Histones are subject to a vast array of posttranslational modifications including acetylation, methylation, phosphorylation, and ubiquitylation. The writers of these modifications play important roles in normal development and their mutation or misregulation is linked with both genetic disorders and various cancers. Readers of these marks contain protein domains that allow their recruitment to chromatin. Interestingly, writers often contain domains which can read chromatin marks, allowing the reinforcement of modifications through a positive feedback loop or inhibition of their activity by other modifications. We discuss how such positive reinforcement can result in chromatin states that are robust and can be epigenetically maintained through cell division. We describe the implications of these regulatory systems in relation to modifications including H3K4me3, H3K79me3, and H3K36me3 that are associated with active genes and H3K27me3 and H3K9me3 that have been linked to transcriptional repression. We also review the crosstalk between active and repressive modifications, illustrated by the interplay between the Polycomb and Trithorax histone-modifying proteins, and discuss how this may be important in defining gene expression states during development. In eukaryotes, DNA is packaged in the form of chromatin. The basic unit of chromatin, the nucleosome, is comprised of 147 bp of DNA wrapped around a histone octamer made of two dimers of H2A and H2B and a tetramer of H3 and H4 proteins. The N- and C-terminal histone tails protrude from the nucleosome core and have the potential to interact with adjacent nucleosomes and the linker DNA. All histones can be posttranslationally modified, and the sites of modification are often on the histone tails. These modifications can regulate chromatin structure directly and frequently act as binding sites for the recruitment of other non-histone proteins to chromatin. The most abundant histone modifications are acetylation, phosphorylation, methylation, and ubiquitylation, although many other modifications have been reported . In yeast, H2BK123u1 is established by the ubiquitin ligase RAD6/BRE1 during transcriptional initiation and localizes to the TSS and along the bodies of active genes H2BK123 lies in close proximity to H3K79 on the exposed nucleosome surface, and the H3K79 methyltransferase DOT1 in yeast has been shown to be influenced by deletion and mutation of residues on the H2B tail The crosstalk between H2BK120u1 and H3K4me3 is conserved in mammals, as knockdown of the BRE1 homologs RNF20/40 leads to global reduction in H3K4me3 in vitro transcription through stimulating histone eviction Histone lysine acetylation is a highly abundant mark and is known to regulate many cellular processes including transcription. Acetylation of histones H3 and H4 is highly correlated with gene expression. A unique structural motif, the bromodomain, specifically recognizes acetylated lysines and is present in many proteins involved in transcriptional regulation Dictyostelium discoideum model shows that upon knockout of SET1 and loss of H3K4me3, dynamic H3 acetylation was lost H3K4me3 and H3/H4 acetylation coexist at the promoter and TSS of active genes, and there are many studies that suggest H3K4me3 promotes downstream H3/H4 acetylation through recruitment of HATs  is responsible for the methylation of lysine 27 and contains four core subunits, EZH2/1, SUZ12, EED, and RBAP46/8 In ES cells, H3K27me3 is present at the promoters of several thousand genes, including the Hox gene clusters, where it is associated with heritable gene silencing The Polycomb repressive complex 1 (PRC1) is an E3 ubiquitin ligase complex that can modify chromatin by monoubiquitylation of H2A on lysine 119. All PRC1 complexes contain the catalytic RING1A/B subunit, and one of six PCGF proteins in vitro, this PRC2.2 complex is more active on an H2AK119u1 nucleosome substrate compared with unmodified nucleosomes Both PRC1 and PRC2, along with their associated chromatin modifications, H2AK119u1 and H3K27me3, have been shown to co-localize at many regions of the genome, such as the promoters of developmentally regulated genes and the inactive X chromosome In summary, the establishment of Polycomb repressive domains may require these enzymes to read not only their own mark, for example, EED-binding H3K27me3 or RYBP-binding H2AK119u1, but also the marks placed by their partner complex. In this way, H3K27me3 can establish or reinforce H2AK119u1 modifications, and H2AK119u1 can establish or reinforce H3K27me3 deposition Fig. Which mGenerally, methylation of H3K9 is associated with constitutive heterochromatin formation and transcriptional silencing. Recently, there has been some evidence that H3K9 methylation can crosstalk with the Polycomb H3K27me3 modification to cooperate in gene repression or as mutually exclusive pathways present at constitutive heterochromatin.de novo DNA methyltransferases (DNMT3A/B) The heterodimeric complex of G9a and GLP catalyzes H3K9me1 and H3K9me2 modifications Several reports have demonstrated that H3K9me3 and H3K27me3 modifications are mutually exclusive In vitro there is no difference in PRC2 activity on nucleosomes that contain H3K9 methylation compared to WT nucleosomes Despite reports that H3K27me3 and H3K9me3 are mutually exclusive, a number of ChIP-sequencing studies in ES cells The mutually exclusive distribution of H3K27me3 and H3K9me3 described above has predominantly been shown in studies of constitutive heterochromatin loci, and in all cases, this clear separation of domains has been seen for trimethylation marks. There are also many examples in which the H3K9me2 modification, and in some cases H3K9me3, can act in concert with H3K27me3, suggesting positive crosstalk between these two mechanisms of heterochromatin formation. In the best-documented examples, this positive crosstalk appears to be between H3K27me3 and H3K9me2. Crosstalk may also vary in different cell types or different differentiation states, in which chromatin structures or the balance of different enzymes may be altered. What is clear is that at least in some circumstances through, for example, CDYL, HP1, or DNA methylation, both the Polycomb system and the H3K9 methylation systems are able to read the chromatin state placed by each other and to write their own modifications accordingly.cis-regulatory elements and the binding of insulator proteins such as CTCF can also determine boundaries. For instance, H3K27me3 domain boundaries within the Hox gene cluster are bound by CTCF; deletion of these CTCF-binding sites results in the infringement of Pol II and H3K4me3 into adjacent heterochromatin territories and disruption of Hox gene silencing As discussed above, both H3K27me3 and H3K9me3 repressive marks can exist as extensive domains within cells. These domains can spread through positive feedback of the writers and their ability to recognize and propagate their marks. Given this, the establishment of boundaries is critical to isolate heterochromatin from euchromatin domains. These boundary elements have been found in multiple eukaryotic organisms, ranging from yeast to human  as the target H3K27. It has been suggested that the allosteric inhibition of the catalytic subunit EZH2 occurs through the recognition of the H3K4me2/3 and H3K36me2/3 marks by the SUZ12 VEFS domain Schmitges cis by H3K4me2/3 and H3K36me3, PRC2 is active on nucleosomes harboring these modifications on only one of the two H3 tails, thereby allowing the formation of asymmetrically modified nucleosomes in vivo and may represent the nucleosomes present at bivalent promoters in mESCs .It is important to note that although PRC2 is inhibited in C. elegans. Normally H3K36me3 and H3K27me3 occupy mutually exclusive domains on the autosomes This negative feedback mechanism has also been shown to operate at a chromosomal level in et alet al shows that upon RING1A/B knockout and subsequent depletion of H2AK119u1, H3K4me3 levels at several PcG target genes increase As well as the inhibition of Polycomb activity by Trithorax marks, Polycomb marks have also been shown to inhibit the activity of some Trithorax proteins. The best-studied examples are the inhibition of H3K36 methyltransferases by the PRC1 modification H2AK119u1. Yuan in vivo by ChIP-sequencing These studies provide good evidence that PcG and TrxG marks mutually inhibit the writers associated with the opposing group. It was therefore unexpected when several groups showed that the tudor domains of the PRC2-associated PCL1/2/3 proteins can specifically bind H3K36me3 H3K27me3 is associated with gene repression, while H3K27ac is associated with gene activation and active enhancers. Since they act on the same lysine residue, these marks are mutually exclusive, and the switch between methylation and acetylation has been well established. The removal of H3K27ac by the NURD complex facilitates the recruitment of PRC2 and accumulation of H3K27me3 at promoters leading to gene repression Upon loss of H3K9 or DNA methylation, PRC2 accumulates at the pericentric heterochromatin as discussed earlier. In this condition, BEND3 recruits NURD to the pericentric heterochromatin Activation of Polycomb-repressed genes requires a methylation to acetylation switch at H3K27. The phosphorylation of H3S28 on the residue neighboring H3K27 has been shown to mediate this switch. H3S28p inhibits H3K27me3, allowing an accumulation of H3K27 acetylation It is clear that specific histone modifications are associated with the transcriptional state. For many modifications, it is not well established whether they directly influence transcription or their placement is simply a consequence of the transcriptional state present at a particular gene. There are reports that acetylation can directly alter chromatin structure to a more accessible state allowing the recruitment of transcription factors and the transcription machinery. In addition, ubiquitylation of H2A has been shown to inhibit the elongating form of Pol II, suggesting direct effects on the transcriptional state. Conversely, there is substantial evidence to support the idea that transcription factors determine and initiate gene expression, and writers recognize this state and aid in the maintenance of this state through multiple feedback mechanisms. One such mechanism is the recruitment of the H3K4 HMT SETD1 and the H3K36 HMT SETD2 by Pol II itself to deposit histone modifications across the promoter and gene body. The emerging idea that PcG and TrxG can sample CpG islands genome wide and establish domains of repression or activation depending on the transcriptional state at the target promoter also supports the idea that the transcriptional state defines the chromatin modification landscape. This mechanism requires extensive positive and negative crosstalk between these modifications that we have discussed.Many writers of chromatin modifications are positively regulated by the marks that they place, as well as other marks associated with the same transcriptional