In a previous paper, we showed that the methylation of H3K4 and consequent promoter nucleosome deacetylation repress ste11 induction and cell differentiation  formation leads to high expression. Therefore, H2Bubi represses gametogenesis by opposing the recruitment of RSC at the promoter of the master regulator ste11 gene.In fission yeast, the ntiation  but the DOI:http://dx.doi.org/10.7554/eLife.13500.001 PHO5, PHO84 and GAL1 expression, suggesting that H3K4me creates a repressive chromatin configuration (The RNA polymerase II (PolII) subunit Rpb1 C-terminal Domain shows a stereotypical pattern of CTD phosphorylation with phospho-S5 (S5P) peaking near the transcription start site (TSS) and phospho-S2 (S2P) accumulating towards the 3’-end of the transcribed region . The disguration . Most geguration . The PHDguration . Similarguration .Histone H2B is monoubiquitylated (H2Bubi) on the conserved lysine 119 in fission yeast. In yeast, Rad6 and Bre1 function as conjugating enzyme (E2) or ligase (E3) . The patFrom the SWI/SNF-class remodeling complexes, RSC is ten-fold more abundant and is essential for viability, in contrast the SWI/SNF complex . RSC is Ste11 in fission yeast  is required for the completion of cytokinesis in response to perturbation of the actomyosin ring by latrunculin A (LatA)  , and theentified  as downrd  mutant strains. Surprisingly, the htb1 K119R mutant completely suppressed the lsk1 sensitivity to the drug  behaved similarly  for ubp8 deletion strain but not in the absence of ubp16 that encodes the closest fission yeast orthologue to ScUBP10  led to an elevated level of H3 onto the promoter region while the htb1 K119R mutant, or a strain lacking hos2  had a reduction of histone occupancy at the ste11 promoter , snf22  and arp9 (shared subunit) on the induction of ste11. The absence of either rsc1 or arp9 clearly affected the expression of ste11 while snf22 had no effect . We therefore tested the effect of inactivating NuA3 and NuA4 by combining a deletion of mst2, encoding the catalytic subunit of NuA3 . These data support a genome-wide mechanistic connection between CTD phosphorylation on serine 2 and remodeling by the RSC complex.In order to test if the proposed connection between the phosphorylation of serine 2 within the CTD and the RSC remodeling complex could be expanded to more genes, we performed genome-wide mapping of nucleosome position by MNase-Seq in the as noted  when selcategory , confirmcategory  behaved  deleted . Remarkaste11, requires an unusual requirement of S2P nearby its promoter for proper induction to counteract the repressed state imposed by the Set1-H3K4me3-HDAC pathway  and the htb1 K119R mutant (and therefore absence of ubiquitylation) resulted in derepression of ste11. This is at first sight hard to explain as removal of histone modifications are expected to oppose the effect of their addition, such as in the acetylation/deacetylation process. However, a similar case was reported at the GAL1 locus  , we specbolished .ste11 very similarly to S2P and indeed both S2P and histone acetylation are required for high occupancy of RSC at this locus. Considering that RSC recognizes acetylated nucleosomes through its bromodomains-containing subunits , anti-H2Bubi (Active Motif #39623), PAP (Sigma #P1291) and anti-tubulin (Sigma #T5168) antibodies. Iodine staining was performed as described (cdc14 plasmid and the cdc14-118 ts mutant were gifts of Viesturs Simanis. Latrunculin A (LatA) was purchased from Enzo Life Sciences (#76343-93-6) and used at 0.5 μM. Anhydrotetracycline (AnTet) was purchased from Sigma (#37919) and used at 2.5 μg/ml. Plasmids used to generate the switch off snf21 strain , anti-H2Bubi (Active Motif #39623), anti-H3K4me3 (Millipore #07–473), anti-H3K14ac (Millipore #07–353) anti-H3 (Abcam #1791) and PAP (Sigma). For all ChIP experiments, each column represents the mean percentage immunoprecipitation value ± SEM (n = 2–4). Note that a 10 min crosslinking in formaldehyde was used as routine but was extended to 15 min in the case of Rsc1-TAP.Chromatin Immunoprecipitations were performed using a Bioruptor (Diagenode) and Dynabeads (Invitrogen). Precipitated DNA was purified on Qiagen. Quantitative RT-PCR was performed using the ABI high capacity RNA-to-cDNA . The unt2 5 mM, CaCl2 1 mM, NP-40 0.75%) supplemented with 7.6 ml NP buffer + 0,5 µl β mercaptoethanol + 400 µl spermidine 10 mM) and split into 2 Falcon tubes . Add 