Trypanosoma brucei evades the host immune system through antigenic variation of its variant surface glycoprotein (VSG) coat. Although the T. brucei genome contains ∼1500 VSGs, only one VSG is expressed at a time from one of about 15 subtelomeric VSG expression sites (ESs). For antigenic variation to work, not only must the vast VSG repertoire be kept silent in a genome that is mainly constitutively transcribed, but the frequency of VSG switching must be strictly controlled. Recently it has become clear that chromatin plays a key role in silencing inactive ESs, thereby ensuring monoallelic expression of VSG. We investigated the role of the linker histone H1 in chromatin organization and ES regulation in T. brucei. T. brucei histone H1 proteins have a different domain structure to H1 proteins in higher eukaryotes. However, we show that they play a key role in the maintenance of higher order chromatin structure in bloodstream form T. brucei as visualised by electron microscopy. In addition, depletion of histone H1 results in chromatin becoming generally more accessible to endonucleases in bloodstream but not in insect form T. brucei. The effect on chromatin following H1 knock-down in bloodstream form T. brucei is particularly evident at transcriptionally silent ES promoters, leading to 6–8 fold derepression of these promoters. T. brucei histone H1 therefore appears to be important for the maintenance of repressed chromatin in bloodstream form T. brucei. In particular H1 plays a role in downregulating silent ESs, arguing that H1-mediated chromatin functions in antigenic variation in T. brucei.The African sleeping sickness parasite  Trypanosoma brucei causes African sleeping sickness, endemic to sub-Saharan Africa. Bloodstream form T. brucei is covered with a dense coat of variant surface glycoprotein (VSG). Only one VSG is expressed at a time out of a vast repertoire of ∼1500 VSGs. The active VSG is transcribed in a telomeric VSG expression site (ES), and VSG switching allows immune evasion. Exactly how monoallelic exclusion of VSG ESs operates, and how switching between ESs is mediated remains mysterious, although epigenetics and chromatin structure clearly play a major role. The linker histone H1 is thought to orchestrate higher order chromatin structure in eukaryotes, but its exact function is unclear. We investigated the role of histone H1 in the regulation of antigenic variation in T. brucei. We show that histone H1 is associated with chromatin and is required for higher order chromatin structure. Depletion of histone H1 results in derepression of silent VSG ES promoters, indicating that H1-mediated chromatin functions in antigenic variation in T. brucei. Trypanosoma brucei is a unicellular parasite causing African sleeping sickness, which is transmitted by tsetse flies in sub-Saharan Africa. As an extracellular parasite of the mammalian bloodstream, T. brucei has evolved a sophisticated strategy to antigenically vary its major surface coat protein, variant surface glycoprotein (VSG) T. brucei genome contains a vast repertoire of silent VSG genes and pseudogenes, most of which are located in tandem arrays at subtelomeric locations VSG repertoire varies in both size and composition between different T. brucei strains, with the exact sizes still unclear due to the technical complications of cloning, sequencing and assembling these subtelomeric sequences T. brucei 927 strain contains more than 1500 VSGs, of which only one VSG is expressed at a time The African trypanosome VSG is located in one of about 15 telomeric VSG expression sites (ES). ESs are transcribed by RNA polymerase I (Pol I) VSG is expressed at a time, and the extensive repertoire of VSGs is kept transcriptionally silent. These restrictions need to operate within the context of a T. brucei genome which is primarily organised as very extensive polycistronic transcription units constitutively expressed by Pol II The active S. cerevisiae, Tetrahymena or mammalian cells affects transcription of a relatively small subset of genes in these different organisms, and does not have a major effect on global transcription In eukaryotes DNA is packaged into nucleosomes, whereby ∼146 bp of DNA is wrapped around a histone octamer consisting of two histone H2A/H2B dimers and two histone H3/H4 dimers. A linker histone H1 (H1) typically interacts with both the nucleosome and the linker DNA to stabilize higher order chromatin structure T. brucei has several unusual properties. The core histones of T. brucei are divergent compared with those of higher eukaryotes, particularly at the N-termini which can be post-translationally modified T. brucei chromatin has a more open conformation, does not form 30-nm fibres in vitro, and chromosomes fail to condense prior to nuclear mitosis T. brucei chromatin are typically influenced by the linker histone H1 in other eukaryotes arguing that T. brucei H1 could play a different role The chromatin of T. brucei are distinct from those in other eukaryotes, in that they lack the central globular domain thought to be responsible for interaction with the nucleosome Histone H1 proteins in T. brucei, as well as in the maintenance of higher order chromatin states. Depletion of histone H1, while having a minimal effect on cell growth, causes significant changes in chromatin structure. H1 knock-down resulted in increased sensitivity to endonucleases in bloodstream T. brucei, but not in the procyclic form of the parasite which replicates in the midgut of the insect vector and does not express VSG. In particular, reduced levels of H1 result in the formation of a more open chromatin structure in the vicinity of silent ES promoters in bloodstream T. brucei, which is correlated with an increase in transcription at silent ESs. Knockdown of a number of chromatin proteins in T. brucei results in disruption of VSG ES silencing or VSG switching T. brucei.We have investigated the role of histone H1 in the regulation of antigenic variation in T. brucei 927 genome sequence contains five predicted histone H1-like genes arranged in two clusters on chromosome 11 , very basic proteins (pI of ∼12) with at least one serine or threonine residue at the N-terminus. We raised antibodies using peptides which theoretically should allow recognition of all five T. brucei histone H1 isoforms. Histone H1 proteins have characteristic biochemical properties including the ability to be extracted with perchloric acid T. brucei using perchloric acid extraction. Several proteins were enriched, and Western blot analysis confirmed that a subset reacts with our H1 antibody  to investigate the genomic distribution of H1 in  control . We firs VSG ESs  [49]. H1T. brucei  and spliced leader (SL) gene loci (T. brucei (Sup. We subsequently used quantitative PCR (qPCR) to determine H1 distribution on different transcription units in bloodstream form . brucei . Statistene loci . Significei Sup. .VSG221 or VSGVO2 ES, we showed that H1 is depleted from the active VSG221 ES (hygromycin and VSG221 genes) compared with the silent VSGVO2 ES (neomycin and VSGVO2). Note that the primer pair for VSGVO2 will also detect an additional non-ES located copy of VSGVO2. This H1 distribution is similar to that of the core histones H2A, H3 and H4 VSG118 gene located at a chromosome internal VSG basic copy array. The distribution of H1 in procyclic form T. brucei is similar, with the exception that H1 does not appear to be depleted from the SL promoter . MNase preferentially digests DNA in between nucleosomes, such that an open chromatin structure is more readily cleaved than closed chromatin. Bloodstream form parental T. brucei and cells in which H1 RNAi had been induced were treated with increasing concentrations of MNase  were enriched in the mononucleosomal fraction compared with sequences in the transcriptionally silent VSG221 ES    than Pol I transcribed regions. This is similar to as observed for transcriptionally inactive sequences such as VSG118  . This ins VSG118 , and is T. brucei genome became more accessible to MNase. This effect was most pronounced at the promoter of the silent VSG221 ES (puromycin resistance and eGFP genes) (P = 0.0007 and P = 0.0043 respectively) . These data indicate that depletion of H1 results in a general opening of chromatin structure, however this effect is particularly clear at the silent ES promoters. Although depletion of H1 caused changes in chromatin structure upstream of the procyclin promoter, we did not detect increases in the levels of procyclin transcript following H1 knockdown  . H1 knocdown Sup .T. brucei, or cells where H1 RNAi had been induced for 48 hours (T. brucei has particularly darkly stained areas (with an electron density comparable to that of the nucleolus) which presumably correspond to heterochromatin. These dark areas are interspersed with more lightly-stained areas which are likely to contain euchromatin T. brucei.Knockdown of histone H1 did not result in obvious changes in the structure or staining intensity of the nucleus as monitored by fluorescence microscopy using DNA staining with DAPI (data not shown). To investigate the effect of histone H1 depletion at the ultrastructural level, we performed transmission electron microscopy (TEM) analysis on parental bloodstream form 48 hours . The nucT. brucei RYT3 reporter cell line has eGFP immediately downstream of the promoter of the inactive VSG221 ES, allowing ES derepression to be monitored using GFP fluorescence . GCV resistance can also arise from mutations in the TK gene As histone H1 depletion results in transcriptional derepression of silent ESs, we also investigated the consequences of H1 knockdown on VSG switching frequency. We used a similar strategy to promoter . VSG swiVSG gene conversion (GC), the TK and VSG221 genes are lost, while the switched cell remains GFP positive. After a switch mediated by a telomere exchange, cells are also GFP positive but retain VSG221. A transcriptional (in situ) switch results in