state, contributing to the reinforcement of gene expression or silencing. This mechanism could also account for the spreading of marks such as the repressive modifications H3K9me3 and H3K27me3 over large domains in differentiated cells. These reinforcing mechanisms may also play a role in cellular memory by faithful propagation of the histone modifications that allow gene expression profiles to be maintained epigenetically through cell division. There is evidence to suggest that some writers remain associated with chromatin during DNA replication, but the exact molecular mechanisms of this epigenetic memory have yet to be fully elucidated.Negative histone crosstalk plays an equally important role in dictating distinct chromatin environments. Writers, especially in the case of PcG and TrxG proteins, are often negatively regulated by marks associated with the opposing transcriptional state. As we have seen in the case of H3K27 methylation and acetylation, negative crosstalk is also involved in the switching of gene expression states. It is known that in certain circumstances, marks associated with positive and negative transcription can coexist. In ESCs, bivalent promoters contain both H3K27me3 and H3K4me3, albeit on different histone tails of the same nucleosome, and may represent a chromatin profile amenable to switching between transcriptional states. Genes coding for master transcription factors often have bivalent promoters in ESCs, and their expression is dynamically regulated through development.The ability of writers to read and place histone modifications is important for the maintenance and regulation of specific transcriptional states throughout development. This is evident by the fact that mice deficient in chromatin-modifying enzymes display severe developmental defects. Several of these enzymes have been known to be involved in various genetic disorders such as Sotos syndrome and Wolf–Hirschhorn syndrome related to translocation of several members of the NSD family of H3K36 methyltransferases In conclusion, the ability of chromatin writers to read preexisting histone modifications contributes in two major ways. First, it allows the maintenance of distinct and robust transcriptional states, which could potentially be propagated through cell division, and therefore act as epigenetically inherited features. Second, crosstalk between modifications and enzymes of opposing transcriptional states can allow the establishment of bistable switches that allow the dynamic regulation of gene expression states. In the future, we hope to understand the extent to which histone crosstalk plays a role in defining the epigenetic landscape of a cell , and addHow do chromatin modifications regulate gene transcription? Although there is a clear correlation between chromatin modifications and gene expression states, it will be important to establish the role that modifications play in regulating transcription and indeed whether they are a cause or an effect of transcription.What is the role of highly prevalent chromatin modifications such as H3K36me2 or H3K27me2 that mark up to 50% of H3 in mESCs and differentiated tissues What is the role of histone variants? H3.3 and H2A.Z are enriched over active genes and may have more specialized regulatory roles compared to their more abundant canonical counterparts How are epigenetic profiles established within a cell? What are the relative contributions of direct targeting of histone-modifying activities and crosstalk between histone modifications or transcriptional state? For example, are chromatin writers able to sample and read the preexisting chromatin state to determine their activity or binding profiles, or are they directly recruited to their sites of action by sequence-specific DNA-binding factors?To what extent are epigenetic modifications maintained through cell division, and do self-reinforcing feedback loops provide a model for the mechanism of such inheritance? Unlike writers and readers, the genomic location of histone modifications can be easily transmitted through both mitosis and meiosis because they are an integral part of the packaging of DNA. However, self-reinforcing loops might become essential after replication to overcome the dilution of old, modified nucleosomes with new nucleosomes and maintain an epigenetic code.in vitro binding and enzymatic assays, and importantly determining the changes that occur when the system has been perturbed.Do the reciprocal feedback loops between positively and negatively acting histone marks provide the basis for a bistable switch, in which each state is positively reinforced and stable once the initial decision has been made? In order to properly generate and validate such mathematical models, it will be critical to obtain quantitative data on the kinetics and dynamics of the catalytic and binding processes that are involved. This will involve experiments such as FRAP and


Trypanosoma brucei is a protozoan parasite that lacks many transcription factors found in other eukaryotes, such as those whose binding demarcates enhancers. T. brucei retains histone variants and modifications, however, and it is hypothesized that it relies on epigenetic marks to define transcription-related boundaries. The histone H3 variant (H3.V) and an alternate nucleotide, base J (ß-D-glucosyl-hydroxymethyluracil), are two chromatin marks found at both transcription termination sites (TTSs) and telomeres. Here, we report that the absence of both base J and H3.V result in transcription readthrough and the appearance of antisense transcripts near TTSs. Additionally, we find that maintaining the transcriptional silencing of pol I-transcribed telomeric Variant Surface Glycoprotein (VSG) genes appears to be dependent on deposition of H3.V alone. Our study reveals that gene expression depends on different epigenetic cues depending on chromosomal location and on the transcribing polymerase. This work provides insight into how these signals may have evolved into the more nuanced and fine-tuned gene regulatory mechanisms observed in other model systems. In eukaryotes, DNA is wrapped around histones to form chromatin. Modifications on the DNA itself, or on the canonical histones around which the DNA is wrapped, can lead to changes in gene expression. Alternate histones are also used to regulate gene expression. The African trypanosome, the causative agent of African sleeping sickness, transcribes its genes in long arrays called polycistronic transcription units (PTUs). In this study, we describe how the African Trypanosome uses two chromatin marks to regulate genes that lie close to the end of the PTU or close to the ends of chromosomes, called telomeres. One mark, base J, is on the DNA itself, while the other utilized mark is an alternate histone protein called H3.V. In the absence of these marks, there is an increase in antisense RNA that corresponds to genes that lie near the ends of the PTUs, and an increase in sense RNA for genes near telomeres. Since trypanosomes are evolutionarily distant from humans, these findings shed light on how gene expression mechanisms that are modulated by chromatin marks may have evolved to produce the complex gene regulatory networks found in our own tissues. Trypanosoma brucei, a eukaryotic parasite that causes African sleeping sickness, belongs to the order Kinetoplastida and diverged from mammals ~500 million years ago. T. brucei is heteroxenous, requiring two obligatory hosts to complete its life cycle: a mammalian host and an insect host . Trypanosomes proliferate in the tsetse gut as procyclic forms (PF) [T. brucei infects a mammalian host, it differentiates into the bloodstream form (BF) and proliferates in the host’s extracellular spaces. The cycle is completed when BF T. brucei reaches a quiescent state and gets ingested by a tsetse.rms (PF)  and diffT. brucei branched early in eukaryotic evolution, and differs substantially from other eukaryotes in its regulation of gene expression. It is thus of great evolutionary interest. One prominent difference concerns transcription of protein-coding genes by RNA polymerase II (pol II), which diverges in two important ways from other well-studied eukaryotes. First, transcription occurs polycistronically, and most genes are arranged in polycistronic transcription units (PTUs). Mature mRNAs are then generated post-transcriptionally by coupled trans-splicing and polyadenylation reactions that trans-splice a 39-nucleotide leader sequence onto every mRNA [T. brucei [cis-regulatory sequences, and cis-regulatory sequences themselves have not been well characterized. The lack of these regulatory factors, and the fact that histone modifications and histone variants are deposited at sites of putative transcription initiation and termination, has led to the idea that chromatin marks are important for demarcating PTUs [3, H4K10ac, and H2 variants H2.AZ and H2.BV at transcription start sites (TSSs)) or transcription termination sites (H3.V and H4.V at transcription termination sites (TTSs)) [ery mRNA . Second,. brucei , but theing PTUs . Prior e (TTSs)) ,7. HowevLeishmania results in readthrough transcription at sites of transcription termination [T. brucei does not cause readthrough at convergent strand switch regions, though it does affect termination at specific sites proximal to J deposition [T. brucei. Based on these results, we hypothesized that while base J may be the dominant mark for transcription termination in L. major, H3.V may compensate for the lack of base J in T. brucei, and its role in the epigenetic network of T. brucei may differ from that of L. major. We surmised that the effects of base J and H3.V in T. brucei might best be assessed by removing them simultaneously, and sought to ask whether perturbation of chromatin marks at sites of transcription termination would have any functional consequence on gene expression.In addition to these distinct chromatin features, pol II transcription initiation and termination regions contain a kinetoplastid-specific DNA modification, known as base J (ß-D-glucosyl-hydroxymethyluracil) . It arismination ,14, whilmination . In contposition . DeletioT. brucei. Variant Surface Glycoprotein (VSG) genes, encoding the major T. brucei surface antigen, are frequently found near telomeres. One genomic location for telomere-proximal VSGs is the Bloodstream-form Expression Site (BES), which contains a polycistronic transcription unit that is transcribed by RNA pol I. Fifteen BESs have been found in the Lister 427 strain [VSG. Another class of telomere-proximal VSGs is found in metacyclic expression sites (mVSGs). These sites are also transcribed by pol I and mVSG is expressed during the metacyclic stage in the tsetse salivary gland. Minichromosomes have also been found to contain VSG genes, but these sites lack obvious pol I promoter sequences. A large number of other VSG genes are scattered throughout the genome, but not all of their genomic locations are known [VSG expression units in BF trypanosomes, taking advantage of the