50 µl MNase (32 units) to one tube and incubate 20 min at 37°C without agitation. Add 500 µl Stop buffer, 200 µl RNase A (0.4 mg/ml) and 225 µl proteinase K (20 mg/ml) and incubate at 65°C overnight. Potassium Acetate was added (1.25 ml of a 3M solution) and the mix was incubated 5 min on ice. After phenol extraction, 200 µl NaCl 5 M, 1.7 µl Glycogene (20 mg/ml), and 3.5 ml of isopropanol were added. After precipitation and ethanol wash, the pellet was resuspended in 200 µl of TE buffer. The samples were run on agarose gel (1.5%) and the bands corresponding to the mononucleosomes were cut and purify with Qiagen. Q-PCR with the primer pairs of the set of overlapping amplicons were performed. Nucleosomal DNA enrichment calculated as the ratio between the amounts of PCR product obtained from DNA samples generated from the mononucleosomal gel purification to that of the input  DNA.A culture of 500 ml of fission yeast cells was grown to OD 0.5 at 32°C and crosslinked with 7 ml of Formaldehyde 37% for 20 min at 25°C, 60 rpm. The crosslink was stopped by the addition of 27 ml of Glycine and cells were pelleted. The pellet was resuspended in preincubation solution  supplemented with 100 µl β mercaptoethanol/50ml and incubated 10 min at 30°C. The cells were centrifugated and resuspended in 10 ml of Sorbitol 1 M / Tris pH 7.4 50 mM buffer containing 200 µl Zymolase  and incubated 20 min at 30°C (40 min when EMM medium was used). After centrifugation, the pellet was resuspended in 7.5 ml NP buffer .Total RNA was prepared as described  and puriThe preparation of mononucleosomal DNA and the sequencing of mononucleosomal DNA were previously described in details . The nuchttps://code.google.com/p/danpos/). Clonal reads  were removed from the reads previously mapped on the S.pombe genome with BWA (bio-bwa.sourceforge.net). Variation in size resulting from MNase treatment were compensated by shifting each read toward the 3’ direction for half of the estimated fragment size. Nucleosome occupancy was then calculated as the quantile-normalized count of adjusted reads covering each base pair in the genome. Afer this processing, DANPOS calculates the differential signal at single nucleotide position based on a Poisson test. Dynamic nucleosomes are then identified by peak calling on these signals. TSS associated NDR lengths were quantified as the length of the longest DNA segment whose proximal border is located closer than 65 bp from the TSS with nucleosome occupancy levels lower than an arbitrary treshold (mean(occupancygenome) – standard_deviation(occupancygenome)) at any point.MNase-seq data was processed and dynamic changes were detected using DANPOS . H2B ubiquitin protease Ubp8 and some components of RSC and SWI/SNF complex were identified, which suggests potential link between H2B-ub1/chromatin remodeling and S2P pathway. In fission yeast gametogenesis, ste11 in fission yeast. Most interesting is that the typically activating chromatin modification, H3K4me3, is thought to be repressive via recruitment of HDAC complex and, in the new data here, resisting Rsc remodeling of promoter. In light of the previous paper, which made the basic observation of this pathway regulating ste11, there are two key questions for this to increase broad interest:The overall findings are interesting with respect to the master regulatory ste11?1) Are there general mechanisms to control gene expression in fission yeast, which can be addressed by genome wide studies in addition to specific analyses of ste11?2) Is there an effect on gametogenesis itself, not only on expression of Additional specific technical questions:ste11 expression in fission yeast, do RSC mutants affect H2Bub levels at ste11 promoter?1) Since H2Bub and RSC are both regulating 2) KR mutagenesis is not perfect to mimic non-ubiquitylated lysine, which may cause side effects. Authors should test the rhp6 deletion strain in at least one key experiment, e.g. in ste11 represses transcription by increasing H3K4-3me', There is no evidence to support "by increasing H3K4-3me".3) Results: 'We conclude that H2B ubiquitylation at the promoter of ste11 promoter via Set1-H3K4me3-SET3C HDAC pathway. So does KR mutant and rhp6 deletion affect H3K14ac at ste11 promoter.4) Authors conclude that H2Bub opposes RSC to remodel chromatin at 5) Quantification: no p-values – need to be added. How many biological repeats were performed for each experiment? I could not find