GFP negative cells without loss of VSG221. Finally, a gene conversion can occur which initiates at or near the ES promoter, resulting in the duplication of an entire new ES into the active ES, thereby resulting in its deletion. These switched cells would be GFP negative and would have lost all sequences present in the old VSG221 ES. Alternatively, as previously observed, the same phenotype can result from an ES in situ switch coupled with deletion of the active ES The presence of the ES-located single copy marker genes allows the mechanism of VSG switching to be deduced from the genotype and phenotype of a clone after a VSG switch event . After aVSG221 ES for 48 hours, and H1 RNAi was induced in one culture of TK-expressing cells. Cultures were subsequently serially diluted in 96-well plates in the presence of GCV, and positive wells were scored after 7 days. We observed an average 4-fold increase in the frequency of generation of GCV resistant clones per generation after the induction of a block in H1 synthesis for 48 hours in five independent experiments  or had not been induced (n = 4 independent cultures). Using immunofluorescence microscopy we established if the GCV resistant clones had indeed switched their VSG221 coat, monitored for GFP expression, and determined the presence or absence of the SG221 ES . We alsoVSG ES deletion events are frequently picked up in VSG switching experiments with T. brucei 427. VSG switching experiments performed with T. brucei have shown variability regarding the predominant VSG switch mechanism used. Some reports have found that VSG gene conversion is the most common switching mechanism VSG221 ES, which is often deleted during switch events involving a switch to another ES. The VSG221 ES is unusually large with extensive duplications and triplications. In addition, it contains unusually short regions of 70 bp repeats which normally facilitate gene conversion VSG221 ES has been hypothesised to have a particularly low frequency of inactivation, it has been postulated that if the rate of switch off of this ES drops low enough, telomere deletion events resulting in its loss are an alternative way of resolving an unfavourable double-expressor state VSG221 ES deletion event is not a consequence of the TK negative selection system, as these events have also been uncovered in experiments using positive selection for ES activation using drugs or negative selection against VSG221 using VSG RNAi We still do not know why VSG. We repeated our VSG switching assays in the presence of puromycin selection to maintain expression of the VSG221 ES. This procedure selects for cells that continue to transcribe the active VSG221 ES, but have silenced (through telomere exchange) or lost (through VSG gene conversion) the HYGTK fusion gene. As with the previous switching assay, this procedure will also select for cells with mutations in the TK gene T. brucei H1 RNAi cells (n = 21), and cells induced for H1 RNAi for 48 h (n = 15). Here we found that our protocol had enriched for cells that continue to express VSG221, and are probably TK mutants  changes in chromatin structure were nonetheless occurring at the URA3 locus which could be affecting DNA recombination, we used an assay described in T. brucei expressing URA3 is sensitive to 5-fluoroorotic acid (FOA). We replaced one allele of URA3 with the hygromycin resistance/thymidine kinase fusion gene (HYGTK) in bloodstream form T. brucei that either did (H1 RNAi) or did not (Par) contain the H1 RNAi construct. Removal of cells from hygromycin selection allows gene conversion at the URA3 locus to occur. This results in cells with either two copies of the URA3 gene (which are sensitive to FOA but resistant to GCV), or two copies of the HYGTK gene (which are sensitive to GCV but resistant to FOA).We next investigated if the rate of DNA recombination in the Pol II-transcribed on units . HoweverT. brucei cells were expanded for 48 hours in the absence of hygromycin selection. During this period, one of the H1 RNAi cultures was induced with tetracycline (H1 RNAi 48 h). We next placed cells under either FOA or GCV selection, and scored for positive wells after eight days. Validation of a selection of these clones by PCR confirmed that, as expected, GCV resistant clones lacked the HYGTK gene while FOA resistant clones lacked the URA3 gene (data not shown). The frequency of positive wells in both FOA and GCV-containing plates was divided by the number of generation times in the absence of hygromycin to obtain the total frequency of gene conversion per generation for each sample  or uninduced cells, compared with cells in which histone H1 had been depleted for 48 hours (n = 3).Parental and H1 RNAi URA3/HYGTK h sample . We did T. brucei. H1 depletion results in a general disruption of electron dense material in the nucleus which appears to correspond to heterochromatin. In addition, H1 knock-down results in an increase in the accessibility of chromatin to MNase in bloodstream T. brucei, as well as a marked opening of chromatin structure at the silent ES promoters. In agreement with this, H1 knockdown results in derepression of silent ES promoters, and also appears to lead to an increase in VSG switching. These data all argue that H1 could be one of the layers of control allowing the parasite to tightly control expression of its extensive VSG repertoire. H1 knockdown in procyclic form T. brucei resulted in only moderate derepression of ESs, and no obvious changes in global chromatin structure as assessed using MNase accessibility. It is possible that the chromatin of wild-type procyclic form T. brucei is in a more open state than that found in bloodstream form T. bruceiT. brucei.We find that the single-domain histone H1 proteins play an essential role in the maintenance of higher order chromatin structure in bloodstream form T. brucei histone H1 has unusual properties. While H1 proteins normally have a tripartite domain structure including a central globular domain flanked by relatively unstructured N- and C-terminal domains, kinetoplastid H1 proteins have only a single domain rich in lysine, alanine, and proline residues T. brucei H1 proteins, while missing domains important for function in other eukaryotes, can still facilitate higher order chromatin structure. It is possible that the mechanism by which single-domain H1 proteins compact chromatin is different from that used by the tripartite H1 proteins T. brucei, both bloodstream and procyclic form life-cycle stages still grow at reduced rates even if histone H1 is depleted down to very low levels. Histone H1 is nonessential in a number of unicellular eukaryotes T. brucei results in a mild growth phenotype, it is possible that here too these proteins are not essential. However, in the absence of a true genetic H1 knockout (complicated by the fact that there are at least five histone H1 genes) it is not possible for us to establish this point definitively. It is also unclear if the different isoforms of T. brucei H1 perform distinct roles or have overlapping functions.T. brucei, T. cruzi, L. major, and most recently L. braziliensis, it has not yet been thoroughly investigated how these proteins function in vivo. T. cruzi has three H1 genes, and phosphorylation sites of the encoded proteins have been mapped T. cruziLeishmania species have at least two H1 proteins (L. brasiliensis has three) L. major have been shown to be developmentally regulated While detailed biochemical analysis of H1 proteins has been performed in T. brucei. Genomic areas that are actively transcribed by Pol I including the rDNA, active ESs, and the procyclin loci are highly depleted of nucleosomes, and have an unusually open chromatin structure T. brucei, where procyclin loci are transcriptionally silent. This could indicate the presence of an open chromatin structure associated with ‘paused’ Pol I complexes. Pol I is thought to initiate transcription at procyclin promoters in bloodstream form T. brucei, which is not fully processive URA3 locus and other single copy genes, the chromatin structure as investigated using histone H1 distribution and MNase accessibility in parental cells appears comparable to that of silent ESs. This indicates that a highly open chromatin structure is not a prerequisite for processive Pol II transcription in T. brucei.In addition to providing insight into the role of H1, this study also provides information on the chromatin structure of T. brucei nucleus is heterogeneously stained in EM thin sections T. brucei. Our ChIP experiments show that H1 is enriched at transcriptionally silent regions, including repeat regions and the VSG basic copy array, which can be assumed to be present in the form of heterochromatin. As our MNase accessibility experiments indicate that H1 knockdown causes the chromatin in these areas to become more open, it is therefore highly likely that the darkly stained regions of the T. brucei nucleus visualized by EM are in fact areas of more tightly packaged DNA. It is possible that H1 knockdown also has an effect on other aspects of subnuclear organization, including telomere clustering and the distribution of minichromosomes, thereby leading to some of the functional consequences we observe. Interestingly, knock-down of NUP-1, a lamin-like protein important for nuclear structure in T. brucei, has also been shown to affect VSG switching frequency It has previously been found that the T. brucei, this effect was not observed in procyclic form T. brucei. It is clear that one function of H1 could be to facilitate different processes required for efficient antigenic variation in T. brucei. We also find that histone H1 plays a role in silencing inactive ESs particularly in bloodstream form T. brucei. In contrast, depletion of H1 in S. cerevisiae had no effect on telomeric silencing T. brucei is critical for complete ES silencing in bloodstream form T. brucei. In addition to being enriched on silent VSG ESs, H1 in T. brucei