H3K9ac (p<0.0001) and negatively correlates with H3K9me3 (p<0.0001) at both stages. P-value and spearman correlation coefficient (r) are indicated. A total of 36 genes were analysed, including 20 (0.40 MB TIF)Click here for additional data file.Figure S7var gene dominantly transcribed in ICAM-selected 3D7 parasites. (A) qPCR analysis of ICAM selected parasites identifies PFL0020w as the most abundantly transcribed var gene. (B) Northern Blot analysis confirms PFL0020w as the dominant transcript in ICAM selected parasites. A PFL0020w specific probe labels a band of about 10 kb in ICAM selected rings stage parasites (3D7-ICAM), but not in unselected parasites (3D7). A probe representing the conserved exon 2 hybridizes to multiple transcripts in 3D7, but only one dominant transcript in 3D7-ICAM which corresponds to PFL0020w. (C) Model of the PFL0020w gene. The predicted transcription start site (TSS) at approximately −400 bp upstream of the start codon is represented by the arrow. Coordinates of loci amplified by qPCR are indicated below the graph. (D) ChIP analysis of PfH2A.Z distribution along the PFL0020w gene in 3D7-ICAM at ring stage (light grey bars), schizont stage (mid grey bars) or in unselected 3D7 ring stage parasites (dark grey bars). PfH2A.Z is enriched near the TSS at −400 bp at ring stage of 3D7-ICAM but not unselected parasites, and not in 3D7-ICAM schizonts. PfH2A.Z is also enriched in the intron of PFL0020w in both ICAM selected and unselected lines and slightly enriched in the sequence amplified by qPCR from DBL5 which is directly adjacent to the intron. Error bars represent two biological replicates.PfH2A.Z is enriched in the upstream region of a (0.89 MB TIF)Click here for additional data file.Figure S8var genes in 3D7ΔSir2A, 3D7ΔSir2B and 3D7 parasites at ring stage. Multiple var genes are expressed at high levels in 3D7ΔSir2A parasites (red bars) and 3D7ΔSir2B parasites (blue bars). Only marginal var gene expression is detected in 3D7 (black bars). Levels of each var gene sequence in cDNA were normalised using the gene arginyl-tRNA synthetase and expressed relative to the level of the same var gene sequence detected in a constant amount of 3D7 strain gDNA using 2−ΔΔCt analysis. Var groups according to Transcription profiles of (0.39 MB TIF)Click here for additional data file.Figure S9var genes in 3D7ΔSir2A trophozoites. Enrichment of PfH2A.Z (X-axis) positively correlates with H3K4me3 (upper panel) and negatively correlates with H3K9me3 (lower panel). P-value and spearman correlation coefficient (r) are indicated. 11 var genes were analysed in one experiment.Correlation between PfH2A.Z and histone modifications in the upstream region of (0.09 MB TIF)Click here for additional data file.Table S1Gene accession numbers and oligonucleotide sequences used for qPCR.(0.08 MB PDF)Click here for additional data file.Video S1PfH2A.Z shows a polarized distribution in the nucleus. 3D reconstruction using serial Z-stacks of confocal images was performed. The animation shows a 360° rotation. The blue area depicts DAPI-stained DNA, the green area depicts PfH2A.Z labelled with rabbit anti-PfH2A.Z antiserum. PfH2A.Z labelling is concentrated towards one side of the nucleus.(0.04 MB MP4)Click here for additional data file.