telomeric location of VSG genes to interrogate telomeric silencing in general.In addition to TTSs, J and H3.V (but not H4.V) marks coincide over telomeric regions in 7 strain , and eacre known . In BF cVSG genes. Thus our experiments suggest that perturbations of the chromatin landscape in different regions of the genome (transcription termination sites vs. telomeres) have functional effects on gene expression, and that the dependence on a particular chromatin mark differs depending on chromosomal location and/or specific features of the transcribing polymerase (RNA pol II vs. pol I).Using high-throughput, strand-specific sequencing of poly-A+ mRNA (RNA-seq) on genetic mutants that lack H3.V, base J, or both marks, we demonstrate that simultaneous deletion of base J and H3.V increases the amount of antisense transcripts for genes proximal to transcription termination sites, where both marks are found. We also demonstrate that of the two marks, H3.V appears to be more dominant than base J with respect to maintaining silencing of telomeric T. brucei using previously published methods [JBP1, then both alleles of JBP2. In both cases, we used deletion-cassettes containing hygromycin- or puromycin-resistance genes fused to Herpes simplex virus thymidine kinase (HYG-TK or PUR-TK) and flanked by loxP sites, allowing the markers to be removed by transient expression of Cre-recombinase and reused. JBP1Δ/Δ JBP2Δ/Δ double mutants, which we will henceforth refer to as JΔ mutants, are completely null for base J [H3.V either from a WT or JΔ background using the deletion cassette H3.VΔPUR (pJEL76) and H3.VΔHYG (pJEL38) [To study the roles of base J and H3.V, we generated knockout cell lines in BF  methods . To maker base J . To gene(pJEL38) .JΔ H3.VΔ trypanosomes were viable. Growth curves revealed that there was no obvious defect in growth for any of the mutants , we asked whether base J and H3.V are important for maintaining silencing of inactive telomeric VSGs by assessing expression levels of these telomeric VSGs in cells lacking one or both chromatin marks. We also assessed expression levels for minichromosomal VSGs (MC VSGs), which are assumed to be proximal to telomeres. We isolated polyA+ RNA from each of the mutants described above, as well as from the parental cell line (referred to as WT). Libraries for high-throughput sequencing were generated using 3 independent cultures from each genotype . Reads from each library were uniquely aligned allowing for two mismatches to the megabase chromosomes. Unmapped reads were then uniquely aligned allowing for two mismatches to the VSGnome [2(RPKM) (Reads Per Kilobase Per Million mapped reads) value was generated for reads aligning to each VSG and these values were averaged across the 3 replicates. We then compared WT and mutant log2(RPKM) values for VSGs with known subtelomeric locations in Lister427 cells  as well as those known to be associated with metacyclic promoters [2(RPKM) values of BES, metacyclic, and minichromosomal VSGs to elucidate the overall distribution of the data  of BES-associated VSGs, and 4 of the 5 metacyclic VSGs were >4-fold upregulated (2(RPKM) values for BES and metacyclic VSGs were higher in the H3.VΔ mutant and the distribution of log2(RPKM) values was shifted upward (JΔ H3.VΔ mutant (VSGs >4-fold upregulated in these cells (2(RPKM) values were also higher in the JΔ H3.VΔ mutant  and detected its presence by western blot analysis  were upregulated in the absence of both base J and H3.V. Of 56 MC VSGs, 13 minichromosomal VSGs showed >4-fold upregulation in the JΔ H3.VΔ mutant (VSGs were >4-fold upregulated in the JΔ and H3.VΔ strains respectively (2(RPKM) values in the JΔ H3.VΔ mutant  values increased slightly in the absence of base J or H3.V, with the greatest difference found in the JΔ H3.VΔ mutant   . All VSGVSGs at known chromosomal locations described above, these results highlight a role for both base J and H3.V in maintaining a chromatin structure that promotes silencing of a subset of VSG genes. The additive effect of removing base J and H3.V on transcription of the set of VSGs with uncharacterized locations suggests that collaborative reading of these chromatin marks may be more common in regions further from the telomeres. The effect observed upon removal of both marks implies that the factors responsible for reading these marks and maintaining silencing may operate in different pathways that have some functional redundancy [VSG genes appears to differ depending on chromosomal context.Combined with the analysis of dundancy . OverallVSG levels could indicate either derepression of silent VSGs or an increase in VSG switching rates, possibly due to effects on telomere length [VSG switching rate was not significantly elevated in H3.VΔ or JΔ mutant cells, and was only slightly elevated in JΔ H3.VΔ cells . Sites where both PTUs terminate coming from opposite directions are putative sites of transcription termination, termed convergent strand switch regions (cSSRs). Sites where one PTU ends and another starts are referred to as head-to-tail or HT . Previou. brucei , but did. brucei , and it 2(RPKM) values were generated for each of these regions and the three values for each replicate within one genotype were averaged. Polyadenylation sites have previously been shown to vary quite widely both within a gene and across different genes, and can extend up to 6,000 nucleotides past the end of the coding region of the gene, with the median length estimated at ~400 bp [JΔ H3.VΔ mutant (2(RPKM) levels and inner quartile ranges for these regions were also increased in each of the mutants, and this increase was significant in JΔ H3.VΔ cells according to a Mann-Whitney U test (2(RPKM) values in the RNA-seq dataset and are used here as negative controls).Stranded RNA-seq libraries were prepared from 3 independent cultures from each genotype, and sequencing reads were aligned uniquely to the megabase chromosomes allowing for 2 mismatches. We investigated polyA+ transcript levels extending past the coding regions of the genes that flank cSSRs. We obtained polyA+ reads that mapped to regions between the coding regions of genes that flank convergent PTUs , which w ~400 bp . OverallΔ mutant . Median y U test . We were2(RPKM) values in the JΔ H3.VΔ mutant, while 42% and 7% of these sites showed significant changes in log2(RPKM) values in the JΔ and H3.VΔ mutants, respectively  and at head-to-tail sites, and these regions are also bound by Bdf3 . We definscripts . Taken tJΔ H3.VΔ mutant cells is found immediately following the end of the last 3´ UTR for the last gene at the end of the PTU  values between the WT and mutant cells for all the genes within each window (2(RPKM) value for genes within each window is very subtle, but is greater at closer distances to the cSSR  values for all reads generated from both sense and antisense transcripts, and separately calculated log2(RPKM) values specifically from reads generated from sense strand transcripts or from antisense transcripts. A list of genes that are significantly up- or down- regulated by >4-fold, as measured by a T-test followed by Benjamimi and Hochberg correction in the mutants with associated p adjusted values is provided in 2(RPKM) values for genes that fall within 1,000 bp of a cSSR (2(RPKM) values for each genotype revealed an increase in the median log2(RPKM) for genes falling within 1,000 bp of the cSSR in each of the mutant lines for both sense and antisense reads, with the most pronounced increase in median log2(RPKM) found in the JΔ H3.VΔ mutant (JΔ mutant and the JΔ H3.VΔ mutant (2(RPKM) in each of the mutant lines, none of which were significant (2(RPKM) value and a shift upward in the interquartile range for each of the mutant cell lines. The largest effect was once again seen in the JΔ H3.VΔ mutant cells  was again unaltered between the WT and the mutant lines for reads generated from sense transcripts , while both marks are important for proper transcription termination at the ends of PTUs. In the absence of these marks, the number of antisense transcripts following the end of a PTU is increased in the neighboring PTU. Previous work on the role for base J in pol II transcription termination was done using small RNA-seq on cells treated with dimethyloxalylglycine (DMOG) [JBP1Δ JPB2Δ mutants using total RNA-seq. Our experiments on readthrough transcription were performed slightly differently, using polyA+ enriched libraries for genetic mutants. In contrast to previous results, we did find evidence for transcriptional readthrough at cSSR regions in JΔ mutant cells, which was exacerbated in the absence of H3.V  may have led to differences in the results from these two analyses. The total of number of genes where sense transcript abundance changes by > 2-fold in the JΔ is relatively low , most of these changes represent an increase in expression in the mutant, and many of the genes affected are VSGs, ESAGs, and RHS hotspots. This is in agreement with previous results using total RNA-seq on JΔ genetic mutants [JΔ H3.VΔ mutant is ~3 times higher than that of J alone, and ~13 times higher than H3.VΔ alone. However, these changes may be the result of secondary effects, since we did not find a specific pattern for changes in sense transcription immediately proximal to cSSRs.Here, we have investigated the effects of base J and H3.V in maintaining the transcriptional landscape surrounding their sites of deposition, which include cSSRs and telomeres. In general, we find that H3.V appears to be required for the maintenance of silencing of telomeric genes (e (DMOG) , and thu3.V Figs  and 5. I mutants . The numT. brucei and L. major possibly due to a third chromatin mark that remains in BF JΔ H3.VΔ cells  to demarcate the ends of PTUs. At subtelomeres however, H3.V appears to be the dominant mark for suppressing transcription of inactive VSG genes by an unknown mechanism. Functional effects for the loss of base J and H3.V during infection remain to be studied in T. brucei, but studies in T. cruzi indicate that loss of JBP1 and JBP2 has functional effects on host cell invasion and egress [T. brucei biology. For example, JBP1/2 are members of a larger family of α-ketoglutarate and Fe2+-dependent dioxygenases whose mammalian homologs are the ten-eleven translocation (TET) proteins . TET priewed in . A numbeiewed in ,30,31). me3 mark ). In ligTrypanosoma brucei bloodstream forms (strain Lister 427) of the ‘single marker’ (SM) background expressing T7 RNA polymerase and Tet repressor (TetR) [r (TetR)  were culr (TetR) . Stable Equal numbers of cells were collected and suspended in Laemmli buffer and separated on an SDS-PAGE gel. Following transfer to nitrocellulose, the proteins were analyzed using anti-VSG3 or anti-tubulin antibody.BSD) was placed at the active BES promoter. Cells were then allowed to switch in the absence of selection for 3–4 days to ensure the same number of population doublings. Switchers were enriched using a MACS-column method. Flow-through enriched with switchers was collected and