this in Figure Legends.6) In 7) In 8) 9) Reviewer #2:eLife by the authors. In that study, they presented genome-wide nucleosome mapping data in fission yeast, S. pombe, showing that the absence of phosphorylation of the CTD of RNA polymerase II (RNAP II) on serine 2 (S2) led to increased nucleosome occupancy and repositioning in the promoters of a subset of yeast genes. The affected promoters generally had a larger nucleosome depleted region (NDR) than the majority of genes, and included the ste11 gene, which the authors previously found had a peak of S2 phosporylation (S2P) near the promoter during its activation. They showed that the peak of S2P at ste11 and a second promoter reduced nucleosome occupancy and increased histone acetylation during gene activation. The take home message from these and other studies in this first manuscript was that during activation S2P counteracts repressive histone deacetylation by SET3C, which is targeted to promoters via its interaction with RNAP II-S5P/Set1-mediated H3K4 methylated histones. The dual presence of S2P and S5P was also found to block Set1 interaction with the CTD.This manuscript follows up on studies published earlier in ste11 gene to investigate the role of other regulators in this pathway of promoter repression. Their examination was restricted to H2B monoubiquitylation (H2B-ub1) and the RSC nucleosome-remodeling complex, which were identified in a screen for mutants that relieved the drug sensitivity of lsk1, the fission yeast S2 CTD kinase. By analyzing loss of function mutations coupled with RNA analysis and ChIP, the authors report that an htb1KR mutation suppressed the transcription defect of a lsk1 deletion and that H2B-ub1 was localized to the ste11 promoter under basal (repressed) conditions. As expected, its promoter occupancy positively correlated with the occupancy of H3K4me3. Consistent with a role in promoter repression through its trans-regulation of H3K4me3, an htb1-KR mutant showed reduced nucleosome occupancy in the ste11 promoter. In contrast, mutations that decreased RSC expression had the opposite phenotype – ste11 transcription was reduced and nucleosome occupancy was increased in the promoter. The authors propose that H2B-ub1 represses transcription of ste11 by opposing RSC-dependent nucleosome remodeling.In the present manuscript, the authors focused on the ste11 activation, thus extending the author's initial definition of this pathway and adding some interesting new information about the interplay between RNAP II phosphorylation and chromatin remodeling at promoters. However, gaps in the analysis remain and should be addressed, as outlined below.The phenocopying of S2A and H3K4me3 mutants with rsc and htb-K119R mutants, respectively, supports the placement of Rsc and H2B-ub1 in the pathway leading to ste11 transcription as they did for an htb-K119R lsk1∆ double mutation.1) It is unclear why RSC mutants were identified in the screen for suppression of drug sensitivity of Lsk1. The authors should examine the effects of a rsc1∆ lsk1∆ double mutation on ste11 promoter.2) The authors used Arp9-TAP to perform ChIP experiments in 3) Nucleosome scanning should be included in the analysis of the phenotypes of the htb-K119R mutant.ste11 promoter, for example what is the role of S5-P, and what is the status of Bre1 occupancy at the promoter?4) An important missing gap in the pathway is the relationship of H2B-ub1 to S2-P; for example, does H2B-ub1 affect Lsk1 recruitment to the promoter and subsequent S2-P? Along the same lines, what is responsible for the deposition of H2B-ub1 at the ste11 promoter and that H3ac levels remain high, consistent with H2Bub1 being upstream of H3K4me3 in the pathway.5) Present evidence confirming that the htb1-KR mutant does not show HDAC recruitment to the 6) This study would be significantly enhanced if the authors extended their MNase mapping experiments genome wide in rsc∆ mutants, and then comparing the results to those reported for an S2A mutant. This would broaden the authors' conclusions and complement their in-depth analysis of a single gene.Reviewer #3:eLife. 2015. 