also appears to play a role in maintenance of the closed chromatin state found at the silent basic copy VSGs. We find that H1 is enriched on silent VSGs including the VSG118 basic copy gene, and H1 knock-down results in a more open chromatin structure at this locus.What is the role of H1 mediated chromatin structure in an organism that has little transcriptional control? Although H1 knock-down resulted in an increase in accessibility of chromatin to nucleases in bloodstream form VSG repertoire. VSG switching rates of the laboratory adapted T. brucei 427 strain are significantly lower than in strains which are closer to field isolates For antigenic variation to be effective, rates of DNA recombination need to be suppressed to levels that prevent unnecessarily rapid depletion of the VSG ES silencing, but also appears to result in an increase in the frequency of VSG switching. It is possible that depletion of H1 from the silent ESs causes an increase in homologous recombination. This is consistent with a proposed role for H1 in suppressing recombination events and maintaining genome stability in yeast VSG switching frequency, presumably through disruption of homologous recombination pathways T. brucei RTR complex, which is thought to resolve recombination intermediates, causes an increase in the VSG switching frequency VSG ESs, as has been found in S. cerevisiae in the rDNA transcription units We find that depletion of H1 not only disrupts VSG gene. It is possible that in the absence of H1 other factors (such as RAP1) are still affecting chromatin structure in the region adjacent to the telomere VSG located in a basic copy array.Depletion of H1 had a much more dramatic effect on chromatin structure in the region immediately downstream of the silent ES promoters compared with at the telomeric URA3 locus. Since H1 knockdown does not alter chromatin structure at the URA3 locus, it is perhaps unsurprising that rates of homologous recombination at this locus do not increase after H1 depletion. Although there was some effect of H1 depletion on the chromatin of other Pol II loci including actin or γ-tubulin, H1 depletion had a less dramatic effect on the chromatin structure at these loci compared with at the ES promoter regions. In S. cerevisiae, histone H1 has been shown to suppress homologous recombination at the repetitive Pol I transcribed rDNA loci, but had no effect on homologous recombination at another locus outside of the rDNA Although our ChIP and MNase accessibility data indicate that H1 could affect chromatin structure in at least some Pol II transcription units, knockdown of H1 did not result in increased MNase accessibility at the VSG ES promoters, but could also be involved in suppressing VSG switching. Our studies on T. brucei histone H1 provide insights into the unique structure and role of chromatin in these parasites, and these data therefore provide additional evidence for the key role played by chromatin structure and remodeling in the processes involved in antigenic variation in these important human pathogens.Here, we show that H1 containing heterochromatin is not only involved in silencing T. brucei brucei strain 427 was cultured in HMI-9 medium 2. During the course of these experiments the amount of FCS in the HMI-9 was reduced from 20% to 15%, with no observable effect on cell growth or the kinetics of the histone H1 RNAi phenotype. Procyclic form (PF) T. brucei was cultured in SDM-79 medium supplemented with 10% FCS, 5 µg/ml hemin, and the appropriate drugs at 27°C. Cell densities were determined using a haemocytometer.Bloodstream form (BF) T. brucei RYT3-H1 cell line was generated by transfection of the p2T7-H1hy histone H1 RNAi construct into the T. brucei RYT3 cell line. This parental cell line is based on the BF T. brucei ‘single-marker’ VSGT3 ES, and a 221GP1 puromycin/eGFP construct integrated behind the silent VSG221 ES promoter T. brucei RYT3-H1 cell line was used for experiments monitoring the general phenotype after the induction of histone H1 RNAi including growth, protein and transcript levels, micrococcal nuclease (MNase) sensitivity, and global nuclear architecture as investigated using electron microscopy (EM).The BF T. brucei ‘single marker’ cells were transfected with the 221GP1 construct (containing the puromycin and eGFP genes) which integrates immediately downstream of the active VSG221 ES promoter VSG221 ES between the 70 bp repeat sequences and the VSG221 gene itself.For the experiments analysing VSG switching, BF T. brucei, the PF T. brucei DsRed-H1 cell line was created. To do this, the p2T7-H1ph construct was transfected into the 221BsrDsRed parental T. brucei line T. brucei 29-13 line VSG221 ES. Phleomycin selection was used to select for stable integration of the construct, and clonal cell lines were obtained. ChIP experiments were performed using the BF T. brucei HNI 221+ T. brucei 221BsrDsRed cell lines To examine the effect of histone H1 depletion in procyclic form T. brucei brucei genome with the TriTrypDB accession numbers: Tb11.42.0005, Tb11.42.0006, Tb11.42.0007, Tb11.55.0001, and Tb11.39.0008. Protein sequence alignments were performed using ClustalW. To produce double-stranded RNA (RNAi) homologous to the different polymorphic members of the histone H1 gene family in T. brucei, two histone H1 fragments (∼600 bp each) corresponding to different histone H1 genes were PCR amplified from the T. b. brucei 427 genome, and cloned in tandem into the p2T7-177 RNAi vector which targets T. brucei minichromosomes GGATTCAGACAACTGCTGTCCCCAAG-3′5′-TAT (BamHI link) and HisH1Con1_884as AAGCTTGAGCAGCAGATGCCTTCG-3′5′-TAT (HindIII link). The second histone H1 fragment includes the intergenic region between Tb11.39.0008 and the coding region of Tb11.55.0001, and was amplified using primers HisH1Con2_524s AAGCTTCCCGCTATTAGACACGCTATG-3′5′-TAT (HindIII link) and HisH1Con2_1173as CTCGAGGATGCGCTCACGCCTTCT-3′5′-TAT (XhoI link). These two histone H1 fragments were inserted by triple ligation into the p2T7-177-Hygro or p2T7-177-Phleo constructs to generate the p2T7-H1hy and p2T7-H1ph constructs. These contained a ∼1.2 kb fragment producing dsRNA capable of RNAi-mediated knockdown of all five predicted T. brucei histone H1 genes.Five genes have been annotated as histone H1-like in the VSG221 ES, we employed a strategy similar to that of Kim and Cross, 2009. First, we cut out a ∼2.5 kb region of 70 bp repeat sequence from the construct RM3173 VSG221 ES between the 70 bp repeats and the VSG221 gene using primers VSG221TAR_55928s GGATCCGACGAATACAAACCATAAATAAATGC-3′5′-TAT (BamHI link) and VSG221TAR_56433as GCGGCCGCCAAGACGTGGTGCAATCATC-3′5′-TAT (NotI link) and cloned it into the same vector. We finally inserted the HYGTK fusion gene (a generous gift of Nina Papavasiliou and George Cross) flanked by an upstream tubulin intergenic region containing an RNA splice site and a downstream actin intergenic region containing a polyadenylation signal in between the 70 bp repeat fragment and the VSG221 ES targeting fragment. The vector was cut with HindIII and NotI prior to transfection, and correct integration confirmed by PCR linking.In order to integrate a construct containing the HYGTK fusion gene into the T. brucei histone H1 proteins, two histone H1 peptides were designed, synthesized, and injected into rabbits (Eurogentec). The T. brucei histone H1 peptide sequences are: N-KKVAPKKVAGKKAAA-C (amino acids 59–73 of Tb11.55.0001) and N-AAPKKAVAKKAAPKK-C (amino acids 6–20 of Tb11.55.0001). Affinity purification of antibodies specific for these peptides was carried out by Eurogentec. The specificity of the T. brucei histone H1 antibodies was confirmed using Western blots comparing lysates from wild-type T. brucei with lysates from T. brucei after RNAi-mediated knockdown of histone H1. To create antibodies specific for VSG221, a ∼500 bp fragment was amplified by PCR using primers VSG221Ab_243s 5′-GCAAGTATATACGCTGAAATAAATCAC-3′ and VSG221Ab_741as 5′-TGTTTGGCTGTTCGCTACTGTGAC-3′ and cloned into the pRSETA vector (Invitrogen) for expression of an N-terminally tagged 6×-His-tagged protein. Protein was purified under denaturing conditions and was used for immunisation of rabbits (Eurogentec).To create antibodies specific for Tris-Tricine SDS-PAGE electrophoresis was performed with 16% polyacrylamide gels before being transferred to PVDF membrane  using standard protocols. Blots were probed with affinity-purified anti-histone H1 peptide antibodies, anti-histone H3 antibody , anti-La antibodies (a gift from Elisabetta Ullu), or anti-BiP (a gift from Jay Bangs). Detection of the appropriate peroxidase-coupled secondary antibody was performed using ECL Plus (Amersham) detection kit.T. brucei strain (BF) HNI 221+ or (PF) 221BsrDsRed, 1×107 cells per sample were centrifuged at 1000 rcf for 7 min. Cell pellets were washed twice and the supernatants removed. For “total” samples, cell pellets were resuspended in 200 µl hot Tricine SDS-PAGE sample buffer. For other samples, cell pellets were resuspended in PBS+1% Triton X-100 containing either 0, 100, 200, 400, or 800 mM NaCl. Roche protease inhibitors (−EDTA) were also included in each lysis solution. Lysates were incubated on ice for 20 min, followed by centrifugation at 16000 rcf for 20 min at 4°C. 25 µl of each supernatant was removed to a fresh tube. Hot 2× Tricine SDS-PAGE buffer (25 µl) was then added to each supernatant and samples were boiled for 5 min. The rest of the supernatant was discarded from each sample, and the pellets resuspended in 200 µl hot Tricine SDS-PAGE sample buffer. All other protein lysates were prepared by resuspension of a washed cell pellet in hot Tricine sample buffer to a final concentration of 105 cell equivalents per µl.To determine the affinity of histone H1 for chromatin in et al 2011 RNA isolation, cDNA production and qPCR analysis of steady state transcript levels was performed as described in Narayanan T. brucei PF 221BsrDsRed cells (1×109) were centrifuged