stained with anti-VSG2 antibody and propidium iodide (PI) for dead cell exclusion and analyzed by flow cytometry. Cells that lost VSG2 signal and are PI negative are live cells that have switched. Switching mechanism was assessed as described previously [Switching assays were performed as described previously ,36. Briehttp://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and aligned with Bowtie [VSGnome (http://129.85.245.250/index.html) using the same parameters. Reads were quantified using SeqMonk (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk) from Babraham Bioinformatics or, in the case of VSG aligned reads, custom python scripts.RNA was extracted from treated or control cells using RNA Stat-60 (Tel-Test) according to the manufacturer’s protocol and quantified on a NanoDrop2000c and further cleaned using RNase easy kit (Qiagen). Poly-A sequencing libraries were prepared by Rockefeller University Genomics Resources Center using the Illumina platform. Stranded sequencing libraries were prepared using the NEBNext Ultra Directional Library Kit (E7420). Sequencing was performed on an Illumina HiSeq 2000 sequencer using 50 bp reads. Reads were trimmed for quality using the TrimGalore program from Babraham Bioinformatics (h Bowtie  to the r2(RPKM) values for each gene were included in the analysis if they fell within a defined length of the cSSR. This length was defined as within 1,000 bp, 5,000 bp, or 10,000 bp upstream of the 3´ end of the last gene in the (+) strand PTU or within 1,000 bp, 5,000 bp or 10,000 bp downstream of the 5´ end of the first gene in the (-) strand PTU (see the diagrams in 2(RPKM) value between mutant and WT cells were computed for genes that fell within 5 kb windows proceeding upstream or downstream of the halfway point within the cSSR, defined as between the 3´ end of the last gene in the (+) strand PTU and the 5´ end of the first gene in the (-) strand PTU. For 2(RPKM) values were computed for the region that lies between the 3´ end of the last gene in the (+) strand PTU and the 5´ end of the first gene in the (-) strand PTU for each analyzed cSSR. For 2(RPKM) values between mutant and WT cells were computed for windows in each cSSR and cSSR-flanking region. Median log2(RPKM) differences were then averaged for each window across all cSSR and cSSR-flanking regions. Notched boxplots were computed using python’s matplotlib library with the following command pylab.boxplot.For the plots shown in VSG expression (2(RPKM) counts in WT and mutant cells. Once significance was established across groups, a Mann-Whitney U test (command wilcox.test) was performed between median log2(RPKM) counts for each mutant genotype and the WT. Significance values in Figs P < 0.05 was considered significant for all tests. For all graphs, P < 0.05 is indicated by * and P < 0.01 is indicated by **. Test statistics and P values are reported in Differences between WT and mutant cells were summarized using descriptive statistics performed in R, version 3.2.0 . To compare pression , expresspression , expresspression , and exppression  a KruskaTotal mRNA was prepared using RNA Stat-60 (Tel-Test) as described in manufacturer’s protocol and cDNAs were generated using random hexamer and reverse-transcriptase (Stratagene). RNA was amplified using primers specific to individual selected TTS regions by quantitative PCR using the LightCycler 480 (Roche). Amplified double-stranded DNA product during 40 cycles was detected by SYBR Green I. All measurements were in triplicate and compared with a 4,000-fold range of serially diluted standard genomic DNA prepared from the wild-type strain. The sequences of primers are available upon request.Stranded RT-PCR was performed as described above with the following differences. Gene specific primers were used to generate cDNAs from antisense or sense transcripts. These cDNAs were amplified using two sets of primers specific to the 5´ or 3´ region of each gene by quantitative PCR using the LightCycler 480 (Roche). Amplified double-stranded DNA products during 40 cycles were detected by SYBR Green I. All measurements were in quadruplicate and compared with a 16,000-fold range of serially diluted standard genomic DNA prepared from the WT strain. The sequences of primers are available upon request.JΔ, H3.VΔ, and JΔ H3.VΔ cells were transfected with pSY37, which targets the HYG selection marker right downstream of the active VSG2 and leaves about 200 bp telomere seed at the end of the chromosome. Over time, telomere seeds should elongate and elongation rate of telomeres was measured by Southern blot. About 200 million cells were collected and genomic DNA was prepared using QIAmp kit from Qiagen. Genomic DNA was digested with AflII to measure the lengthening of BES-VSG2-telomere or with BglI to measure the length of silent BES-VSG3 telomere, and separated on an agarose gel. Specific VSG2 or VSG3 probes were used for detection.To examine telomere growth, WT, S1 FigAsterisk indicates non-specific band.(EPS)Click here for additional data file.S2 Fig2(RPKM) values for all VSG genes for WT and indicated mutant cells. Dotted grey lines indicate changes that are 4-fold up or down. Comparisons between log2(RPKM) values WT and JΔ cells are show in (A), WT and H3.VΔ cells in (B), and WT and JΔ H3.VΔ cells in (C). (D). Notched boxplots of log2(RPKM) values were generated for all VSGs in WT and indicated mutant cells. Boxplots are displayed as in Scatter plot showing log(EPS)Click here for additional data file.S3 FigVSG2 locus. Integration of the plasmid was confirmed by PCR and telomere length was analyzed by Southern blot using VSG2-specific probe. (D) and (E) Telomeres at the silent BES were maintained stably in all strains. Diagram shows the silent VSG3 BES and Southern blot probes. The probe also recognizes the 17.8 kb fragment (marked with asterisk).(A) Observed switching frequency in WT and indicated mutant cells. (B) and (C) Telomeres grow at a normal speed in all mutants. Diagram shows the targeting strategy. The plasmid pSY37 swaps the original telomere with the 200 bp telomere seed at the active (EPS)Click here for additional data file.S4 FigHT, ‘head-to-tail’ site.(EPS)Click here for additional data file.S5 Fig2(RPKM) values were generated from stranded RNA-seq reads at HT sites in WT and indicated mutant cells. HT sites were defined as regions bound by Bdf3 that were not dSSRs. Boxplots are displayed as in Notched boxplots of log(EPS)Click here for additional data file.S6 Fig2(RPKM) values are calculated in WT or mutant cells for each gene that falls within 1,000 bp of a cSSR. In this example, 2 genes upstream of this cSSR and one gene downstream of this cSSR (indicated by solid black frames around the gene) would be included in the analysis. Bottom left, boxplot generated from log2(RPKM) values of genes that flank cSSRs, defined as within 1,000 bp upstream of the 3´ end of the last gene in the (+) strand PTU or within 1,000 bp downstream of the 5´ end of the first gene in the (-) strand PTU in WT and mutant cells as indicated. Boxplots are displayed as in (A) Top, schematic of computed values. log(EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableP values < 0.05 indicated by * and P values < 0.01 indicated by **.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableP values < 0.05 indicated by * and P values < 0.01 indicated by **.(XLSX)Click here for additional data file.S5 TableP values < 0.05 indicated by * and P values < 0.01 indicated by **.(XLSX)Click here for additional data file.S6 TableOnly genes with fold-change >4 between mutant and WT cells are listed. Distance from the start of the gene to the nearest cSSR and to each adjacent head-to-tail site is also provided.(XLSX)Click here for additional data file.S7 TableP values < 0.05 indicated by * and P values < 0.01 indicated by **.(XLSX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.


I110 (TBP-associated factor 110) at a single threonine residue (T852). Upon exit from mitosis, T852 is dephosphorylated by Cdc14B, which is sequestered in nucleoli during interphase and is activated upon release from nucleoli at prometaphase. Mitotic repression of Pol I transcription correlates with transient nucleolar enrichment of the NAD+-dependent deacetylase SIRT1, which deacetylates another subunit of SL1, TAFI68. Hypoacetylation of TAFI68 destabilizes SL1 binding to the rDNA promoter, thereby impairing transcription complex assembly. Inhibition of SIRT1 activity alleviates mitotic repression of Pol I transcription if phosphorylation of TAFI110 is prevented. The results demonstrate that reversible phosphorylation of TAFI110 and acetylation of TAFI68 are key modifications that regulate SL1 activity and mediate fluctuations of pre-rRNA synthesis during cell cycle progression.Mitotic repression of rRNA synthesis requires inactivation of the RNA polymerase I (Pol I)-specific transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of TAF I110 subunit, which impairs the interaction with UBF and the assembly of pre-initiation complexes. Here we show that hCdc14B, the phosphatase that regulates Cdk1/cyclin B activity and progression through mitosis, promotes reactivation of rDNA transcription by dephosphorylating TAFI110. In addition, the NAD+-dependent deacetylase SIRT1 becomes transiently enriched in nucleoli at the onset of mitosis. SIRT1 deacetylates TAFI68, another subunit of SL1, hypoacetylation of TAFI68 destabilizing SL1 binding to the rDNA promoter and impairing transcription complex assembly. The results reveal that modulation of SL1 activity by reversible acetylation of TAFI68 and phosphorylation of TAFI110 are key modifications that mediate oscillation of rDNA transcription during cell cycle progression.In metazoans, transcription is arrested during mitosis. Previous studies have established that mitotic repression of cellular transcription is mediated by Cdk1/cyclin B-dependent phosphorylation of basal transcription factors that nucleate transcription complex formation. Repression of rDNA transcription at the onset of mitosis is brought about by inactivation of the TBP-containing transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of the TAF Posttranslational modification of transcription factors is critical for cell cycle progression in a unidirectional and reversible manner. Cell cycle-dependent oscillation of transcriptional activity is governed by a complex network of regulatory proteins and signaling pathways that respond to various intra- and extracellular stimuli by influencing the activity and tertiary structure of proteins, controlling subcellular distribution, and regulating interactions with other proteins. Global repression of gene expression starts at prophase and is accompanied by release of most transcriptional regulators from mitotic chromatin –3, 4. MiIs), TAFI110, TAFI68, TAFI48, TAFI41, and TAFI12 , 5 mM MgCl2, 0.2 mM EDTA, 0.5% Triton X-100 and protease inhibitors . After capture on GFP-trap beads , associated hTAFI68 was monitored on Western blots. To examine the