Amycolatopsis mediterranei U32. Under nitrogen-rich conditions, the AlaDH pathway is the major route for ammonium assimilation, while the GS/GOGAT pathway takes over when the extracellular nitrogen supply is limited. The global nitrogen regulator GlnR was previously characterized to activate the transcription of the GS encoding gene glnA in response to nitrogen limitation and is demonstrated in this study as a repressor for the transcription of the AlaDH encoding gene ald, whose regulation is consistent with the switch of the ammonium assimilation pathways from AlaDH to GS/GOGAT responding to nitrogen limitation. Three transcription initiation sites (TISs) of ald were determined with primer extension assay, among which transcription from aldP2 contributed the major transcripts under nitrogen-rich conditions but was repressed to an undetectable level in response to nitrogen limitation. Through DNase I footprinting assay, two separate regions were found to be protected by GlnR within ald promoter, within which three GlnR binding sites  were defined. Interestingly, the major TIS aldP2 is located in the middle of a2 site within region II. Therefore, one may easily conclude that GlnR represses the transcription of ald via specific binding to the GlnR binding sites, which obviously blocks the transcription initiation from aldP2 and therefore reduces ald transcripts.Ammonium assimilation is catalyzed by two enzymatic pathways, i.e., glutamine synthetase/glutamate synthase (GS/GOGAT) and alanine dehydrogenase (AlaDH) in  For most bacteria, like Escherichia coli, Corynebacterium glutamicum and Streptomyces coelicolor, the glutamate dehydrogenase (GDH) pathway is the major route for ammonium assimilation when bacteria are growing under ammonium-excess conditions, and the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway then takes over when the environmental nitrogen supply is poor Amycolatopsis mediterranei, an important actinomycete for industrial production of rifamycin, although the assimilation pathway is the same as most bacteria under nitrogen limitation, i.e. using the GS/GOGAT pathway, the alanine dehydrogenase (AlaDH) pathway, instead of GDH pathway, is responsible for ammonium assimilation under high ammonium conditions m value for ammonium, and thus the AlaDH pathway is less efficient than the energy-consuming GS/GOGAT pathway in ammonium assimilation and is only suitable for bacteria growing in ammonium-excess conditions Nitrogen is present in nearly all bacterial metabolites and can be assimilated from inorganic or organic sources. To cope with variations of nitrogen sources  was used as a template to prepare probes by random-primed α-32P-dCTP labeling. Hybridization and washing procedures were performed at 42°C at the presence of 50% de-ionized formamide.A. mediterranei U32 was grown in Bennet liquid medium supplemented with different extra nitrogen sources and total RNA was extracted for primer extension assay. According to the method previously mentioned 32P-labeled primer ald_pe, which was complementary to the −3th to the 17th nucleotides of the ald protein coding sequence (CDS). At the same time, radio-labeled ald_pe was also used for preparation of the sequencing ladder, with the usage of the fmol DNA Cycle Sequencing System (Promega). The reverse transcribed products together with the sequencing ladders were analyzed on 6% ployacrylamide sequencing gels containing 7 M urea and analyzed with a phoshorimager.ald promoter region was PCR amplified, purified with gel purification kit and then labeled with γ-32P-ATP using T4 polynucleotide kinase (PNK) (NEB). Production of purified recombinant A. mediterranei GlnR as well as the EMSA conditions were the same as reported 32P-ATP using T4 PNK, and then a 527-bp DNA fragment was PCR amplified using primer U32aldp_F1 and γ-32P-labeled primer ald_pe. Proper probe  was incubated at 30°C for 30 min with different amounts of purified His6-GlnR, 1 µg sheared salmon sperm DNA in a total volume of 40 µl in the same buffer as EMSA and 0.3 Unit DNase I  was used for digestion at room temperature for 1 min. The preparation of DNA sequencing ladders and analyses of the digests were the same as reported before The A. mediterranei U32 chromosomal DNA was used as a template for PCR amplification with paired primers aldbs_F2 and ald_r and the products (fragment AFM_Ald) were then purified for further AFM experiments. In a volume of 15 µl, 600 ng of fragment AFM_Ald was incubated