at 1000 rcf for 10 min and washed twice with 10 ml trypanosome wash solution , at 4°C The sample was then centrifuged at 16000 rcf for 20 min at 4°C, and the supernatant transferred to fresh tubes (100 µl/1.5 ml tube). Proteins were precipitated using acetone, and dried pellets were resuspended in Laemmli SDS-PAGE sample buffer. Samples were boiled for 5 min before loading on Tris-glycine SDS-PAGE gels for analysis using standard protocols.T. brucei BF HNI(221+) and PF 221BsrDsRed cells were used. Histone H1 was detected using the affinity purified T. brucei anti-histone H1 peptide antibody and an Alexa-594-conjugated anti-rabbit secondary antibody (Invitrogen). The T. brucei nucleolus was detected using the monoclonal L1C6 antibody (a gift from Keith Gull) and an anti-mouse Alexa-488-conjugated secondary antibody (Invitrogen). Slides were visualized on a Zeiss Axio Imager.M1 microscope equipped with a Zeiss AxioCam MRm camera using AxioVision Rel 4.8 software. Images were cropped and brightness and contrast uniformly adjusted using Adobe Photoshop.For localization of histone H1 proteins, immunofluorescence microscopy was performed essentially as described Chromatin immunoprecipitation (ChIP) experiments were performed as described 7 cells per sample were permeabilized with digitonin and treated with either 0.0625, 0.125, 0.25, or 0.5 units of MNase . Isolated DNA was then loaded on a gel, and bands quantitated using ImageJ. For MNase treatment followed by sucrose gradient fractionation, 2–3×108 cells per sample were permeabilized and treated with 2.5 units MNase per 1×108 cells. Solubilised chromatin was then loaded onto a 5–30% discontinuous sucrose gradient, and the different nucleosome species were fractionated by ultracentrifugation at 40,000 rpm (Beckman SW41 Ti rotor) for 16 h at 4°C. Fractions were treated with proteinase K and RNase A, and DNA isolated by phenol-chloroform extraction followed by ethanol precipitation. After visualization of some of the isolated DNA on a gel, the remaining DNA fractions were pooled into mono-, di-, di/tri-, tri-tetra-, and >tetra nucleosomal fractions. The amount of each DNA target present in each fraction was determined by qPCR using primer sets described in Micrococcal nuclease (MNase) treatment of chromatin was performed essentially as described Cells were fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide and stained with 2% uranyl acetate before dehydration through a series of different ethanol concentrations and embedding in Agar 100 epoxy resin. Thin sections (90 nm) were stained with lead citrate and examined using an FEI Tecnai 12 transmission electron microscope at 80 kV according to VSG221 ES in the T. brucei BF RYT3 and PF 221BsrDsRed cell lines was monitored by flow cytometry as described 6 cells were centrifuged, washed, and fixed in 2% paraformaldehyde for one hour at room temperature. Fixed cells were then washed and analysed by flow cytometry using a Becton-Dickinson FACSCalibur and CellQuest software (BD). For each time point, fold derepression was calculated by dividing the mean FL1-H or FL2-H fluorescence value of induced cells by the mean fluorescence value of the uninduced cells.Derepression of the silent T. brucei 221pGFPhyTK cell line, which contains a construct containing a puromycin and eGFP gene immediately downstream of the promoter in the active VSG221 ES, and a construct containing the HYGTK fusion protein gene downstream of the 70 bp repeats in the same ES. The T. brucei 221pGFPhyTKH1 cell line contains the same marker genes as 221pGFPhyTK with the addition of the p2T7-H1ph RNAi construct. We maintained both T. brucei 221pGFPhyTK  and T. brucei 221pGFPhyTKH1 cells under puromycin and hygromycin selection to select for a homogenous population expressing the VSG221 ES. We subsequently removed these cells from drug selection and allowed them to switch for 48 hours. Tetracycline (1 µg/ml) was added to an additional culture of T. brucei 221pGFPhyTKH1 cells to induce RNAi of histone H1, also for 48 hours. Cells were then plated in 3-fold serial dilutions in the presence of 4 µg/ml ganciclovir (GCV) (Sigma), and VSG switching frequencies were calculated based on the number of positive wells (containing growing cells), the number of cells plated for each dilution, and the number of generations undergone in the absence of drugs. We have determined the plating efficiency of the different cell lines using different experimental conditions (i.e. in the presence or absence of various drugs) and did not find a clear difference in plating efficiency (data not shown). We therefore did not normalise our data for this variable. However as the T. brucei lines capable of histone H1 RNAi have a longer generation time than parental cells, we expressed our data as switch/recombination events per generation. However, the effect of H1 depletion is also apparent even when the data is not normalised for number of generation times.To determine VSG switching frequencies and mechanisms, we employed a strategy similar to that used in VSG221 gene (primers VSG221_243s 5′-GCAAGTATATACGCTGAAATAAATCAC-3′ and VSG221_741as 5′- TGTTTGGCTGTTCGCTACTGTGAC-3′). PCR amplification of the large subunit of RNA polymerase I was performed as a positive control (Pol1_4102s GGATCCAGCGCCGTTCCACGCGAGA-3′5′-CT and Pol1_4554as CTCGAGCTATCCCCAATCCGTGCCGTCCCG-3′5′-GA). Pulsed-field gels were performed essentially as described VSG221 and VSG1.8 using standard protocols. The probes were amplified by PCR using VSG221_121s 5′- TGCCAGGTCTCCGAG-3′, VSG221_1056as 5′-GCTGCTCGGATATGAGCTTTT-3′, VSG1.8_179s 5′-CAATCTCGAGGCTCACAAAAGTCTG-3′, and VSG1.8_1022as 5′-GCTGGGATCCTAGCCTCGAAAAATG-3′.Individual switched clones were analysed for GFP fluorescence, VSG221 expression (using immunofluorescence microscopy), and PCR for the T. brucei URA3 locus was performed as described T. brucei 221pGFP cells or BF T. brucei 221pGFPH1 cells (containing the histone H1 RNAi construct). Correct integration of the construct was confirmed by PCR amplification using primers URA3uplink_9228s (5′-AGAAAGAACCGTACCGCAGA-3′) and Hygro_125as (5′-CCTACATCGAAGCTGAAAGCAC-3′) for upstream linking and TKlink_2370s (5′-TTTACGGGCTACTTGCCAAT-3′) and URA3dnlink_12374as (5′-AGGGGGAAACAGCGTAAGTT-3′) for downstream linking. Cells were grown in the absence of hygromycin selection for 48 hours. Cells were then plated (5×105 cells per sample) in the presence of either 6 µg/ml 5-Flouroorotic acid (Sigma) or 30 µg/ml GCV. Plates were scored 8 days later, and the gene conversion frequency was calculated by dividing the frequency of positive wells for each selection method by the number of doubling times undergone by each culture in the absence of hygromycin.Determination of the frequency of gene conversion at the Figure S1Association of histone H1 proteins with chromatin in procyclic form (PF) T. brucei.T. brucei 221BsrDsRed cells were lysed with 1% Triton X-100 in the presence of increasing concentrations of NaCl (molarity indicated above the lanes). Total indicates total lysate. Pellet and supernatant (Sup.) fractions were analysed by Western blotting with antibodies against histone H1, histone H3 or the RNA binding protein La. The size markers in kiloDaltons (kDa) are indicated on the left.(TIF)Click here for additional data file.Figure S2Genomic distribution of histone H1 in procyclic form T. brucei.A. Schematic of the procyclic form (PF) T. brucei PF 221BsrDsRed cell line used for ChIP experiments indicated as a large box containing two relevant ESs. The blasticidin (Bla) resistance gene and the DsRed gene are inserted immediately behind the promoter (white flag) of the VSG221 ES. The VSGVO2 ES is shown below. B. Representative slot blots showing the association of histone H1 and histone H3 proteins with 50 bp repeat sequences flanking ESs, or 177 bp repeat sequences which comprise the T. brucei minichromosomes. Experiments were performed with no antibody (No ab) or pre-immune serum (Pre-imm.) as negative controls. For each sample, 10% of the ChIP material was loaded on a slot blot and compared with 0.1% of the total input. C. Quantitation of material immunoprecipitated (% IP) using anti-histone H3 (H3) or anti-histone H1 (H1) in the slot blots shown in panel B. Bars show the mean of three experiments with standard deviation indicated with error bars. Two negative controls were used, no antibody (No ab) or pre-immune serum (pre-im) from the rabbit used to produce the histone H1 antibody. D. Distribution of histone H1 within the genome of procyclic form T. brucei as determined using qPCR analysis of immunoprecipitated material. The bars indicate the amount precipitated (% IP) using the anti-histone H1 antibody (H1) or the pre-immune serum (Pre-imm.) with the standard deviation from three experiments indicated with error bars. Statistically significant amounts of histone H1 (P<0.05) were found at all loci. The regions analysed include the actin, γ-tubulin (γ-tub) and spliced leader (SL) gene loci. The SL intergenic region (int.), promoter region (pro.), or the SL gene itself (SL) are indicated. The ribosomal DNA (rDNA) regions analysed include the rDNA intergenic region (int.), promoter (pro.) or the 18S rDNA gene (18S). The EP procyclin locus analysed includes the region upstream of the EP promoter (up.), the promoter (pro.), or the EP procyclin gene (EP). A higher level of histone H1 was immunoprecipitated upstream of the rDNA promoter compared with at the promoter region itself, with the statistical significance indicated with asterisks (** indicates P<0.01). ES sequences analysed include a region immediately upstream of the ES promoter (up.) as well as the ES promoter itself (pro.) with these primer pairs expected to recognise most if not all ESs. Sequences analysed within the VSG221 ES include the blasticidin resistance gene (Blast) and the telomeric VSG221. VSG118 is located in the silent chromosome internal VSG arrays.