association of TAFI110 with Cdc14B, Flag-tagged hTAFI110 co-expressed with GFP-hCdc14B in HEK293T cells was bound to M2-agarose in the same buffer containing 120 mM KCl, and co-purified GFP-hCdc14B was visualized on immunoblots. Co-immunoprecipitation of Flag-tagged hTAFI110 with endogenous TAFI68 and TBP was performed in the presence of 250 mM KCl.2 μg of GST or GST-tagged proteins immobilized on Glutathione-Sepharose 4B were incubated with  control . To moni2 comprising human rDNA sequences from -410 to +378 (with respect to the transcription start site) and 40 μg of extract from HeLa cells [In vitro transcription reactions contained 40 ng of linearized plasmid pHrPLa cells . DepletiLa cells . SL1 was2, 1% Triton X-100), high salt buffer containing 500 mM NaCl, followed by LiCl buffer  and TE buffer. After reversal of the crosslink and proteinase K digestion, DNA was purified and analyzed by qPCR (Roche LightCycler480). Precipitated DNA was calculated as the percentage of DNA in the immunoprecipitates compared to input DNA. The primers used for PCR are listed in Cells were fixed with 1% formaldehyde , quenched with 0.125 M glycine, and sonicated to yield 250–500 bp DNA fragments. After 5-fold dilution with IP dilution buffer  and preclearing with protein A/G Sepharose, lysates were incubated overnight with the respective antibodies, and protein-DNA complexes were captured on protein A/G Sepharose followed by washes in low salt buffer  in 50 mM ammonium bicarbonate and lyophilisation, the peptides were resolved on cellulose thin-layer plates by electrophoresis for 25 min at 1000V in 1% (w/v) ammonium carbonate (pH 8.9), followed by ascending chromatography in a buffer containing 62.5% isobutyric acid, 1.9% n-butanol, 4.8% pyridine, and 2.9% acetic acid [Recombinant TAFtic acid , 41. Photic acid .S1 FigI110 was phosphorylated in vitro with 32P and immunopurified Cdk1/cyclin B in the absence or presence of the Cdk1/cyclin B inhibitor roscovitine, and subjected to acid hydrolysis and phosphoamino acid analysis. The letters at the right indicate the position of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y). (B) Sequence of the synthetic tryptic phosphopeptide (marked in yellow) encompassing the carboxy-terminal region of human TAFI110 (UniProtKB accession number Q15572). The peptide was used for co-migration with 32P-labeled tryptic phosphopeptides of hTAFI110 in 2-dimensional tryptic peptide maps  Mitotic repression of rDNA transcription is relieved by the ATP analogue AMP-PNP. Transcription by Pol I was assayed in vitro using 40 μg of extracts from asynchronous (as) or nocodazole-arrested mitotic (M) HeLa cells and 40 ng of the reporter template pHrP2 linearized with Nde I. Reactions contained either 200 μM ATP or 200 μM AMP-PNP. (E) Cdc14B counteracts Cdk1-mediated mitotic repression of Pol I transcription. Transcriptional activity was assayed in extracts from mitotic HeLa cells in the presence of ATP (200 μM)-/+ 2.5 mM DMAP, or the non-hydrolysable analog AMP-PNP. Where indicated, the assays were supplemented with similar units of calf intestine phosphatase (CIAP) or purified GST-hCdc14B. Run-off transcripts were analyzed on native polyacrylamide gels and visualized by PhosphorImaging. An internal control demonstrates equal loading. (F) Ccd14B is released from rDNA during mitosis. ChIP of Cdc14B and UBF from asynchronous (as) or nocodazole-treated (M) HeLa cells. Increasing amounts of precipitated DNA were analyzed by semi-quantitative PCR, using the indicated primer pairs . (G) Evaluation of the specificity of the anti-Cdc14B antibody used for ChIP. The ChIP results shown in (A) Flag-hTAFmaps see . (C) Thr(EPS)Click here for additional data file.S2 FigI110/WT (clone WT17) or Flag-hTAFI110/T852A (clone TA4). Flag-hTAFI110 was detected on immunoblots using anti-Flag or anti-TAFI110 antibodies. (B) Quantitative measurement of fluorescence signals in single interphase and mitotic cells presented in (A) Nuclear extracts were prepared from HeLa cell lines which stably express Flag-hTAF(EPS)Click here for additional data file.S3 FigI68 peptide containing the acetylated lysine residues 438 and 443. (B) Mutation of K438 and K443 abolishes acetylation by PCAF. Flag-tagged wildtype hTAFI68 and the point mutants K438R and K438/443R were immunopurified from HEK293T cells and acetylated with purified Flag-PCAF expressed in Sf9 cells. Acetylation of hTAFI68 was detected on Western blots with antibodies specific for acetylated lysine (acet. TAFI68). Equal amounts of TAFI68 in the assays and similar expression of PCAF were verified by re-probing with anti-TAFI68 and anti-PCAF antibodies, respectively.(A) Sequence of the hTAF(EPS)Click here for additional data file.S4 Fig1-phase cells from Quantitative analyses of the fluorescence microscopy images presented in (EPS)Click here for additional data file.S1 TableThe sequences of DNA oligonucleotides are shown in 5’ to 3’ orientation.(DOCX)Click here for additional data file.S2 TableThe sequences are shown in 5’ to 3’ orientation, mutated nucleotides are underlined.(DOCX)Click here for additional data file.


Drosophila melanogaster, piRNAs are produced both in the ovarian germline cells and in their surrounding somatic cells. Accumulating evidence suggests that germinal and somatic piRNA pathways are far more different than previously thought. Here we review the current knowledge on piRNA production in both these cell types, and explore their similarities and differences.Transposable elements (TEs) are major components of genomes. Their mobilization may affect genomic expression and be a threat to genetic stability. This is why they have to be tightly regulated by a dedicated system. In the reproductive tissues of a large range of organisms, they are repressed by a subclass of small interfering RNAs called piRNAs (PIWI interacting RNAs). In  Drosophila melanogaster proteins of the PIWI clade bind piRNA populations with a length that peaks at 26, 25 and 24 nucleotides (nt) for Piwi, Aub and AGO3 respectively [Eukaryotic genomes contain large numbers of transposable elements (TEs) whose activity represents a constant threat to genome stability. Protection mechanisms have evolved that limit their mobilization. The molecular nature of these protecting mechanisms came to light with the discovery of RNA silencing pathways. One of these pathways, the piRNA pathway (PIWI interacting RNAs) is more specifically active in gonads and protects the germline from TE mobilization. In this pathway, piRNAs produced from genomic regions referred as piRNA clusters or from TE mRNAs are loaded onto a PIWI protein belonging to the Argonaute family: Piwi, Aubergine (Aub) or Argonaute 3 (AGO3). In most species, the length distribution of piRNAs is relatively broad. For example, the ectively . The resectively .Drosophila melanogaster that have provided a better understanding of this major protecting pathway active against external and internal genomic invaders from unicellulars to human.In this review, we discuss the latest results from studies mainly performed in et al. reported piRNA clusters, they identified 142 genomic locations in the Drosophila genome as sites of abundant piRNA production [Most piRNAs derive from discrete regions called piRNA clusters. When, for the first time, Brennecke oduction .Drosophila melanogaster somatic cells surrounding the germline, piRNAs are mainly produced from two piRNA clusters located in pericentromeric regions: traffic jam [flamenco (flam) [. Of the two, flam is the best studied [Drosophila strains evidenced its highly dynamic nature which results in the loss and gain of TEs [ZAM and Idefix [flam acts not only as a trap for endogenous TEs but also for TEs coming in by horizontal transfer from other Drosophila species.In ffic jam  and flamo (flam) . Of the  studied . It is ln of TEs . This std Idefix . It alsoDrosophila ovaries, flam is transcribed from a polymerase II promoter as a long single-stranded precursor RNA that is a substrate for piRNA biogenesis. It is referred to as a uni-strand piRNA cluster. In flam, most of the retrotransposons are anti-sense oriented copies which results in the production of an anti-sense transposon RNA precursor giving rise to antisense piRNAs capable of silencing active transposon mRNAs.Like most piRNA clusters expressed in the somatic follicular cells of Drosophila to primates [Drosophila, they mostly produce piRNAs from both genomic strands and, therefore, must be transcribed in both directions. They are referred to as dual-strand piRNA clusters. They do not show the canonical features of polymerase II transcribed genes as uni-strand clusters. Moreover, in these clusters, TEs or their vestiges are inserted in both orientations as the Drosophila piRNA clusters at cytological positions 42AB, 38C and 80 F.piRNA clusters expressed in the germline have been identified in several species from primates ,6-8. In In mammals, mapping of piRNAs has shown that they are highly clustered in distinct genomic loci and mostly produced from uni-strand clusters. piRNAs are processed either exclusively from a single strand or from two non-overlapping anti-sense transcripts ,7,9,10.Drosophila transposon I-element become de novo piRNA-producing clusters that are reminiscent of native dual-stranded clusters [Caenorhabditis elegans in which 21U piRNAs are independently transcribed, an 8 nt motif located 40 nt upstream of the piRNA sequence has been shown to promote their transcription [Drosophila (see below). Cubitus interruptus (Ci) drives the transcription of flam and potentially several other piRNA clusters expressed in Drosophila follicle cells [It is still unknown how a genomic locus becomes a piRNA cluster. Many on-going studies are attempting to decipher the underlying mechanism of the process. It was found that integration of TEs into the 3′ UTR of actively transcribed genes may induce piRNA production towards the 3′ end of these transcripts. Such insertions induce the formation of genuine piRNA clusters active in the germline . Similarclusters . Howevercription ,14. In ole cells . In micele cells . HoweverThus, how some transcripts are distinguished from mRNAs and directed for piRNA processing is still an open question.flam have been recently discovered in Arabidopsis thaliana by a HI-C approach [et al. reported a nuclear structure named KNOT in which genomic regions of all five Arabidopsis chromosomes interact at high frequency. These KNOT Engaged Element (KEE) regions are significantly enriched in TE. They act as traps for Ds transposons that preferentially insert in the proximity of KEEs. On the basis of numerous similarities with Drosophila piRNA clusters, the authors hypothesize that KNOT is a conserved nuclear structure that plays a role in TE defense. They anticipate that nuclear structures analogous to KNOT will be discovered in other eukaryotes. This new study raises the possibility that nuclear organization and genomic interactions could play a role in the identification and/or maintenance of piRNA clusters.Interestingly, regions