for 15 min at room temperature with 1 µg of purified His6_GlnR in the same binding buffer as used in EMSA. The reaction mixture was firstly diluted by 15 to 20 times with deionized water, and was then deposited on a piece of mica modified by Ni2+ and incubated for about 2 min. After gently washed with deionized water, the resulting sample was imaged with AFM. All images were performed in tapping-mode™ using a Multi-mode AFM with a Nanoscope IIIa controller  Co., Santa Barbara, CA, USA). AFM images were collected using commercialized silicon tip  with a spring constant of 3.5 N/m and a resonant frequency of 75 kHz. All AFM experiments were conducted in an ambient environment.The ald in response to extracellular nitrogen supplies, U32 was cultured in minimal medium supplemented with different nitrogen sources, including increasing concentrations of ammonium and nitrate, and total RNA was then extracted for Northern blot analysis .Similar results were obtained with RT-PCR assays when U32 was grown in rich Bennet medium supplemented with either 120 mM ammonium or 80 mM nitrate, ald, the transcriptional regulation of other nitrogen metabolism-related genes, including glnA, nas operon amtB (data not shown), in Bennet medium with ammonium (or nitrate) is the same as that in minimal medium with ammonium (or nitrate). Therefore, Bennet medium, which is easier to prepare, is usually used for analysis of gene transcription in U32 and Bennet medium supplemented with 120 mM ammonium is designated “nitrogen-rich” medium for U32 while supplemented with 80 mM nitrate is designated “nitrogen-limited” medium for U32.Besides glnR null mutant Rk, the transcription of ald remained at a high level under nitrogen-limited conditions , the −46A (P2) and −67G (P3)  for ondition , suggestald promoter region, EMSA was employed to find that GlnR could specifically bind to the ald promoter in vitro  were manually defined within the ald promoter region  is separated from the a1-b1 sites. In nas promoter, the internal distance between b1 and b2 sites is 16 bps b1 and a2 sites are separated by 76 bps in ald promoter.Three GlnR binding sites  to coordinate the global metabolisms, helping the bacteria to cope with the environmental stress. A. mediterranei is an important actinomycete for rifamycin production and has been studied for decades in our lab. In A. mediterranei, GlnR has been characterized to positively regulate the transcription of glnAnas operon amtB (unpublished data) under nitrogen-limited conditions; however, because the GDH activities do not change significantly in A. mediterranei (data not shown), transcription of gdhA is unlikely to be regulated. Interestingly, unlike most bacteria, A. mediterranei uses AlaDH pathway instead of the GDH pathway for ammonium fixation when the extracellular ammonium concentration is high. Therefore, a simple question raised is whether GlnR plays a repressor role in ald transcription in A. mediterranei, just as its homologues do in many other phylogenetically close actinomycetes? Based on the experiments exemplified in this study, we have proved that GlnR negatively regulates ald transcription and switches the ammonium assimilation route from the AlaDH pathway to GS/GOGAT pathway in response to nitrogen limitation, facilitating the bacterial adaption to the nutritional stress. Therefore, GlnR may be considered a global nitrogen regulator in A. mediterranei.So far, GlnR has been well characterized as a central regulator for governing global nitrogen metabolisms in several important actinomycetes, including cis-elements have been studied for years by several groups around the world. Through aligning several promoter sequences of the under-regulated genes, Fink et al. firstly proposed a 44-bp S. coelicolor GlnR binding motif et al.a-site” of gTnAc and a “b-site” of GaAAc with a fixed distance of 6 bases in between A. mediterranei GlnR is not only structurally homologous to that of S. coelicolor GlnR S. coelicolor glnR null mutant in transa1, b1 and b2) in the nas promoter, which are of high similarity to the GlnR binding sites characterized in S. coelicolorald for further investigation. Notably, these sites in ald promoter are highly similar to those in nas promoter, in not only their primary DNA sequences but also their configurations. Although the mechanism for negative regulation of ald transcription by GlnR is obvious and clear, the in-depth depiction of the action of GlnR (e.g. how GlnR binds to these three