(TIF)Click here for additional data file.Figure S3Statistically significant chromatin immunoprecipitation (ChIP) using anti-histone H1 antibody compared with pre-immune serum. Different T. brucei genomic regions are indicated in the table. ChIP was performed in both bloodstream form and procyclic form T. brucei. The P values are shown for the ChIP performed using anti-histone H1 anti-serum compared with the pre-immune serum, with the significance of the results indicated with asterisks.(TIF)Click here for additional data file.Figure S4Histone H1 depletion in procyclic T. brucei does not result in an increase in open chromatin. Parental procyclic form (PF) T. brucei 221BsrDsRed (Par) or cells in which histone H1 had been depleted using RNAi for four days (H1+4 d) were permeabilized, and the chromatin digested with increasing amounts of micrococcal nuclease (MNase). Lanes contain material digested with different MNase concentrations . Purified DNA was subsequently analysed on agarose gels stained with ethidium bromide. DNA corresponding to mono-, di- and tri-nucleosome species are indicated, along with undigested DNA. A representative gel is shown.(TIF)Click here for additional data file.Figure S5Depletion of histone H1 has a minimal effect on steady-state transcript levels derived from the γ-tubulin, EP procyclin and 18S rRNA gene loci. RNA was isolated from indicated time points in hours (h) following induction of histone H1 RNAi, and used as templates for cDNA production. Quantitative PCR (qPCR) was then performed. Relative levels of each transcript were determined, first after normalization to actin, and next in comparison with the level of each transcript at the 0 hour timepoint. Error bars indicate the standard deviation from three independent experiments.(TIF)Click here for additional data file.Figure S6Confirmation of VSG switching mechanisms through genotype analysis.A. The presence of the single copy VSG221 gene was determined using PCR. Representative PCR reactions were analysed on 1% agarose gels with the gene encoding the RNA polymerase I (Pol I) large subunit used as a positive control. Genomic DNA from single-marker BF T. brucei cells was analysed as a positive control for the presence of the VSG221 gene (lane 1). DNA from a cell line which has deleted the VSG221 ES as a result of a switch event was used as a negative control (lane 2). DNA from clones which have undergone an in situ switch is shown in lanes 7, 11 and 17. DNA from a clone which has switched as a result of a gene conversion is shown in lane 13. DNA from clones which have switched as a consequence of an ES gene conversion or deletion event is shown in lanes 2–4, 6, 8–10, 14–16 and 18. DNA from clones which have not switched their VSG, but contain a mutation in the TK gene is shown in lanes 5, 12 and 19. B. Pulsed field gel electrophoresis (PFGE) analysis confirms the presence or absence of the VSG221 gene in a representative selection of switched clones as determined using PCR in panel A. An ethidium bromide (EtBr) stained PFGE gel of T. brucei chromosomes is shown with a DNA marker (L) indicated in megabases (Mb) on the left. The gel was subsequently blotted and probed for VSG221 or VSG1.8. DNA from the unswitched parental control is shown in lane 1 compared with various clones which have switched their VSG. DNA from clones which have undergone an ES gene conversion or deletion (ES GC/ES del) (lanes 2–5), an in situ switch (lanes 6–8) or a VSG gene conversion (VSG GC) (lane 9) are shown in comparison. The blots were probed for both VSG1.8 and VSG221, which appear to be located on the same chromosome. This allows us to monitor the VSG221 containing chromosome, even in clones where the VSG221 gene has been deleted. This can reveal changes in chromosomal size resulting from deletions of large regions of the chromosome after VSG switching Click here for additional data file.Figure S7Analysis of VSG switching using fluorescence microscopy. Fixed T. brucei was subjected to immunofluorescence using an anti-VSG221 antibody. Cells were also monitored for GFP fluorescence, and DNA stained with DAPI. A differential interference contrast (DIC) image is shown for reference. For each channel, a similar exposure time was used to image the different clones. Scale bar is 10 µm.(TIF)Click here for additional data file.


Recent evidence indicates that the mycobacterial response to DNA double strand breaks (DSBs) differs substantially from previously characterized bacteria. These differences include the use of three DSB repair pathways , and the CarD pathway, which integrates DNA damage with transcription. Here we identify a role for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response. Arr is transcriptionally induced following DNA damage and cellular stress. Although Arr is not required for induction of a core set of DNA repair genes, Arr is necessary for suppression of a set of ribosomal protein genes and rRNA during DNA damage, placing Arr in a similar pathway as CarD. Surprisingly, the catalytic activity of Arr is not required for this function, as catalytically inactive Arr was still able to suppress ribosomal protein and rRNA expression during DNA damage. In contrast, Arr substrate binding and catalytic activities were required for regulation of a small subset of other DNA damage responsive genes, indicating that Arr has both catalytic and noncatalytic roles in the DNA damage response. Our findings establish an endogenous cellular function for a mono-ADP-ribosyltransferase apart from its role in mediating Rifampin resistance. Mycobacterium tuberculosis and Mycobacterium leprae, most of these stresses are derived from the host immune system, whereas for environmental mycobacteria like Mycobacterium smegmatis, these stresses may arise from toxins and chemicals secreted by other organisms, UV radiation, osmotic stress, and heavy metal pollutants. Many of these stresses cause damage to a range of macromolecules, including the bacterium's genomic DNA. DNA double strand breaks (DSBs) are the most dangerous form of DNA damage and represent the greatest threat to genome integrity and cell survival. Prior studies have provided evidence that DNA damage responses are important for intracellular pathogens, including mycobacteria Mycobacteria are ubiquitous environmental and pathogenic bacteria that must withstand a range of stresses present in their respective habitats. In the case of pathogenic mycobacteria like M. smegmatis to DSBs and implicates the mono-ADP-ribosyltransferase Arr in this response. ADP-ribosylation is a reversible covalent modificationin which the ADP-ribose moiety of NAD+ is attached to its target + binding core of 5 β-strands arranged as two adjoining sheets M. smegmatis Arr-catalyzed ADP-ribosylation is rifampin, an antimicrobial agent that inhibits RNA polymerase (RNAP) M. smegmatis to rifampin in comparison to mycobacteria that do not express Arr. Other mycobacteria that encode Arr homologs include the pathogens M. marinum and M. ulceransM. tuberculosis does not encode an Arr homolog and is, therefore, more sensitive to rifampin, which is a first line agent in treatment of Tuberculosis.This manuscript describes the transcriptional response of Pseudomonas aeruginosa exotoxins A and S, cholera toxin, pertussis toxin, and Escherichia coli LT-I and LT-II, which all target proteins in the host cellto facilitate pathogenesis M. smegmatis Arr lacks an obvious secretion signal and thus is expected tomodify targets within the mycobacterial cell. Endogenous mono-ADP-ribosylation, in which the ADP-ribosyltransferase and the protein to be modified originate from the same cell, has been described in M. smegmatisM. smegmatis DSB response and demonstrate a role for Arr in this response. These experiments provide a physiologic role for mono-ADP-ribosylation in mycobacteria apart from its function in rifampin resistance.Beyond its role in ADP-ribosylating rifampin, endogenous protein or small molecule targets of Arr have not been identified. The most well-characterized prokaryotic ADP-ribosyltransferases are secreted toxins, including diphtheria toxin, M. smegmatis gene expression during DNA double strand breaks (DSBs) caused by the I-SceI homing endonuclease M. smegmatis strains: mgm181 and mgm182. Anhydrotetracycline (ATc) treatment of both strains induces expression of a hemagglutinin (HA) epitope-tagged I-SceI homing endonuclease, which cuts at a single site engineered into the mgm182 genome. Thus, only the mgm182 genome is cleaved by I-SceI, while mgm181 expresses the endonuclease without suffering a chromosomal break. For the remainder of the paper we will refer to mgm182 asthe site(+) strain and mgm181 as the site(−) strain. The advantage of the I-SceI system is that the DNA damage induced is limited to DSBs, unlike similar microarray experiments that have been performed using genotoxins like UV radiation, which causes many different types of DNA damage as well as damage to other macromolecules To better understand the mycobacterial pathways that respond to double strand DNA damage, we used whole genome transcriptional profiling to detect changes in To detect HA-I-SceI expression, cultures were harvested before (T = 0) and after addition of ATc for protein extraction and western blot analysis with HA specific antibodies . To veriM. smegmatis ORFs. M. smegmatis does not simply involve the regulation of DNA repair pathways, but is actually a coordinated response ofmany metabolic processes including transcription, translation, replication, sugar metabolism and lipid biogenesis. Most of the genes upregulated in either growth phase were genes of unknown function, highlighting the amount that remains to be characterized about the mycobacterial DNA damage response. Most functional classes of genes that were upregulated were evenly represented between stationary and log phase cells, except for membrane transport, lipid metabolism, and cell envelope pathways, which collectively made up 30% of the genes upregulated