acting as a TE trap similar to approach . Grob etDrosophila melanogaster ovaries, the piRNA pathway was found to differ in the somatic supporting follicle cells and the developing germline [When studied in germline . piRNA bflam cluster [flam transcripts, initiated from the RNA polymerase II promoter, appear to undergo differential alternative splicing. This could help to generate diverse RNA precursors that all share the first exon at their 5′ end before being processed into piRNAs [flam precursors. Their transfer to cytoplasmic structures, called Yb bodies, in which the processing machinery is present, was recently analyzed by two groups [et al. reported that flam piRNA precursors, together with transcripts coming from other somatic piRNA clusters, are addressed to a single nuclear structure in ovarian follicle cells  experiments as both labs used the same protocol and the same probes. Further experiments are needed to resolve the discrepancy between these two studies. However, it has to be pointed out that one study used fly ovaries [flam transcripts accumulate either in the nucleus or in the cytoplasm depending on the developmental stage of the follicular epithelium. One can imagine that an unknown factor, responsible for the targeting of flam transcripts to nuclear Dot COM, is missing in OSS cells. This would lead to the export of flam transcripts in the cytoplasm, followed by their capture and redirection to flam-bodies by cytoplasmic components. Alternatively, the nuclear accumulation of flam transcripts may vary along oogenesis depending upon the efficiency of the piRNA biogenesis machinery. Indeed, a decrease in Armi staining is clearly observed from the earlier to later stages of oogenesis in experiments from Dennis et al. [flam transcripts in Dot COM could then reveal a decrease in their cytoplasmic export/processing. Future studies are required to shed more light on how a cytosolic protein might influence nuclear events. flam RNA transfer through the nuclear membrane to the cytoplasm would require proteins involved in nuclear export. Indeed, the helicase UAP56 or the nuclear RNA export proteins Nxt1, Nxf1, Nxf2, and nuclear pore complex factors like the nucleoporins Nup43, Nup54, Nup58, Nup154, have been shown to be necessary for TE silencing in the soma [Recent papers have provided insights into the synthesis and fate of transcripts produced from the  cluster ,19. Someo piRNAs  , a member of the phospholipase-D family of phosphodiesterases which includes both phospholipases and nucleases ,27. Crys in vivo  Yb  required for transposon silencing in mouse testes is a nuclear Piwi interactor [in vitro with Piwi [mael mutants [When matured, Piwi pi-RISC is imported to the nucleus Figure , right. ilencing ,37. Uponilencing ,57,58. Tilencing . DmGtsf1teractor . Depletiteractor . HP1a, kith Piwi  and histith Piwi . Finallyith Piwi . Loss of mutants  . Their transcription depends on Rhino (Rhi), Cutoff (Cuff) and Deadlock (Del) and, at least for some clusters, is presumed to be initiated at neighboring genes [In the ng genes -64  bias while AGO3-associated piRNAs exhibit preferentially an adenine (A) at the tenth nucleotide from the 5′ end. The slicer activity of PIWI proteins directs their RNA target cleavage between the tenth and eleventh positions, so the primary piRNAs and their corresponding secondary piRNAs exhibit a perfect complementary along their first ten bases .vasa mutants lead to the mislocalization of the other nuage components: Tejas, SpnE, Krimp and Mael, whereas mutation of any of these genes does not affect Vasa localization [Bombyx mori that Vasa could act in a transient Amplifier complex [Bombyx mori ortholog of Aub) and AGO3. The role of this complex could be to transfer the 5′ end of newly cleaved secondary piRNAs to AGO3, thereby protecting them from complete degradation. Vasa contains residues that are targeted for symmetrical dimethyl arginine methylation (sDMA), which is potentially important for protein/protein interactions. A number of factors in the piRNA pathway are TUDOR domain-containing (TDRD) proteins, able to recognize and interact with proteins possessing sDMAs or asymmetrical dimethyl arginine (aDMAS). However, Vasa sDMA does not seem to be required for the Amplifier complex assembly and the interplay between TDRD proteins and members of the PIWI clade, Piwi, Aub and AGO3, which also contain sDMAs, is not yet fully understood.Additional proteins localized in the nuage are required for effective ping-pong amplification and secondary piRNAs production. They are: Vasa, SpnE, Krimp, Papi Qin/Kumo, Tapas and Tejas ,40-45,67lization . It was  complex . This coDrosophila ovary as well as in OSS cells uncovered numerous factors required for piRNA mediated transposon silencing [Piwi, Armi, Zuc, Shu, Vret, Mael, Gasz and Gtsf1; (2) genes necessary for transcription  complex proteins MBD-R2 and Rcd5) and RNA export ; (3) genes encoding components or subunits of general cellular pathways such as the exon junction complex (EJC) , RNA metabolism and/or trafficking, and the SUMOylation machinery . A set of genes whose function remains to be determined has also been identified in both somatic and germinal screens (CG9754). In addition to these shared genes, numerous identified components unique to either germ or somatic cells have been described. From current knowledge, two key differences distinguish the germline piRNA pathway: the process of secondary piRNA biogenesis and the bi-directional transcription of germline piRNA clusters. Accordingly, Aub, AGO3, Vasa, Qin/Kumo, Spn-E and Tejas involved in the ping-pong amplification as well as Rhino and Cuff required for germline transcription of piRNA clusters were found as specific components of the germline piRNA pathway. Although primary piRNA biogenesis and Piwi-mediated silencing are likely to be similar in both germ and somatic cells, several factors were found specific to the somatic pathway. Some genes associated with RNA export (Nxf1), nuclear pore complex  or genes involved in transcriptional elongation and regulation  were identified as important components of the somatic pathway while their derepression has no or only a slight impact on germline silencing [Hen1, responsible of the 2′-O-methylation at 3′ ends of piRNAs, Acinus and Tsunagi that are part of the EJC as well as Asf1, Egg, His2Av involved in transcriptional silencing were only found in the in vivo RNAi screen in Drosophila [Complementary screens carried out in somatic and germ cells of the ilencing -37. Someilencing ,37. Howeosophila  whereas Overall, if shared components are required for piRNA biogenesis in ovarian somatic and germ cells, both cell types have also their own specificities certainly due to the fact that they have to face distinct threats when TEs transpose.Drosophila melanogaster, Dufourt et al. recently reported that piRNA biogenesis factors can be classified according to their temporal requirements for TE silencing [et al. further identified some cells within the germarium in which Piwi protein is down-regulated and piRNA-mediated silencing is weakened. This short developmental window, which has been called the piwiless pocket (Pilp) corresponds to the dividing germline cysts [de novo primary piRNAs biogenesis. Thus, the down-regulation of Piwi in the Pilp could be a way of promoting Aub pi-RISC formation, to boost the ping-pong piRNA amplification so that enough piRNAs is generated and deposited in the oocyte which, in turn, would initiate efficiently TE silencing in the next generation  as ‘sensors’ of the silencing activity exerted on a TE named Idefix from ilencing . A firstne cysts  (Figure ne cysts . Inheritn Figure B. FutureResearch into the piRNA pathway has shown how it fulfills the essential function of preserving the whole genome from the mutagenic effect of TE mobilization. The challenge is now to fill in the gaps that remain in our understanding of piRNA biogenesis and TE silencing understandings. It recently emerged from several studies that piRNAs have broader functions beyond TE silencing, including the regulation of gene expression . There i


Lamins are components of the peripheral nuclear lamina and interact with heterochromatic genomic regions, termed lamina-associated domains (LADs). In contrast to lamin B1 being primarily present at the nuclear periphery, lamin A/C also localizes throughout the nucleus, where it associates with the chromatin-binding protein lamina-associated polypeptide (LAP) 2 alpha. Here, we show that lamin A/C also interacts with euchromatin, as determined by chromatin immunoprecipitation of euchromatin- and heterochromatin-enriched samples. By way of contrast, lamin B1 was only found associated with heterochromatin. Euchromatic regions occupied by lamin A/C overlap with those bound by LAP2alpha, and lack of LAP2alpha in LAP2alpha-deficient cells shifts binding of lamin A/C toward more heterochromatic regions. These alterations in lamin A/C-chromatin interactions correlate with changes in epigenetic histone marks in euchromatin but do not significantly affect gene expression. Loss of lamin A/C in heterochromatic regions in LAP2alpha-deficient cells, however, correlated with increased gene expression. Our data show a novel role of nucleoplasmic lamin A/C and LAP2alpha in regulating euchromatin. The confined space of metazoan cell nuclei requires chromatin to be tightly packed while maintaining a high degree of organization to provide genome stability and coordinated operation of the transcriptional machinery . ConceptThe nuclear lamina, a scaffold structure at the periphery of metazoan nuclei, has a major role in chromatin organization by anchoring heterochromatin . Lamins,Tmpo, also known as LAP2). The major LAP2 isoform in the INM, LAP2beta binds chromatin-associated BANF1 and DNA via its LEM and LEM-like domains, respectively  and LAP2alpha-deficient (Tmpo KO) murine dermal fibroblasts (imMDFs), we found that A-type lamins not only associate with heterochromatic LADs as reported (Tmpo KO cells resulting in changes in gene expression and epigenetic histone marks.Using various protocols for chromatin immunoprecipitation (ChIP) in immortalized wild-type  signal, display wide-range chromatin interactions of all proteins  identified by DamID . WhereasElucidating the disparity between our data and the reported lamin A/C binding to LADs , we noteOur data showed that sonication conditions are a critical parameter for the nature of recovered lamin A/C-associated chromatin regions: moderate sonication (12 cycles) enriching for euchromatic regions, and extended sonication (30 cycles) for heterochromatic LADs. Intriguingly, this effect was more pronounced in the lamin A/C-3A6 compared to lamin A/C-N18 ChIP samples. Whereas in the 12-cycle ChIP, the lamin A/C-associated chromatin regions retrieved by the two lamin A/C antibodies coincided by nearly 80%, they considerably differed in the 30-cycle ChIP  cells, lamin A/C-3A6 and lamin B1 detect primarily the peripheral lamina in both cell types  