sites) seems a mission impossible at present.The GlnR binding nas regulation, two complexes of CI and CII were found during the process of GlnR binding to nas promoter and two distinct steps were proposed. First, GlnR binds to a1 and b1 sites to form CI; then, on the basis of CI formation, GlnR further binds b2 site to produce CII, which is required for the activation of nas transcription b1 and b2 are required for the formation of CII, it is unclear whether GlnR binds to a1 site in CII. Recently, based on the results of DSS-crosslinking assay and crystal structural analysis, Lin et al. demonstrated that GlnR mainly works as a homodimer b1 and b2 sites are bound by a homodimer GlnR in the complex of CII. Due to the high similarities between the GlnR binding cis-elements in nas and ald promoters, we presume here a similar procedure for GlnR binding to the ald promoter, and GlnR may first bind to a1-b1 sites and then to b1–b2 sites with a final release of a1 site  to directly examine the change of DNA configuration after binding of GlnR to the r region  in vitro of GlnR . However of GlnR , indicat of GlnR  and a Gab1 and a2 in ald promoter is only 76 bps, other protein(s) may probably participate in bending of the DNA between the sites and facilitate GlnR binding. Although possible loops were observed with merely GlnR addition in the in vitro AFM assay, the concentration of GlnR added was obviously higher than in vivo and thus may be unable to fully reflect the in vivo situation. And the suspects include a group of Nucleoid-Associated Proteins (NAPs), e.g. Fis (Factor for Inversion Stimulation), IHF (Integration Host Factor) and HU (Heat-Unstable Nucleoid Protein), which have been shown to be able to bend DNAs Double-stranded DNA is semi-flexible and DNA fragments shorter than 150 bps are difficult to form a DNA looping


In silico analysis of P1 promoter revealed presence of two evolutionary conserved RUNX motifs, 0.6kb upstream of the transcription start site, and three RUNX motifs within 170bp of the 5’UTR. Transcriptional contribution of these RUNX motifs was studied in myeloid and T-cells. RUNX1 genomic fragment containing all sites show very low basal activity in both cell types. Mutation or deletion of RUNX motifs in the UTR enhances basal activity of the RUNX1 promoter. Chromatin immunoprecipitation revealed that RUNX1 protein is recruited to these sites. Overexpression of RUNX1 in non-hematopoietic cells results in a dose dependent activation of the RUNX1 P1 promoter. We also demonstrate that RUNX1 protein regulates transcription of endogenous RUNX1 mRNA in T-cell. Finally we show that SCL transcription factor is recruited to regions containing RUNX motifs in the promoter and the UTR and regulates activity of the RUNX1 P1 promoter in vitro. Thus, multiple lines of evidence show that RUNX1 protein regulates its own gene transcription.RUNX1 a member of the family of runt related transcription factors (RUNX), is essential for hematopoiesis. The expression of RUNX1 gene is controlled by two promoters; the distal P1 promoter and the proximal P2 promoter. Several isoforms of RUNX1 mRNA are generated through the use of both promoters and alternative splicing. These isoforms not only differs in their temporal expression pattern but also exhibit differences in tissue specificity. The RUNX1 isoforms derived from P2 are expressed in a variety of tissues, but expression of P1-derived isoform is restricted to cells of hematopoietic lineage. However, the control of hematopoietic-cell specific expression is poorly understood. Here we report regulation of P1-derived RUNX1 mRNA by RUNX1 protein.  RHD is also required for nuclear import, interaction with core binding factor β (CBFβ) for an efficient binding to target DNA, and physical and functional interaction with several other proteins to regulate gene transcription [Runt-related transcription factor 1 (RUNX1) belongs to a family of three transcription factors. All three members share homology in a 128 amino acids region designated as runt homology domain (RHD), which mediates the binding to consensus core sequence cription –6, RUNX2cription –9 and RUcription –13. ThusGlobal deletion of RUNX1 gene results in embryonic lethality at midgestation due to hemorrhages in the central nervous system , 5. In ade novo acute myeloid leukemia [In leukemia RUNX1 gene is one of the most frequent targets of mutations and chromosomal rearrangements. In human, rearrangements of RUNX1 locus are associated