during DSBs in stationary cultures and only 12% in log phase cells.To determine the transcriptional response to DSBs, we prepared cDNA libraries from either exponential or stationary phase site(−) and site(+) cultures before and after the addition of ATc. Fluorescently labeled cDNAs were co-hybridized to microarrays spotted 4 times with oligos representing 6746 Since we had observed a low level of I-SceI expression even in the absence of ATc , we alsoM. smegmatis genes were upregulated more than 2 fold in both log and stationary cultures during both low and high levels of I-SceI expression (arr (MSMEG_1221), which encodes a mono-ADP-ribosyltransferase and was transcriptionally upregulated as high as 15 fold in stationary cultures and 5 fold in logarithmic cells  and nutrient deprivation before measuring changes in arr transcription by qRT-PCR .The only known target of Arr catalyzed ADP-ribosylation is rifampin, a chemical inhibitor of RNAP. To determine the role of Arr in stress responses, a function that is presumably independent of its function in rifampin resistance, we took a genetic approach and deleted s of arr  [8]. To r strain . The trur strain . The Δararr leads to increased susceptibility of M. smegmatis to killing by rifampin arr strain by measuring the zone of inhibition by a rifampin soaked disk on a lawn of either wild-type M. smegmatis or Δarr , Arr(Y48A), or Arr(D83A), each with an N-terminal FLAG epitope. Overexpression of FLAG-tagged Arr proteins containing any one of these amino acid substitutions failed to confer rifampin resistance in Δarr or wild-type M. smegmatis, even though all Arr alleles were expressed in Δarr , Δarr site(+), and Δarr+Flag-Arr site(+). We found numerous transcripts that were differentially expressed between Δarr site(+) and wild type cells /wt(site+) = 1.5) indicating that arr does not participate upstream of these genes in the DNA damage response. Similar results were obtained for the other DNA repair genes induced by DSBs  compared to wild type site(+) and complemented by expression of FLAG-Arr  strains expressing either FLAG-Arr(H18A) or FLAG-Arr(D83A), which fail to confer rifampin resistance  were restored in the complemented strain but remained deregulated in the Arr(H18A) and Arr(D83A) strains , HA-Arr(Y48A), or HA-Arr(D83A) with HA antibodies. Analysis of the eluates by western blotting with an antibody specific for the RNAP β subunit showed that Arr coprecipitates the RNAP β subunit ppGpp and in E. coli, Bacillus subtilis, Thermus thermophilus, and Streptomyces sp., stringent coupling between translation and transcription is eliminated by mutations in rel genes In previous immunoprecipitation experiments, we have shown that CarD associates with RNAP M. smegmatis rplK with a gene coding for an N-terminally FLAG-tagged L11 protein in our strains expressing HA-Arr. The M. smegmatis ΔrplK::FLAG-L11 strains grew like wild-type, indicating that the FLAG-tagged version of L11 was functional because L11 is predicted to be an essential gene rplK::FLAG-L11 strains expressing HA-Arr or HA-Arr point mutants showed that wild-type Arr and Arr(H18A) were able to co-precipitate similar amounts of FLAG-L11, whereas Arr(D83A) and Arr(Y48A) were not  may be structurally unstable or unfolded ppGpp synthesis is dependent on the N-terminus of L11 and deletion or mutation of this region is enough to confer a relaxed phenotype in transE. coli.In 32P, biotin, or etheno as a cofactor.However, these studies were complicated by the relative instability of purified L11 protein (data not shown). Thus, although Arr interacts directly with the N terminus of L11, we were unable to conclusively determine whether L11 is an ADP-ribosylation target of Arr.Unfortunately,despite extensive efforts, we were unable to demonstrate ADP-ribosylation of L11 by Arr in vitro using NAD that was labeled with M. smegmatis log and stationary cultures in response to both low and high levels of DNA damage. So far, we have described physiological functions for two of these genes, arr and carD, in regulating the levels of transcription of the translation machinery. Taken together, these studies indicate that a major response to double strand DNA damage is the induction of pathways that coordinately repress expression of the translational machinery. These findings highlight that stringent response-like changes in transcription are not only important in the classical case of nutrient deprivation, but also during double strand DNA damage.A total of 14 genes were consistently upregulated more than 2 fold in E. coli by these bacteriophages leads to the expression of three phage mono-ADP-ribosyltransferases, Alt, ModA, and ModB. Each phage ADP-ribosyltransferase modifies a distinct group of host proteins to regulate gene expression during the transition from host to phage protein synthesis. Alt is a structural component of the phage head that ADP-ribosylates one of the RNAPα subunits to enhance transcription from T4 early promoters M. smegmatis arr, are either on mobile genetic elements or clustered with predicted transposase encoding genes, which suggests acquisition from horizontal gene transfer arr genes were derived from lysogenic phages incorporated into their bacterial hosts. If this were the case, it would be expected that phage and prokaryotic ADP-ribosyltransferases would have similar specificities.We have shown that Arr associates with the RNAP and the ribosomal protein L11 and affects the transcript levels of the translation machinery during DNA damage. Arr is not the first mono-ADP-ribosyltransferase proposed to modulate transcription and translation. One of the most well characterized bacterial exotoxins, Diphtheria toxin, ADP-ribosylates host elongation factor Tu, inactivating it and shutting down host protein synthesis. Evidence for endogenous ADP-ribosylation affecting transcription and translation comes from T4 and related bacteriophages. Infection of arr strain during DNA damage and the association of Arr with the RNAP and L11 suggest a role for Arr in the mycobacterial stringent response. There is precedencein other bacteria for ADP-ribosylation during starvation and sporulation, processes tightly linked to the stringent response. ADP-ribosyltransferase activity has been reported to increase in Bacillus subtilis, Myxococcusxanthus, and Streptomyces species in response to adverse environmental conditions that may involve severe DNA stress, nutrient limitation and sporulation B. subtilisB. subtilis, (p)ppGpp levels increase, GTP levels decrease, sporulation is initiated, and ADP-ribosyltransferase activity increases The induction of ribosomal protein and ribosomal RNA in the ΔM. smegmatis showed that Arr mutants deficient in binding L11 retain the ability to repress rRNA and ribosomal protein gene transcription, which suggests that Arr does not need to bind L11 to downregulate these transcripts. We were also unable to demonstrate ADP-ribosylation of L11 by Arr. Therefore, although Arr and L11 may both function to regulate transcription of the translation machinery, this function may not require them to interact physically. This implies that additional components of the Arr and L11 pathways are unaffected by the Arr(D83A) mutation. Arr substrate binding and catalytic activity mutants retain their ability to associate with the RNAP, and thus could conceivably be regulating transcription through this interaction, apart from L11.However, our studies in M. ulcerans, M. marinum, and M. aviumPseudomonas aeruginosa, E. coli, Klebsiella pneumoniae, Vibrio cholerae, and Acinetobacter baumanniiM. tuberculosis, but this activity is clearly not originatingfrom an enzyme with the exact same specificity as M. smegmatis Arr since wild-type M. tuberculosis remains sensitive to rifampin M. tuberculosis chromosome. Identification of novel ARTs in organisms like M. tuberculosis is hindered by the low sequence similarity between the ADP-ribosylating enzymes, but is worth pursuing as it could potentially provide exciting new approaches to treatment.Predicted ADP-ribosyltransferase genes have been identified in the genomes of numerous pathogenic mycobacteria including M. smegmatis strains were isogenic to mc2155 and were grown at 37°C in LB supplemented with 0.5% dextrose, 0.5% glycerol, and 0.05% Tween 80 (broth). MGM181, MGM182, and Tet-CarDstrains were described previously arr gene (MSMEG_1221) was deleted from the M. smegmatis genome via an allelic exchange system arr gene with a truncated null allele containing only the first 10 and last 2 codons, thus yielding the M. smegmatis Δarr strain (M. smegmatis nucleotides 1290611 to 1290611 were deleted). The truncated allele conserves the reading frame of the wild type gene to avoid polar effects on neighboring genes that may be caused by the deletion.All arr were made by overlap extension PCR. The residue designations are based on the annotation of arr from J. Craig Venter Institute Comprehensive Microbial Resource. Our amino acid residue numbers are shifted by one residue from the published description of these mutants rplK::FLAG-L11 was engineered using a specialized transducing phage rplK gene with a FLAG-tagged version followed by a hygR cassette.Point mutations in In mycobacteria, 20 µg/ml kanamycin, 50 µg/ml hygromycin, and 50 ng/ml of ATc were used. H2O2 (Fisher Scientific), MMS (Sigma), bleomycin, Ciprofloxacin (USB), and rifampin (Sigma) were used at the indicated concentrations.Primary antibodies include monoclonal antibodies specific for HA  FLAG , and RNAP β .M. smegmatis ORFs . Experiments were done in triplicate and results were processed with GenePix software and analyzed in either GeneSpring GX or Partek Genomics Suite. Clustering was performed using the hierarchical clustering module on the Gene pattern server http://www.broadinstitute.org/cancer/software/genepattern/index.html.RNA was prepared from 20 ml of mycobacteria cultures with TRIzol (Invitrogen), bead beating 3× , and QIAGEN's RNAeasy RNA clean-up protocol. The RNA was reverse transcribed into cDNA with the Stratagene Fairplay kit. cDNA coupled to fluorescent dyes Cy3 and Cy5  were hybridized to gene chips spotted four times with oligos representing 6746 M. smegmatis sigA-5′-TGCCGATCTGCTTGAGGTAGG-3′ and 5′-TTCGTGTGGGACGAGGAAGAG-3′; M. smegmatis arr- 5′-GGCGCATCATGAACCACGTC-3′ and 5′-CTTCACCGGCAGCAAGTTCG-3′; M. smegmatis rplL- 5′-GCAGTCGGAATTCGACGTCATCCTC-3′ and 5′-CGAGATCCTTGGCTTCCTTCAGGC-3′; M. smegmatis rplX- 5′- GTGAACCGGATCAAGAAGCACACC-3′ and 5′- GAATCGACCACCATCACGTTCGAG-3′; M. smegmatis 16S rRNA- 5′-GTGCATGTCAAACCCAGGTAAGG-3′ and 5′-GGGATCCGTGCCGTAGCTAAC-3′.RNA was prepared from 5 ml of mycobacteria cultures with TRIzol and bead beating. To assure that there was no contaminating genomic DNA, RNA preparations were treated with Ambion's TURBO DNA-free kit. cDNA was created with Invitrogen's SuperScript III First-Strand Synthesis System. Quantitative PCR was performed with NEB's DyNAmo SYBR Green qPCR kit. All qRT-PCR experiments were done in triplicate.The following primer sequences were used for qRT-PCR. For immunoprecipitation, 50 ml cultures were washed and lysed in 500 µl NP-40 buffer  by bead beating. 