4:e09008) that phosphorylation of the RNA polymerase II CTD on Ser2 (S2P) controlled nucleosome dynamics in S. pombe at the promoters of 324 genes, including ste11 that encodes a regulator of mating and gametogenesis. The authors also found that MAP kinase activation of latrunculin-sensitive kinase 1 (Lsk1) mediated the S2P modification. Mechanistically, the authors established that CTD S2P by Lsk1 counters CTD S5P-dependent recruitment of Set1-mediated trimethylation of histone H3 lysine 4 (H3K4me3), which, in turn, recruits the SET3C histone deacetylase (HDAC) complex.Previously, Materne et al. reported , a disruptor of the actinomyosin ring. The screen identified SAGA-associated Ubp8, a ubiquitin (ub) protease that removes monoubiquitin from H2B, as well as shared and non-shared subunits of the chromatin remodelers RSC and SWI/SNF  the authors convincingly show that the level of H2Bub1 at the ste11 correlates with the presence of H3K4me3, and both histone marks decrease on activation  reported that transcript levels of most genes (80%) were downregulated in the absence of Set1 and H3K4 methylation. In contrast, Set1 and H3K4 methylation does silence the rDNA . The statement should be justified or corrected and referenced appropriately. Reviewer #1:1) Are there general mechanisms to control gene expression in fission yeast, which can be addressed by genome wide studies in addition to specific analyses of ste11?We have addressed the question by analysing the genome wide effect of the deletion of rsc1, a non-essential subunit of RSC, on nucleosome positioning, and by comparing it with the CTD S2A mutant. We decided to perform an MNase-Seq experiment, which is time consuming but provides a high resolution map of nucleosome. A new figure  with 5 p“In order to test if the proposed connection between the phosphorylation of serine 2 within the CTD and the RSC remodeling complex could be expanded to more genes, we performed genome-wide mapping of nucleosome position by MNase-Seq in the rsc1 deletion mutant. […] These data support a genome-wide mechanistic connection between CTD phosphorylation on serine 2 and remodeling by the RSC complex.”ste11 can likely be extended to a subset of genes  in the conditions we have tested (growth in standard conditions).To answer the question of the referee, we can therefore conclude that the mechanism we describe in details in the case of 2) Is there an effect on gametogenesis itself, not only on expression of ste11? The referee asks the important question of the biological relevance of our analyses. We have added a panel in ste11 expression  Since H2Bub and RSC are both regulating ste11 expression in fission yeast, do RSC mutants affect H2Bub levels at ste11 promoter?ste11 but leads to a strong decrease of ubiquitylation in the 3’ region. This is not surprising considering that ste11 transcription is strongly impeded in the absence of Rsc1 as judged by Q-RT-PCR  KR mutagenesis is not perfect to mimic non-ubiquitylated lysine, which may cause side effect. Authors should test the rhp6 deletion strain in at least one key experiment, e.g. in We agree with the referee that the KR is not a perfect mimic of the non-ubiquitylated lysine. The referee may have missed that we had already performed the experiment he requested as shown in 3) Results: 'We conclude that H2B ubiquitylation at the promoter of ste11 represses transcription by increasing H3K4-3me', There is no evidence to support "by increasing H3K4-3me".The data presented in ste11 correlates with repressed transcription and high level of H3K4me3, which in turn may favour histone deactetylation by the SET3C complex.”“We conclude that H2B ubiquitylation at the promoter of 4) Authors conclude that H2Bub opposes RSC to remodel chromatin at ste11 promoter via Set1-H3K4me3-SET3C HDAC pathway. So does KR mutant and rhp6 deletion affect H3K14ac at ste11 promoter.ste11 promoter. The text was modified as follows:As mentioned above (point 2), we have tested the issue raised by the referee by chipping the K14 acetylated histone H3  in the strains lacking H2B ubiquitylation, which results an elevated acetylation at the ste11 promoter when H2B ubiquitylation was abolished in either the htb1-K119R or the rhp6 mutant  Quantification: no p-values – need to be added. How many biological repeats were performed for each experiment? I could not find this in Figure Legends.The number of replicates that was previously indicated in the Materials and methods is now mentioned for each experiment in the figures legend, as requested by the referee.When a statistical analysis was performed as in 6) In In all experiments where the occupancy of modified histones was assessed, the signal was indeed normalized to the total