and less repressive H3K9me3 than the lamin A/C-3A6-bound regions in euchromatin-enriched samples, and they had more H3K27me3 marks in the heterochromatin-enriched samples E. These ll types B. Furthell types C. We conHaving shown that lamin A/C binds in a heterochromatic and a euchromatic environment, we next tested to what extent the lamin A/C binding sites correlate with those of LAP2alpha. By overlapping of lamin A/C- and LAP2alpha-associated regions, we found that, in euchromatin-enriched samples (12 cycles), the majority of lamin A/C EDD peaks (85% for 3A6 and 90% for N18) concurred with LAP2alpha EDD peaks D. After Tmpo KO fibroblasts . We assessed changes in lamin A/C EDD peaks between Tmpo WT and Tmpo KO samples on a genome-wide scale in euchromatin-enriched regions. This analysis revealed lamin A/C-associated chromatin regions exclusively in Tmpo WT cells (“lost in KO”) (Tmpo KO cells (“gained in KO”) (Tmpo WT and KO cells (“WT^KO”); the remaining regions were either lost (39% of N18 and 53% of 3A6 peaks) or gained (41% of N18 and 51% of 3A6 peaks) in Tmpo KO cells  and 7% (3A6) overlap   D.Tmpo KO versus WT cells was also detectable in the 30-cycle ChIP analyses , suggesting that, in the absence of LAP2alpha, the association of lamin A/C with chromatin is reorganized at a genome-wide level.Interestingly, partial reorganization of lamin A/C-N18- and lamin A/C-3A6-associated domains in Tmpo KO cells and that of lamin A/C-N18-identified EDD peaks from 27% to 41%   and diminished in regions that lost lamin A/C binding (“loss”) in “WT^KO”) B. IntrigTmpo KO versus WT in imMDFs by RNA-seq, we found 616 genes to be differentially expressed , of which 341 were up-regulated and 275 were down-regulated . Of the 616 de-regulated genes, 239 were located in genomic regions of altered lamin A/C association . As expected, promoters of up-regulated genes showed an increase in active and a decrease in repressive histone marks and vice versa for down-regulated genes  favored lamin A/C complexes containing LAP2alpha, while an antibody to lamin A/C's C-terminal Ig fold (3A6) preferentially precipitated heterochromatin-associated lamin A/C not bound by LAP2alpha. As LAP2alpha interacts with the Ig fold of lamin A/C , it may Mature lamin A/C in the nuclear interior colocalizes with LAP2alpha . As euchIn a LAP2alpha-deficient background, lamin A/C relocalizes toward heterochromatic regions. How can LAP2alpha affect lamin A/C-chromatin interactions? Lamin A/C and LAP2alpha complexes in the nuclear interior are highly mobile  and may Tmpo KO versus WT cells? Interestingly, in euchromatin both repressive and active histone marks were decreased in regions that lost lamin A/C binding and increased in regions that gained lamin A/C, without affecting overall gene expression. Thus, lamin A/C-LAP2alpha complexes may primarily be involved in establishing a chromatin environment permissive for epigenetic regulation, rather than regulating gene expression directly by binding to promoters as suggested for lamin A/C  may contribute to the disease phenotype. In line with this hypothesis, expression of a muscle disease-causing lamin mutant in Caenorhabditis elegans impairs cell-type–specific reorganization of repetitive DNA arrays during muscle differentiation , supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL Streptomycin, 0.1 mM nonessential amino acids.Immortalized murine dermal roblasts  were culChIP was performed as previously described   and sequenced either on an Illumina HiSeq 2000 or HiSeq 2500 at the CSF NGS unit. After de-multiplexing, adapters were clipped with cutadapt (http://broadinstitute.github.io/picard/). Supplemental Table 3 lists the number of sequenced reads and mapped reads after filtering and de-duplication for all samples.Sequencing libraries were prepared from DNA fragments ranging from 100 to 800 bp by the Vienna Biocenter CSF NGS unit (cutadapt . Reads wcutadapt  using deRegions of enrichment (peaks) were identified using EDD version 1.0.2  and SICE2-fold change to fold changes obtained from 10,000 random permutations of differentially expressed genes (P < 0.05).Ten micrograms of purified, rRNA-depleted RNA (>200 bp) were fragmented by hydrolysis  at 94°C for 3 min. First-and second-strand cDNA synthesis was performed as described in detail in the Supplemental Material. Sequencing libraries were prepared by the Vienna Biocenter CSF NGS unit using the NEBNext Library Prep Reagent Set for Illumina (NEB), multiplexed (2 samples/lane), and sequenced on HiSeq 2000 (Illumina) at the CSF NGS unit. RNA-seq data analysis was done as described by https://github.com/CollasLab/edd). Genes shown in the browser view were obtained from the RefSeq data track provided by the UCSC Genome Browser. cLAD, ciLAD, and MEF LAD annotations were downloaded from the GEO database (accession number GSE17051 [ChIP-seq data (peaks and log ratios) were visualized with the Integrative Genomics Viewer . Log ratGSE17051  and GSE3GSE17051 ).Fifty to one hundred micrograms of cross-linked chromatin, diluted 1:10 in ChIP dilution buffer, were incubated with antibodies overnight at 4°C. Antibody-protein complexes were captured with magnetic protein A/G beads, 1/10 of the IP supernatant was put aside for later analysis, beads were washed , and antibody-protein complexes were eluted in 1× Laemmli Buffer .7Tmpo WT imMDFs were harvested in 3 mL IP buffer  and incubated on ice for 10 min. One-twentieth of the lysate was kept as “Input.” One-third of the soluble supernatant fraction was incubated with antibodies overnight at 4°C. Antibody-protein complexes were captured with magnetic protein A/G beads, 1/10 of the IP supernatant was put aside, beads were washed twice with IP buffer (w/o Benzonase) and IP samples eluted in 1× Laemmli buffer.Two times 10Polyacrylamide gel electrophoresis was performed according to 5 cells on coverslips were washed once with PBS++ (1 mM CaCl2 and 0.5 mM MgCl2), fixed for 10 min with 4% formaldehyde/PBS, permeabilized with 50 mM NH4Cl and 0.5% Triton X-100/PBS for 10 min, and blocked with 0.5% gelatine/PBS for 15 min. Primary and secondary antibody dilutions were prepared in 0.5% gelatine/PBS and applied for 1 h at room temperature.One to two times 10Samples were imaged using a Zeiss LSM 700 with a Zeiss Plan-Apochromat 63×/1.4 oil DIC objective and processed with ImageJ and Photoshop CS4.Lamin A/C ; lamin A/C ; lamin B1 ; mouse LAP2alpha ; LAP2alpha ; H3K9me3 ; H3K4me3 ; H3K9ac ; H3K27me3 ; mouse normal IgG ; rabbit normal IgG .Secondary antibodies for WB: Licor IRDye 680RD donkey anti-mouse, 680RD donkey anti-rabbit, 800CW donkey anti-rabbit, 800CW donkey anti-goat; all 1:15000.Secondary antibodies for IF: DyLight donkey anti-mouse 594, donkey anti-rabbit 594, goat anti-mouse 488, goat anti-rabbit 488, donkey anti-goat 549; all 1:500.http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE70149.The data from this study have been submitted to the NCBI Gene Expression Omnibus  (GEO; ht


Bacillus subtilis induces a sophisticated network of adaptation responses. More precisely, the B. subtilis TnrA regulator represses or activates directly or indirectly the expression of a hundred genes in response to nitrogen availability. The global TnrA regulon have already been identified among which some directly TnrA-regulated genes have been characterized. However, a genome-wide mapping of in vivo TnrA-binding sites was still needed to clearly define the set of genes directly regulated by TnrA. Using chromatin immunoprecipitation coupled with hybridization to DNA tiling arrays (ChIP-on-chip), we now provide in vivo evidence that TnrA reproducibly binds to 42 regions on the chromosome. Further analysis with real-time in vivo transcriptional profiling, combined with results from previous reports, allowed us to define the TnrA primary regulon. We identified 35 promoter regions fulfilling three criteria necessary to be part of this primary regulon: (i) TnrA binding in ChIP-on-chip experiments and/or in previous in vitro studies; (ii) the presence of a TnrA box; (iii) TnrA-dependent expression regulation. In addition, the TnrA primary regulon delimitation allowed us to improve the TnrA box consensus. Finally, our results reveal new interconnections between the nitrogen regulatory network and other cellular processes.Under nitrogen limitation conditions,  In bacteria, nitrogen is present in nearly all macromolecules such as proteins, nucleic acids, and peptidoglycan. To provide an optimal supply of this macronutrient, prokaryotes have developed transport and assimilation systems for a variety of nitrogen sources as well as sophisticated control mechanisms to ensure energy-efficient uptake and assimilation. This regulatory network allows an adequate response to situations of nitrogen limitation.Bacillus subtilis, ammonium assimilation occurs via the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway to generate glutamate, the precursor for amino acids and nucleotides biosynthesis           (Brown and Sonenshein gltAB (glutamate synthetase)     to identify the TnrA DNA-binding sites, in vivo, at the genome scale. We provide evidence that TnrA binds reproducibly to 42 regions on the chromosome. In combination with real time in vivo transcriptional profiling using firefly luciferase, our data allowed us to define the TnrA primary regulon, which is now composed of 35 promoter regions. Thanks to this restricted list of genes directly regulated by TnrA, we proposed an improved TnrA box consensus. In addition, we characterized the TnrA secondary regulon, which is composed of promoter regions harboring a TnrA box and bound by TnrA in vivo. However, the growth conditions revealing a TnrA-dependent regulation for this second category of genes remain still unknown. Finally, our results highlight connections between the nitrogen metabolism and other regulatory networks.B. subtilis strains used in this work are listed in TableBacillus subtilis cells were grown in Luria–Bertani (LB) medium or in minimal medium containing 62 mmol/L K2HPO4, 44 mmol/L KH2PO4, 17 mmol/L trisodium citrate, 11 mmol/L K2SO4, 0.6% glycerol, 1 mmol/L MgSO4, 1 mmol/L CaCl2, 100 μmol/L FeCl3 citrate, 112 μmol/L ZnCl2, 5 μmol/L MnCl2, 2.5 μmol/L CuCl2, and 0.3% glutamate. Glutamine was added at the final concentration of 0.3% when needed. Escherichia coli cells were grown in LB medium. Antibiotics were added at the following concentrations when required: 100 μg ampicillin mL−1; 5 μg chloramphenicol mL−1; 60 μg spectinomycin mL−1. Solid media were prepared by the addition of 20 g Agar noble L−1 . Standard procedures were used to transform E. coli . DNA fragments were purified from agarose gels using the QIAquick kit .DNA manipulations and cloning procedures were performed as described elsewhere  was constructed by chromosomal integration of a translational fusion between the tnrA coding sequence and the sequential peptide affinity (SPA) tag sequence  was amplified by PCR with oligonucleotides creating an Acc651 restriction site at the 5′ end and a NcoI