25 µl of lysate was used for input sample and the rest was added to monoclonal anti-HA agarose (Sigma) and rotated overnight at 4°C. The matrix was washed 3× with NP-40 buffer and proteins were eluted with 50 mM Tris-HCl [pH 7.5], 50 mM NaCl, 500 µg/ml HA peptide (Roche), and protease inhibitors. For western blot analysis, cells were lysed in 100 µl of NP-40 buffer by bead beating three times and adding lysates directly to SDS-PAGE loading buffer.Saccharomyces cerevisiae strain EGY48 containing the AD fusion plasmid to strain W303a containing the BD bait plasmid and the lacZ reporter plasmid pSH18-34.pMSG295 and LipQ were used as controls for a positive interaction with each other, and a negative interaction with L11 or Arr Yeast Two Hybrid interaction studies were done as previously described For Table S1I-SceI microarray- Ratio of M. smegmatis gene transcript levels in mgm182 (site(+)) as compared to mgm181 (site(−)) at both growth phases and all timepoints.(XLSX)Click here for additional data file.Table S2I-SceI microarray- Genes organized by functional category that are upregulated 1.5× only in the site(+) strain, and not in site(−), during log growth following ATc addition. The increase in transcript levels is expressed as a ratio of levels at T = 45/T = 0. p-value≤0.5.(XLSX)Click here for additional data file.Table S3I-SceI microarray- Genes organized by functional category that are upregulated 1.5× only in the site(+) strain, and not in site(−), during stationary (stat) growth following ATc addition. The increase in transcript levels is expressed as a ratio of levels at T = 45/T = 0. p-value≤0.5.(XLSX)Click here for additional data file.Table S4I-SceI microarray- Genes upregulated 2× throughout all timepoints in log phase site(+) cultures as compared to site(−). p-value≤0.5.(XLSX)Click here for additional data file.Table S5I-SceI microarray- Genes upregulated 2× throughout all timepoints in stationary site(+) cultures compared to stationary site(−) cultures. p-value≤0.5.(XLSX)Click here for additional data file.Table S6I-SceI microarray- Genes upregulated 2× in both log and stationary site(+) cultures at all timepoints, as compared to site(−).(XLSX)Click here for additional data file.Table S7Δarr I-SceI microarray- Genes upregulated 1.5× in Δarr site(+) as compared to wild-type site(+) and complemented by WT Arr expression, p value<0.05.(XLSX)Click here for additional data file.Table S8Δarr I-SceI microarray- Genes upregulated 1.5× in Δarr site(+) as compared to wild-type site(+) and complemented by WT Arr expression but not by expression of ArrH18A, p value<0.05.(XLSX)Click here for additional data file.Table S9Δarr I-SceI microarray- Genes upregulated 1.5× in Δarr site(+) as compared to wild-type site(+) and complemented by WT Arr expression but not by expression of ArrD83A, p value<0.05.(XLSX)Click here for additional data file.Table S10Δarr I-SceI microarray- Genes downregulated 1.5× in Δarr site(+) as compared to wild-type site(+) and complemented by WT Arr expression, p value<0.05.(XLSX)Click here for additional data file.Table S11Δarr I-SceI microarray- Genes downregulated 1.5× in Δarr site(+) as compared to wild-type site(+) and complemented by WT Arr expression but not by expression of ArrH18A, p value<0.05.(XLSX)Click here for additional data file.Table S12Δarr I-SceI microarray- Genes downregulated 1.5× in Δarr site(+) as compared to wild-type site(+) and complemented by WT Arr expression but not by expression of ArrD83A, p value<0.05.(XLSX)Click here for additional data file.Table S13Δarr I-SceI microarray- Ribosomal protein transcript levels in Δarr I-SceI microarray experiments.(XLSX)Click here for additional data file.


The selection pressure imposed by the parasite has a functional consequence on the immune genes, leading to altered immune function in which regulatory T cells (Tregs) induced by parasites during infectious challenges modulate or thwart T effector cell mechanism.IL2 and its receptor IL2R alpha  in Gabonese individuals exposed to plentiful parasitic infections.We identified and investigated regulatory polymorphisms in the immune gene IL2 and its receptor IL2R alpha gene loci. Also identified were two novel variants, -83 /-84 CT deletions (ss410961576) for IL2 and -409C/T (ss410961577) for IL2R alpha. We further validated all identified promoter variants for their allelic gene expression using transient transfection assays. Three promoter variants of the IL2 locus revealed no significant expression of the reporter gene. The identified novel variant (ss410961577C/T) of the IL2R alpha revealed a significant higher expression of the reporter gene in comparison to the major allele (P<0.05). In addition, the rs12722616C/T variant of the IL2R alpha locus altered the transcription factor binding site TBP (TATA box binding protein) and C/EBP beta (CCAAT/enhancer binding protein beta) that are believed to regulate the Treg function.We identified two reported variants each for The identification and validation of such regulatory polymorphisms in the immune genes may provide a basis for future studies on parasite susceptibility in a population where T cell functions are compromised. Any nucleotide alteration in the promoter region is likely to alter the gene expression, reflecting the level of susceptibility to a parasitic infection as well as Treg expression . PCR primers were designed to amplify the promoter region of the gene using the PRIMER3 Software [http:// www genome.wi.mit.edu/cgi-bin/primer/primer3_http://www.cgi]. The promoter regions of the human IL2 and IL2R alpha (CD25) genes were amplified by polymerase chain reaction (PCR). The primer pairs employed for amplifying promoter regions of the IL2 were IL2F: 5′-TAAATAAGGCCATAGAATGG-3′ and IL2R: 5′- GTTACATTAGCCCACACTTA -3′. The primer pairs employed to amplify promoter regions of IL2R alpha were IL2RF: 5′- GATCCACCCACCTTGGTCTA -3′ and IL2RR: 5′-GGCAGCCAGGCACCATGATGAAC -3′ . In brief: PCR were carried out in a 20 μl reaction volumes with 5ng of genomic DNA, 1x PCR buffer , 0.125mM of dNTPs, 0.5mM of each primer and 1 U Taq DNA polymerase  on a PTC-200 Thermal cycler . Thermal cycling parameters for the amplification of both IL2 and IL2R alpha were as follows: initial denaturation at 94°C for 5min, followed by 40 cycles of 15 sec at 94°C denaturation, 60 sec at 60°C annealing temperature, 60 sec at 72°C extension, followed by a final extension of 10 min at 72°C. PCR products were cleaned up using Exo-SAP-IT  and 1μl of the purified product was directly used as templates for sequencing, using the BigDye terminator v. 2.0 cycle sequencing kit  on an ABI 3130 XL DNA sequencer, according to the manufacturer’s instructions. Polymorphisms in the promoter regions were identified by assembling the sequences, the respective reference sequences being obtained from SNPper database (http://snpper.chip.org) using Codon code Aligner 4.0 software (http://www.codoncode.com/) and were then reconfirmed visually from their respective electropherograms.For purposes of sequencing analysis, gene and genomic sequences of the IL2 and IL2R alpha were amplified with infusion primers (flanks a 15bp homology to the linearised pGL3 vector) and were then cloned to a linearised pGL3 basic vector. In brief, PCR amplifications were carried out in 50 μl reaction volumes under the same program conditions as mentioned above. The amplified PCR products was analyzed by electrophoresis in 1.5% agarose gels, using a 100 bp DNA ladder molecular size marker  and PCR -products were gel eluted and purified using a Nucleospin kit  before being cloned to the pGL3 basic vector using Infusion advantage PCR cloning kit . Plasmids were isolated using QIAprep® Spin Miniprep Kit . To ensure accuracy of the sequenced promoter regions, several independent plasmids containing inserts were sequenced in both directions using appropriate primer pairs. The plasmid exhibiting the confirmed polymorphism was transformed into one shot E. coli . Two independent colonies were selected from these transformations and maxi prep was performed using Endofree plasmid maxi kit .The SNPs identified in the promoter regions had their polymorphism status reconfirmed for their polymorphism status by subsequent cloning procedures. Those genomic DNA sequences identified as having SNPs in the promoter regions of the IL2 and IL2R alpha using Jurkat T cell lines. Basically, four independent transfection experiments for each construct in duplicates were performed with Jurkat T cells . Jurkat T cells (0.8X10 6 cells/μl) were grown in a RPMI 1640  supplemented medium containing 10% FBS, 2mM L-Glutamine and 1% Streptamycin-pencillin substrate . Jurkat cells (0.8X10 6 cells/μl) were transfected with TransIT reagent  as recommended by the manufacturer. In brief: 