corresponding histone. This is now more clearly indicated in the y-axis of the corresponding graphs.7) In We agree with the referee. The scanned blots were quantified in ImageJ and the level of H2Bubi was normalized to H2B with the wild type set as 1.8) The timing of these experiments is very different. As the snf21 gene is essential, it was important to show that the switch-off system we used is sufficiently efficient to ultimately mimic a deletion. Indeed, the strain does not sustain growth when the promoter is turned off .In the experiments presented in 9) ste11. It is difficult to extend it further as the data presented already required more than 30 primers. However, the ChIP experiment presented in The nucleosome scanning experiment presented in We hope that these comments answer the issues raised by this referee and we thank him for his deep analysis of our work.Reviewer #2:1) It is unclear why RSC mutants were identified in the screen for suppression of drug sensitivity of Lsk1. The authors should examine the effects of a rsc1∆ lsk1∆ double mutation on ste11 transcription as they did for an htb-K119R lsk1∆ double mutation.latrunculin sensitive kinase) was shown to be sensitive to latA, most likely because the expression of one or several genes required for survival in the presence of the drug are affected. Our data support that the cdc14 gene encodes such a target gene. If Lsk1 and RSC are required in the same pathway to enforce cdc14 expression, it is not surprising to us that subunits of RSC were identified in the screen, as their deletion results in lower gene transcription. In the case of cdc14, contrary to ste11, both RSC and SWI-SNF seem to be required for proper expression.The referee is surprised that subunits of RSC were identified in the latA screen presented in eLife 2015). Therefore, the experiment cannot be performed.Regarding the experiment proposed by the referee, namely to examine the effect of a double rsc1 lsk1 mutant, the combined deletion of these two genes is synthetic lethal as reported in our previous work  The authors used Arp9-TAP to perform ChIP experiments in ste11 promoter:While the work was in progress, we noticed that the Rsc1 protein was difficult to Chip, resulting in less reproducible results. We therefore had to increase the timing of crosslinking as indicated in the Materials and methods. The Arp9 protein was easier to work with. As pointed by the referee, this raised the question of the specificity. Below is a list of the evidences supporting a specific role of RSC at the ste11 expression are presented, which points to a specificity of RSCste11 expression.ste11 promoter, while Rsc1 and Arp9 does  Nucleosome scanning should be included in the analysis of the phenotypes of the htb-K119R mutant.We have performed the requested experiment, which is difficult in this case due to the slow growth of the mutant. This has technical implications as the time required to digest the cell with zymolyase is critical and affected by many parameters, including the physiological state of the cell. The result is presented below and shows that the H2B K119R mutant behaves very similarly to a wild type, or could even have lower occupancy.4) An important missing gap in the pathway is the relationship of H2B-ub1 to S2-P; for example, does H2B-ub1 affect Lsk1 recruitment to the promoter and subsequent S2-P? Along the same lines, what is responsible for the deposition of H2B-ub1 at the ste11 promoter, for example what is the role of S5-P, and what is the status of Bre1 occupancy at the promoter?ste11 promoter, which supports the model previously reported. The text was modified as follows:The referee points to an important issue, namely the relationship of H2Bubi to S2P. Indeed, previous work from the Berger lab has revealed that H2Bubi acts as a barrier to the S2 kinase nucleosomal recruitment, which we have mentioned in the Discussion. In order to test the effect of H2Bubi on Lsk1 recruitment, we have performed a ChIP experiment of Lsk1 in a wild type and a H2B K119R strains . The resste11 induction.Supporting this possibility, the level of Lsk1 was increased at the ste11 promoter when histone H2B ubiquitylation was abolished (“Considering that H2B ubiquitylation was reported to act as a barrier to Ctk1 (the Lsk1 orthologue of budding yeast) , we speculate that this additional layer of control  may alsobolished .”The referee then asks additional questions regarding