with 30% of all acute leukemia –19. Indeleukemia .Dysregulation of RUNX1 gene also results in development of other hematological disorders such as Myelo Dysplastic Syndrome (MDS), Acute Lymphoblastic Leukemia (ALL) and Familial Platelet Disorder (FPD). Somatic mutations in the RUNX1 gene is one of the major driving factors in the etiology of the MDS which is characterized by 20% blasts in the blood or bone marrow. FPD is characterized by haploid insufficiency mutation of RUNX1 gene with qualitative and quantitative defects in platelet. FPD patients show high frequency (20–50%) of acute myeloid leukemia development –25. ThusRUNX1 gene transcription remains poorly understood.The RUNX1 gene locus spans 260kb on human chromosome 21. RUNX1 expression is regulated by a proximal P2 and distal P1 promoter . The isoIn addition to RUNX1, several nuclear regulators such as GATA1, PU.1 and SCL play important roles in the RUNX1 gene transcription during hematopoiesis. For instance, the SCL, a basic helix loop helix containing transcription factor, is predominantly expressed in hematopoietic tissues . SCL alsIn this paper we have investigated the role of RUNX1 protein in transcriptional regulation of P1 promoter. Through the use of chromatin immunoprecipitation assays, we show in hematopoietic cells that RUNX1 protein is bound to multiple sites situated in P1 promoter and first exon. Site directed mutagenesis revealed that the promoter and UTR RUNX regulatory motifs show a differential response. RUNX motifs in the promoter enhance transcription, whereas RUNX motifs in the UTR inhibit gene transcription. In hematopoietic cells a positive feedback loop regulates RUNX1 gene transcription.2.The human myeloid HL-60 cells and Jurkat T-cells were obtained from The European Collection of Cell Cultures (ECACC), cultured in RPMI media supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. Human cervical carcinoma, HeLa cells, which do not express RUNX1, were obtained from Stein’s lab, grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100mg/mL streptomycin. Cells were maintained at 37°C in a humidified atmosphere with 95% air and 5% COHuman RUNX-P1 promoter was amplified using genomic DNA isolated from HL-60 cells. The ∼1.1kb genomic fragment spans 591bp of P1 promoter, 444bp of the UTR and 41bp of coding sequence. The P1 promoter and UTR fragment (P1+UTR) was amplified using forward and reverse primers indicated in The pGL3 Luciferase reporter plasmid was digested with SmaI to produce a blunt end vector. The 1.1kb and 645bp blunt end PCR products were ligated into the pGL3 Luc vector. The integrity of the sequences and correct orientation of the P1+UTR-Luc and P1-Luc plasmids was confirmed by direct sequencing. Site directed mutagenesis was utilized to introduce mutation in the three RUNX motifs located in the UTR. To introduce the mutation in RUNX site 3, complementary oligonucleotides carrying the two base pair mutation were synthesized. Two independent PCR fragments containing overlapping sequences of mutated RUNX site 3 were generated by using complementary oligos and the forward and reverse primers used for P1+UTR promoter amplification. The two PCR products of 661bp and 434bp were gel purified, combined and hybridized to generate P1+UTR template. The 1.1kb P1+UTR fragment carrying the mutation in RUNX site 3 was then amplified using the same forward and reverse primer as described above. To generate RUNX site 3,4 double mutant complementary oligonucleotides carrying mutations in both RUNX sites see  were syn™ Transfection reagent , as per manufacturer’s instructions. Briefly, the cells were co-transfected with 200ng of promoter reporter, and increasing concentrations of RUNX1 and SCL expression vectors and a fix amount (5ng) of Renilla luciferase plasmid  used as internal control. The transfected DNA was maintained at a constant total amount of 1400ng by using pBluescript plasmid  as a filler DNA.HeLa cells cultured in 12-well plates were transiently transfected with NanoJuice6 cells were re-suspended in 100μl of Neon transfection buffer and electroporated with 10μg of each promoter reporter plasmid and 50ng of Renilla luciferase plasmid. Electroporated cells were seeded in 6-well plates and harvested 18 hours later. Cell pellets were collected by centrifugation and lysed and suspended in 50μL of passive lysis buffer . Luciferase activity was determined in 20μl of cell lysates using the Dual-Luciferase Reporter Assay System . Promoter-luciferase activity was normalized with Renilla luciferase values obtained in each sample.Jurkat and HL-60 cells were electroporated with P1+UTR-Luc and P1-Luc to determine basal activity of RUNX1 promoter. Briefly, 1×106) were fixed with 1% formaldehyde at 37°C for 10 min. Crosslinking was stopped by using a final concentration of 0.125M glycine and cells were collected by centrifugation. Cells were washed with ice cold PBS, and re-suspended in 1mL of lysis buffer  NP-40, 1X Cømplete, 25mM MG132) on ice for 10 min. Following dounce homogenization , nuclei were collected by centrifugation at 750g for 5 min. Nuclear pellets were re-suspended in 1mL sonication buffer  Triton X-100, 0.1%(v/v) Na-deoxycholate, 0.1%(v/v) SDS, 1X Cømplete, 25mM MG132) and sonicated on ice to an average DNA size of 200-300bp. The samples were centrifuged at 16,000 g for 15 min and precleared with A/G plus- agarose beads precoated with 2μg/mL sonicated salmon sperm DNA, and 1mg/mL BSA. For immunoprecipitation 3 units (A260) of pre-cleared chromatin was mixed with 2μg of RUNX1 polyclonal antibody , for 12 hours at 4°C. The immuno complexes were collected by binding to A/G plus- agarose beads for 1h at 4°C. The beads were washed twice with each of the following buffers: sonication buffer, sonication buffer containing 500mM NaCl, LiCl buffer  and 10mM Tris pH 8. The immunocomplexes were eluted in 50mM Tris pH 8, 1mM EDTA and 1% SDS at 65°C for 15 min. To reverse the crosslinking eluted chromatin was incubated with 200mM NaCl at 65°C for 12 hours. The immunoprecipitated DNA was purified by ethanol precipitation and 2μl of DNA was used for PCR reactions. Occupancy of RUNX1 protein was determined on promoter and UTR by independent PCR reactions. The forward and reverse primers for site 1, site 2 and sites 3,4,5 are described in Chromatin immunoprecipitation (ChIP) assays were performed as described earlier  with som6 Jurkat cells were electroporated with either a control or two different RUNX1 siRNA  for 18 hours. Cells were harvested for either ChIP or western blot analysis.Jurkat cells were electroporated with 80nM of siRNA against human RUNX1 using the Amaxa Nucleofector system . Multiple siRNA targeting RUNX1 mRNA  were initially screened. Only the siRNA with greater than 50% knock down efficiency of RUNX1 protein, were used for subsequent experiment. Briefly, 1×10Blots were probed with polyclonal RUNX1 antibody  or SCL antibody . Blots were stripped and re-probed with polyclonal Actin, Lamin B antibody or mouse monoclonal GAPDH antibody . Protein were detected using species matched HPR-conjugated secondary antibodies and chemiluminiscence imagining.®Mini Kit  according to the manufacturer’s protocol. The purified DNA-free hnRNA were used for synthesis of cDNA using SuperScript™ First-Strand Synthesis System  as per manufacturer’s instructions.Jurkat cells were electroporated with siRNA as described above and RNA was extracted using RNeasyThe relative level of P1 derived RUNX1 hnRNA was determined with a forward primer located in exon 1 and a reverse primer located in intron 1 see . RT-PCR in silico analysis. Genomic sequences of the human RUNX1-P1 promoter were screened for putative motifs of DNA binding proteins using TFSEARCH program. Multiple binding sites for hematopoietic transcription factor GATA1 and RUNX1 were identified in both the promoter and the proximal part of the untranslated region  are separated by 338bp. To evaluate independent occupancy of RUNX1 at each site, we initially optimized our sonication conditions . TraditiWe find RUNX1 protein is recruited to both R1 and R2 motifs and exhibit significant enrichment over IgG ranging from two to three fold in Jurkat cells . BindingTo determine transcriptional function of endogenous RUNX1 protein, we depleted RUNX1 protein from Jurkat cells. Several small interfering RNA targeting various regions of RUNX1 RNA were initially screened (data not shown). Two siRNA targeting different coding regions of the RUNX1 mRNA showed consistent knock down of RUNX1 protein by 54% and 56% respectively . ChIP anSeveral transcription factors are critical for development of hematopoietic cells. Here we assessed involvement of basic helix-loop-helix containing protein SCL that binds E box motif in the target DNA. The initial two hundred base pairs of the RUNX1-P1 promoter contain an evolutionary conserved E-box motif . To asseThese results are consistent with increased basal activity of the RUNX1 P1+UTR