restriction site at the 3′ end of the fragment. The PCR product was cloned into plasmid pMUTIN-SPA subsequent to digestion with Acc651 and NcoI  was amplified by PCR with a 24-bp spc fragment at its 3′ end. The region downstream of tnrA (nucleotides 1397671 to 1398742) was amplified with a 24-bp spc fragment at its 5′ end. The three DNA fragments were combined and then a PCR reaction was performed with the two external oligonucleotides. The final product, corresponding to the two regions flanking tnrA with the inserted spc cassette in between, was purified from a gel and used to transform B. subtilis. Integration and deletion were confirmed by PCR and verified by DNA sequencing.The que Wach . The spcB. subtilis chromosome. The used primers contained extremities matching with the pUC18cm-luc plasmid (Table S3). The linear plasmid and the 1-kb fragment were mixed with the enzymes mix according to the assembly Gibson's procedure . A 1-kb fragment ending with the initiating codon of the gene of interest, and containing the promoter, was amplified by PCR from the 600) of 2. Cells were then centrifuged and resuspended in fresh minimal medium, adjusting all the cultures to an OD600 of 2. These precultures were then diluted 20-fold in fresh minimal medium and 200 μL was distributed in each of two wells in a 96-well black plate . Ten microliters of luciferin was added to each well to reach a final concentration of 1.5 mg/mL (4.7 mmol/L). The cultures were incubated at 37°C with agitation in a PerkinElmer Envision 2104 Multilabel Reader  equipped with an enhanced sensitivity photomultiplier for luminometry. The temperature of the clear plastic lid was maintained at 38°C to avoid condensation. Relative Luminescence Unit (RLU) and OD600 were measured at 5-min intervals.For the detection of luciferase activity, strains were first grown in LB medium to an optical density at 600 nm  was used to preferentially purify the DNA regions specifically cross-linked to TnrASPA. The immunoprecipitated DNA (IP) and the control whole cell DNA extract (WCE) were labeled with Cy3 and Cy5, respectively, and co-hybridized to the B. subtilis Roche-NimbleGen tiled microarrays  standard bioinformatic method  . In the resulting tnrA::tnrA-spa strain, expression of the gene encoding the TnrASPA protein is under the control of its native transcriptional signals. In order to verify if the TnrASPA protein (used below in the next experiments) was functional, we compared the transcriptional regulation of known TnrA-regulated genes in the presence of TnrASPA or TnrAWT. The promoter region of the alsT gene, whose expression is known to be inhibited by TnrA . By decreasing the threshold defining a TnrA-binding peak (ChIPScore = 4.0), we did not identify more sites overlapping the so far known TnrA regulon. In total, we retrieved 19 out of 25 well-characterized TnrA-regulated promoters. We did not detect TnrA-binding sites in the promoter regions of alsT, ansZ, degU, ywlFG, ywrD, and yxkC operons.Overall 42 enriched DNA regions ≤200 bp were identified from the ChIP-on-chip signals (Table S1) Fig.. SixteenIn addition, 20 additional promoter-proximal TnrA-binding sites were detected less than 300-bp upstream of the start codon (Table S1), suggesting a TnrA-dependent expression and therefore the existence of new candidates in the TnrA regulon. The presence of predicted TnrA boxes in these regions is discussed below.kamA, purB, tcyJ, yfmF, yobI, and ykoH (Table S1). The location of these sites was intriguing since no TnrA intragenic binding sites have been described so far.Finally, six peaks were located within intragenic regions more than 150 base-pairs downstream of the start codon of appD, dtpT, ydaB, yrbD, ysnD, yuiA, pucH, yvgT, and pgsB translational start sites. It is interesting to notice that two TnrA box motifs have been detected in silico in front of the dtpT, glnR, ycsF, and ysnD genes correlating with the detection of two TnrA-enriched regions by ChIP-on-chip (Table S1). As putative TnrA boxes actually corresponded to experimentally identified in vivo TnrA-binding sites, the associated genes were assumed to be relevant TnrA-regulated genes candidates.To investigate the presence of TnrA boxes within the newly identified 20 promoter-proximal TnrA-binding sites, we compared our data with previous in silico studies available in the RegTransBase database Fig.A Cipria Leyn et. Nine ofThe 11 other promoter-proximal TnrA-binding sites did not display a significant match to the previously proposed TnrA box consensus Fig.. This retnrA cells. Luciferase activity was recorded during exponential growth in minimal medium with glutamate as sole nitrogen source. Two transcriptional fusions containing the nasB and alsT promoter regions were used as controls of these experiments. As expected in the growth conditions used, expression of PnasB::luc was upregulated, whereas expression of PalsT::luc was downregulated in a tnrA mutant strain . In contrast, the transcription rate of luc fused to PappD, PdtpT, PpucR, PyrbD, and PysnD exhibited 3- to 30-fold lower levels in ΔtnrA cells . These results validated the TnrA-dependent regulation of these seven genes. Regulation of pgsB could not be tested as this operon is not expressed in the strain 168 lineage . This result suggested that other transcription factors could be involved in this regulation masking TnrA activity or that the TnrA box identified is not properly positioned with respect to the promoter and thus not functional.We further tested the expression regulation of our nine candidate genes in the same conditions. Transcription rates from Pluc reporter gene. PyfiR showed a slight 1.92-fold activation (±0.08) by TnrA while Phom and PpycA were 8 and 2-fold repressed by TnrA, respectively . These results demonstrated the TnrA-dependent regulation of these three transcription units. On the opposite, the expression from PbraB, PiscS (divergently transcribed from PbrabB), PcodV, Ptdh, PyclM, PyclN (divergently transcribed from PyclM), PykkC, PyflL, PypqP, and PyugK was not affected by the absence of TnrA in the conditions used (data not shown).Eleven promoter-proximal TnrA-binding sites were identified and located in regions without a predicted TnrA box. We tested the ability of TnrA to mediate their regulation with transcriptional fusions between promoter regions and the tnrA deletion on the expression of those 20 promoters was observed in this medium, as expected (data not shown).We observed a full overlap between the set of TnrA-binding sites detected in minimal medium with glutamate and in LB medium. Thus, we examined the regulation of the 20 newly identified promoter regions bound by TnrA in LB medium. No effect of yuiA, yvgT, appD, dtpT, pucR, yrbD, and PysnD. The alignment of all the TnrA boxes is shown in Figure S3. The resulting new TnrA box consensus sequence exhibited a dyad symmetry as previously found in Nakano et al. ByuiA, yvyfiR, hom, and pycA; (ii) the promoter regions of braB, ykkC, yflL, ypqP, and yugK, whose expression was not affected in a tnrA mutant in the tested conditions; (iii) the intragenic binding sites of kamA and ykoH genes .We used this newly defined TnrA box consensus to search TnrA box motifs among the 17 TnrA-binding sites identified in ChIP-on-chip, which did not harbor a previously predicted TnrA box. In this analysis, we considered the genomic regions representing 100 bp centered at each TnrA-binding site. As a result, a TnrA box was detected in: (i) the promoter region of the TnrA-regulated genes Altogether, the results presented above allowed us to identify 10 new promoter regions harboring a TnrA box and directly regulated and bound by TnrA in vivo Table. The comSubsequently, we verified if a different consensus could be detected if the 42 sites were considered using the MEME method. We did not impose a constraint that the motif must be an inverted repeat sequence on the search. When all the sites were used, no difference could be detected in the TnrA box consensus. Moreover, if only the promoters not regulated by TnrA were considered, no consensus at all was found. This suggested that TnrA probably does not directly interact on these sites.pucA, pucI, ycdE, and yumC (Table S1). Expression of the corresponding promoter regions was compared in wild-type and ΔtnrA genetic backgrounds. No effect of TnrA could be detected for the PycdE expression in the conditions used (data not shown). Expression from PpucI and PyumC was 3-fold upregulated in a tnrA mutant, whereas the expression from PpucA was fully downregulated in ΔtnrA cells . Thus, we identified a second set of three TnrA-dependent transcription units encoding seven genes, pucABCDE, pucI, and yumC.In order to obtain a full extent of the TnrA regulon, we also examined the regulation of genes associated with a predicted TnrA box . These results confirmed that TnrA is the major regulator to mediate regulation of these genes in response to glutamine availability.GS enzyme acts as a glutamine sensor, transducting the glutamine signal to TnrA. In the GS sequestered state, TnrA cannot control its regulon . This was in agreement with a previous report, which showed by transcriptomic analysis that hom expression was downregulated by glutamine  . Altogether, our study illustrates the power of ChIP-on-chip approaches, generating pertinent data to finely delineate a transcriptional regulator in vivo binding sites.Among the 42 TnrA-binding sites identified by ChIP-on-chip in this study, 16 belong to the known TnrA regulon Fig.A Yoshid. As someChIP-on-chip data combined with real-time in vivo transcriptional profiling enabled us to validate the functionality of seven in silico predicted TnrA boxes Fig.. MoreoveThe TnrA primary regulon delimitation finally allowed us to refine the TnrA box consensus Fig.B. A sequtnrA strain according to the transcriptional fusions analysis. These unexpected protein–DNA interactions that could not be identified using transcriptional profiling have been observed before . As we did not observe a TnrA-dependent regulation of these genes in the conditions used, we propose that TnrA might also play a regulatory role in specific unknown conditions. It was recently shown that the CodY regulator also binds upstream of the braB and ypqP genes . It is possible that TnrA could bind to these intragenic sites to mediate repression by a roadblock mechanism, as described for the B. subtilis CcpA and CodY regulators . This regulon includes pucA, pucI, and yumC, which exhibit a predicted TnrA box as well as citB , oligopeptides (appDABC), and amino acids (yrbD) . It is worthwhile mentioning that several of the positively regulated TnrA genes are involved in the transport of peptides  + oxidoreductase, which can play a role in protecting cells against oxidative stress (Bianchi et al. yuiAB and yumC genes in the nitrogen metabolism and/or the oxidative stress response.Finally, our study highlights the TnrA-dependent regulation of genes connected to the oxidative stress response. In previous transcriptomic study, the