the regulation  of H2Bubi. Some aspects have been largely covered over the years by previous studies from the Tanny, Gould, Ekwall, Grewal, Winston and Allis laboratories, leading to a pretty good description of the pathway leading the H2B ubiquitylation in fission yeast. In addition, we believe that these experiments are beyond the scope of the current work and message. Moreover, they would require several months of work. For example, we have no experience with Bre1 ChIP and preliminary set up for these type of experiments is always time consuming.5) Present evidence confirming that the htb1-KR mutant does not show HDAC recruitment to the ste11 promoter and that H3ac levels remain high, consistent with H2Bub1 being upstream of H3K4me3 in the pathway.ste11 promoter. This constitutes an evidence that H2B-ubi is upstream of the repressive deacetylation occurring at the ste11 promoter. The text was modified as follows:We present a new experiment where we have tested the issue raised by the referee by chipping the acetylated K14 histone H3  in the strains lacking H2B ubiquitylation, which results an elevated acetylation at the ste11 promoter when H2B ubiquitylation was abolished in either the htb1-K119R or the rhp6 mutant  This study would be significantly enhanced if the authors extended their MNase mapping experiments genome wide in rsc∆ mutants, and then comparing the results to those reported for an S2A mutant. This would broaden the authors' conclusions and complement their in-depth analysis of a single gene. We have responded to this request by analysing the genome wide effect of the deletion of Rsc1, a non essential subunit of RSC, on nucleosome positioning, and by comparing it with the CTD S2A mutant. A new figure . p-value < 0.05 indicated by *, n.s.: non-significant. This is added to the legend.Introduction, first paragraph: "However, in the absence of Set1, most genes are upregulated, suggesting a predominant role in repression." To the best of my knowledge, this is incorrect. In budding yeast, Boa et al.  reported that transcript levels of most genes (80%) were downregulated in the absence of Set1 and H3K4 methylation. In contrast, Set1 and H3K4 methylation does silence the rDNA . The statement should be justified or corrected and referenced appropriately.eLife. 2014 Dec 15;3:e04506). They both revealed a predominantly repressive role of Set1.The referee cites works done in budding yeast. In fission yeast, to our knowledge, two studies have analysed the effect of set1 deletion on gene expression genome-wide


Transcription is affected via the modulation of the concentrations of the different types of holoenzymes, so saturated promoters are only weakly affected by sigma factor competition. However, in case of overlapping promoters or promoters recognized by two types of sigma factors, we find that even saturated promoters are strongly affected. Active transcription effectively lowers the affinity between the sigma factor driving it and the core RNAP, resulting in complex cross-talk effects. Sigma factor competition is not strongly affected by non-specific binding of core RNAPs, sigma factors and holoenzymes to DNA. Finally, we analyze the role of increased core RNAP availability upon the shut-down of ribosomal RNA transcription during the stringent response. We find that passive up-regulation of alternative sigma-dependent transcription is not only possible, but also displays hypersensitivity based on the sigma factor competition. Our theoretical analysis thus provides support for a significant role of passive control during that global switch of the gene expression program.Sigma factors control global switches of the genetic expression program in bacteria. Different sigma factors compete for binding to a limited pool of RNA polymerase (RNAP) core enzymes, providing a mechanism for cross-talk between genes or gene classes via the sharing of expression machinery. To analyze the contribution of sigma factor competition to global changes in gene expression, we develop a theoretical model that describes binding between sigma factors and core RNAP, transcription, non-specific binding to DNA and the modulation of the availability of the molecular components. The model is validated by comparison with  in vitro competition assays and to address the question of indirect or passive control in the stringent response upon amino acids starvation. We show that sigma factor