promoter in HL-60 cells . The abiLike several hematopoietic transcription factors, SCL forms a molecular complex with RUNX1 protein. Therefore, we assessed interplay between RUNX1 and SCL transcription factor for regulation of the RUNX1-P1 promoter. Interestingly, chromatin immunoprecipitation revealed that SCL protein is recruited to both the P1 promoter and the UTR region that do not contain SCL motif . These dIn hematopoietic cells two principal isoforms of RUNX1 mRNA are derived from P1 and P2 promoters respectively , 43, 44.in vivo [The P1 and P2 promoters of the RUNX1 gene in both human and mice are separated by 160kb sequences. Earlier reports have established that a 531bp enhancer region is present in the intron 1 of the mouse RUNX1 gene. This enhancer region located 23.5kb after the RUNX1 translational start site, is highly conserved among different mammalian species. This RUNX1 enhancer can drive hematopoietic cell specific expression of a reporter gene in vivo . To defiin silico analysis of UTR sequences. The UTR region contains three RUNX sites in close proximity. These sites are evolutionary conserved, suggesting a role in the transcriptional regulation of RUNX1 gene. Indeed, chromatin immunoprecipitation revealed that native RUNX1 protein in hematopoietic cells is recruited to these sites. Deletion or mutation of these RUNX motifs results in a 4 to 20-fold increase in activity of the RUNX1-P1 promoter. These data indicates that repressor activity of UTR sequences is due to recruitment of RUNX1 protein to the Runx sites. Interestingly, two evolutionary conserved RUNX motifs are also present within initial 570 bp of the P1 promoter. In hematopoietic cells, endogenous RUNX1 protein is directly recruited to these regulatory motifs. In contrast occupancy of the Runx motifs in the promoter by RUNX1 protein enhances transcriptional activity of the P1 promoter. The ability of RUNX1 protein to serve as transcriptional activator was further confirmed by overexpression and knockdown approaches. Depletion of RUNX1 protein by siRNA in hematopoietic cells, significantly reduces RUNX1 occupancy at these sites, as well as transcription of the RUNX1 gene. Thus, a positive feedback loop is required for expression of RUNX1 gene in hematopoietic cells.Interestingly, UTR of the RUNX1 gene contains potential repressor regions, as removal of these sequences enhances basal transcription of the RUNX1-P1 promoter. To identify factors contributing to repressor response, we performed an Our over-expression results indicate that RUNX protein activates P1 promoter. However, RUNX motifs located in the promoter and UTR showed a differential response in the presence of RUNX1 protein. The two RUNX sites located in the P1 promoter showed an enhanced transcriptional response to RUNX1 protein. In sharp contrast, the three RUNX motifs located in UTR inhibits transcriptional activation by the RUNX1 protein. These findings are consistent with earlier reports demonstrating that based on promoter and cell context, RUNX proteins can act as either transcriptional activators or transcriptional repressors . Most liSeveral transcription factors including members of the SMAD family, GATA1 and PU.1 regulates RUNX1 gene expression in hematopoietic cells. During hematopoiesis, these transcription factors work cooperatively by either forming a physical and/or functional interaction with RUNX1 protein. In this paper we also studied involvement of SCL transcription factor in regulating P1 promoter derived RUNX1 gene transcription. A putative SCL binding motif is present in the RUNX1 P1 promoter. This SCL motif is located between RUNX sites present in the UTR and the P1 promoter. In hematopoietic cells the endogenous SCL protein is recruited to this site. However, SCL occupancy in the promoter site is rather modest when compared with the SCL motif located in the +23 enhancer of RUNX1 gene. Overexpression of SCL protein in non-hematopoietic cells enhances transcriptional activity of the RUNX1-P1 promoter. Interestingly, SCL protein is recruited to both the UTR and the P1 promoter regions. Our findings are consistent with previous reports that showed physical interaction between SCL and RUNX1 protein . HoweverIn summary, our findings demonstrate that in hematopoietic cells RUNX1 protein is recruited to its own promoter to regulate RUNX1 gene transcription in a positive feedback loop. This auto-regulation is also noted for other members of the RUNX gene family , 59. Thu