120μl of TransIT reagent was added to 3ml of RPMI 1640 serum free medium , this being then incubated for 20 min at room temperature. Each of the 24 well plates was then seeded with 0.5ml (0.8X10 6 cells/μl) of Jurkat T cells along with 500ng of plasmid DNA constructs and 20ng of Renilla, before being allowed to incubate for 20 minutes. After incubation, 52μl of TransIT+RPMI serum free medium mix were suspended across each well. The whole procedure was performed in 2X24 well plate formats. After 24 hours, one plate was induced with 20ng/ml Phorbol 12-myristate 13-acetate  as well as 25μg/ml Concanavalin A . After 24 hours cells were harvested by centrifugation, washed twice with phosphate-buffered saline and lysed in 100μl of 1x passive lysis buffer . After incubation for 20 min at room temperature on a rocking platform, 10μl of the lysate was used to measure luciferase activity in the SIRIUS luminometer . We employed the dual luciferase reporter assay system . For each experiment, a plasmid expressing constitutively Renilla luciferase in low amounts was used as a positive control [We tested the activities of the observed polymorphic promoters for both  control ; while ahttp://www.gene-regulation.com/pub/programs/alibaba2) that utilizes TRANSFAC 4.0.An extensive search for transcription factor binding sites for the observed SNPs in the promoter region was performed using a TF-Search online tool (http://www.statview.com). The mean ratio (Luciferase/Renilla) across all measurements was considered for purposes of calculating relative luciferase activity. The luciferase activity of the two different IL2 and IL2R alpha (CD25) promoter variants was comparatively analyzed by a t-test (before and after stimulation). In addition, each constructs activity was compared to the activity of the major allele  in both an induced and a non induced state. The statistically significant level was set as 0.05.Data were normalized and have been analyzed by StatView  and were investigated for described and novel SNPs. For the IL2 promoter region, two predescribed SNPs (rs2069762T/G and rs2067006T/A) were observed. In addition we observed a novel CT deletion at the position −83/-84 in one individual. This novel SNP was submitted to the SNPper database and a corresponding ID was obtained (#ss410961576). The corresponding references SNP ID (#rs); observed allele frequencies from investigated IL2 gene are summarized in Table IL2 promoter variants analyzed by luciferase activities is compared in Figure p-values of the two observed SNP constructs of the IL2 variant remained insignificant in comparison to the major allele before and after stimulation (P > 0.05). When explicitly examined for a transcription factor binding site in the observed variants of IL2, the rs2067006T/A variant was found to be positioned on the transcription site ETS-1, which is a member of the ETS (E-twenty six) family of transcription factors that function as transcriptional activators or repressors in numerous genes believed to be involved in stem cell development, cell senescence, and death. The other two variants (#ss410961576CTdel and rs2069762T/G) do not alter any putative transcription factor binding site.All forty subjects were sequenced for the entire promoter region of the ined #ss40961576. IL2R alpha (CD25), two described variants in the promoter region (rs12722617C/T and rs12722616C/T) were observed. In addition we observed a novel C/T variant at the position −409 in two individuals. This novel SNP was submitted to the SNPper database and a corresponding ID was obtained (#ss410961577). The corresponding references SNP ID (#rs); observed allele frequencies from investigated IL2R alpha (CD25) are summarized in Table IL2 promoter variants analyzed by luciferase activities is compared in Figure rs12722617C/T and rs12722616C/T were observed to be in absolute linkage in all individuals analyzed. The p-values of the SNP construct (rs12722617C/T + rs12722616C/T) of the IL2R alpha variant remained insignificant in comparison to the major allele before and after induction (P >0.05), but remained significant when compared to the new variant in a stimulated state (P<0.0005). The observed novel variant #ss410961577C/T showed significantly increased activity in comparison to the major allele (P<0.05). When looked in explicit for transcription factor binding site in the observed variants of IL2R alpha (CD25) variants, two variants (rs12722617C/T and #ss410961577C/T) were found not to alter any putative transcription factor binding site. However the SNP variant (rs12722616C/T) which was in linkage disequilibrium (LD) with rs12722617C/T is positioned at the transcription factor binding site TBP (TATA box binding protein) and C/EBP beta (CCAAT/enhancer binding protein beta).For the rs2069762T/G and rs2067006T/A) and one novel CT deletion (#ss410961576) at the position −83/-84 when screened for IL2 promoter region. None of the investigated SNP constructs revealed differential luciferase activity compared to the major allele. In the current investigation the IL2 variant (rs2067006T/A) was considered to be the major allele, as all the individuals were found to harbor this particular variant. When compared to the NCBI HapMap database, the frequencies of the observed SNP variants (rs2067006T/A and rs2069762T/G of IL2) were in accordance with Yoruba population of Nigeria which represents a Sub Saharan African group. The rs2067006T/A variant is positioned on to the transcription site ETS-1, a member of the ETS (E-twenty six) family of transcription factors. The ETS-1 DNA-binding domain recognizes the core consensus DNA in target genes and acts either as transcriptional activators or repressors [ETS-1 belongs to a large protein complex which binds to the Treg-specific demethylated region (TSDR) in the FOXP3 locus, thereby restricting the stable FOXP3 expression in the Tregs [FOXP3 is described as a master regulator of natural Tregs development and function [FOXP3 lack any suppressor function [FOXP3 expression leading to a subtle T effector cell function.The underlying idea of the current study is to understand how regulatory SNPs in populations that are naturally exposed to array of parasites contribute to immune outcome. A significant impact on the diversity among immune gene families is believed to be attributable to invasion maneuvers performed by the parasites. Therefore variations in the promoter regions of these immune system genes can potentially amend the gene expression levels either by changing specificity of transcription binding sites or by altering the kinetics of transcription initiation . In our pressors . Recent he Tregs . FOXP3 ietion #ss10961576 function . Since ars12722617C/T and rs12722616C/T) along with one novel C/T variant (#ss410961577) at the position −516 when screened for the IL2R alpha (CD25) promoter region of the IL2R alpha. Both the predescribed SNP variants of the IL2R alpha (rs12722617C/T and rs12722616C/T) were observed in linkage in all studied individuals. We did not observe a differential luciferase activity compared to the major allele for the construct (rs12722617C/T + rs12722616C/T). However the identified novel variant (#ss410961577C/T) revealed significant increased activity in comparison to the constructs with the major allele. This particular novel variant did not alter any putative transcription factor binding site. However the variant rs12722616C/T which was in LD with rs12722617C/T altered the transcription factor binding site TBP (TATA box binding protein) and C/EBP beta (CCAAT/enhancer binding protein beta). The TATA box is a type of promoter sequence that indicates the transcriptional start site whereby a genetic sequence can be read and decoded. It is named after the conserved DNA sequence TATAAA. TBP (TATA binding protein) together with TATA associated factors (TAFS) make the transcription factor TFIID which binds the TATA box in combination with other transcription factors  [TATA box in the construct rs12722616C/T ma contributes to the transcription initiation in vitro in the Jurkat cells. The other transcription factor C/EBP beta (CCAAT/enhancer binding protein beta) is believed to modulate inflammatory processes. A recent study has demonstrated in an experimental autoimmune encephalomyelitis model that C/EBP expression by dendritic cells (DC) influences Th17 versus Treg differentiation but has little or no impact upon Th1 development [IL2R alpha loci were found to be in accordance with the frequencies in the Yoruba population.In our study we also identified two known SNPs  . All theelopment . ExaminiIL2 and its receptor IL2R alpha are described as constituting a potent T cell growth factor and are mainly produced by activated CD4+ T cells, though also by naïve CD8+ T cells, dendritic cells, and thymic cells [+CD25+ regulatory T cells [IL2 are associated with many diseases including auto immune diseases. Recent studies have demonstrated that the studied IL2 promoter variant (rs2069762T/G) is associated with Helicobacter pylori infection [IL2 and IL2R alpha in this study needs to be validated in terms of the specific role these play in different parasitic diseases.Human ic cells . Human I T cells . Also st T cells ,39. Studnfection , multiplnfection , pathogenfection , pathogenfection , measlesnfection , a highenfection  and in anfection . HoweverIn summary, the regulatory SNPs identified in this current study will provide useful information for understanding the relevance of sequence polymorphisms in populations exposed to many parasitic diseases and may serve as a basis for backup studies examining disease susceptibility.The authors declare that they have no conflicting interests.BL designed the field study; VTP and JFJ Kun designed and supervised the experiments; XH and VK performed the experiments; PTS and VTP analyzed the data; BL and VTP contributed materials/analysis tools; XH and VTP wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/13/117/prepub