competition provides a mechanism for a passive up-regulation of the stress specific sigma-driven genes due to the increased availability of RNA polymerase in the stringent response. Moreover, we find that active separation of sigma factor from the RNA polymerase during early transcript elongation weakens the sigma factor-RNA polymerase equilibrium constant, raising the question of how their in vitro measure is relevant in the cell.Bacteria respond to changing environmental conditions by switching the global pattern of expressed genes. A key mechanism for global switches of the transcriptional program depends on alternative sigma factors that bind the RNA polymerase core enzyme and direct it towards the appropriate stress response genes. Competition of different sigma factors for a limited amount of RNA polymerase is believed to play a central role in this global switch. Here, a theoretical approach is used towards a quantitative understanding of sigma factor competition and its effects on gene expression. The model is used to quantitatively describe  Escherichia coli this competition is indicated by the re-distribution of ribosomes between protein classes upon changes in cell growth conditions During recent years, much effort has been made towards the quantitative characterization of gene regulation and regulatory networks E. coli, B. subtilis) is required for most transcription during growth, while other sigma factors act as master regulators for stress responses such as heat shock or entry to stationary phase (E. coli) or for developmental programs such as growth of flagella (E. coli) and sporulation (B. subtilis). In addition some phages carry genes for sigma factors that direct transcription to phage genes in vitro transcription assays At the level of transcription, such coupling appears to be weaker, as RNA polymerase core enzyme is available in excess of the numbers needed for transcription As a result of competition, any increase in activity of one sigma factor indirectly represses binding of other sigma factors to core RNAP and thus transcription of the genes they control. Such passive control has been proposed to contribute to the switch of the global gene expression program et al.in vitro competition experiments from the literature σ70-dependent transcription against passive effects such as an increased RNA polymerase concentration due to the stop of ribosomal RNA transcription. By contrast, we show here that non-specific binding does not buffer alternative σ-dependent transcription against such passive effects, supporting a role for passive up-regulation of alternative σ-dependent stress response genes In this article, we develop a model for sigma factor competition to quantitatively analyze different situations. Our model is based on and extends previous theoretical work on sigma factor competition by Grigorova et al.To analyze sigma factor competition, we have developed a quasi-steady state model based on earlier work by Grigorova For simplicity, we focus on the case of only two competing sigma factors, the housekeeping sigma factor E. coli cell, and modulate the concentration of in vitro experiments and mimics the accumulation of alternative sigma factors during the transition from exponential to stationary phase. First, we study the formation of holoenzymes in the absence of transcription (i.e. no DNA present) as in We consider fixed concentrations of core RNAP and  factors . The stain vitro competition experiment between We next used the model described so far to analyze an in vitro competition experiment among the seven sigma factors of E. coli performed by Maeda et al.et al. from the same data using a fit that assumed a saturation condition.The concentration of a certain species of holoenzyme can be written as a function of the concentration of the holoenzymes of a competing species, their relative dissociation constants, and the total number of sigma factors asin vitro experiments, but may not hold in the cell; the latter case will be discussed below. σ70-dependent promoter as a function of the increasing amount of Next, we examine the transcription rates. Each holoenzyme species transcribes a set of cognate genes with a transcription rate that depends on the holoenzyme concentration and on the parameters of the promoter, which is described with a Michaelis-Menten model see . We assuHσ-dependent PdnaK promoter was mixed with fixed concentrations of RNAPs and The prediction for the transcription rate can be compared to another in vitro competition experiment, this time between σ70-driven transcription , we find that non-specific binding strongly reduces the concentration of free holoenzymes and, thus, specific binding to promoters. In in vitro transcription if different experimental conditions are used.In our model, using parameters expected for the situation in the cell  and alternative sigma-driven promoters . Thus, the stop of rrn transcription releases 2600 core RNAPs, so that the total number of available cores to transcribe mRNA is increased to rrn-transcribing cores in the stringent response  and the maximal sensitivity is found for Altσ-dependent transcription by repeating the analysis above for a wide range of Altσ-dependent transcription, for large numbers of core RNAPs and rrn transcription, either a sizeable pool of e.g. by release from anti-sigma factors or by a stop of turnover. Once a level of Altσ-dependent transcription are similar. The response factor of the transcription rate of Altσ-dependent genes to an increase of core RNAPs is shown by the density plot of In addition to the release of core polymerases from the ribosomal genes, the response to stress such as amino acid starvation also involves the accumulation of alternative sigma factors via their increased synthesis and reduced degradation as well as through release of sigma factors sequestered by anti-sigma factors In this study, we have analyzed sigma factor competition in bacterial transcription, a mechanism by which gene can be controlled “passively” either through the cross-talk with another set of genes that are specifically regulated or by a change in the availability of the components of the transcription machinery, core RNAP and sigma factors. Extending previous work in vitro competition experiments We have used the model to describe several e.g., to stress responses, where sigma factor competition is induced.  and initiates transcription upon activation by an ATPase activator, which typically binds at distance from promoter and contacts the holoenzyme via DNA looping gln from Salmonella typhimurium) has recently been determined using single-molecule fluorescence Nσ-driven transcription from competition may be related to the special role of Nσ-driven promoters are saturated and protected from competition. Effects of sigma factor competition have been reported for several weaker E. coli) in vitro and in vivoE. coli and have specific non-housekeeping functions in their native hosts. In E. coli transcription from these promoters is induced during transition to stationary phase, similar to stress response transcription The paradigmatic case of a saturated promoter in bacteria is a promoter under the control of A key condition for competition is that sigma factors are in excess of core RNAP. When binding between sigma factors and core RNAP is strong, as experimentally observed with dissociation constants in the nM range and the competing sigma factors have approximately the same affinity for core, this condition is very intuitive: when core is in excess, all sigma factors are found in holoenzymes and no competition is obtained; competition sets in when the total number of available sigma factors is larger than the number of available core enzymes (not counting “unavailable” sigma factors and cores that are for example sequestered by anti-sigma factors or tied up in transcript elongation). This conditions is a general property of systems with one-to-one stoichiometry and competition for binding and similar observations have been made for small regulatory RNAs in vitro, they should have a bigger impact in vivo, where there transcription does perturb the pool of free holoenzymes, thus calling into question how relevant the measured equilibrium dissociation constant are for the cell. These effects could be tested experimentally, e.g. by implementing the scheme of transcription studied in in vitro transcription assays.The hierarchy of sigma factor binding may also be affected by the transcriptional activity of the different holoenzymes, because transcription affects sigma factor competition in complex ways. Transcript elongation sequesters core RNAPs and, to a lesser extent, sigma factors, thus modulating the availability of these components. In addition, transcription also serves as a pathway for the effective dissociation of holoenzymes, effectively increasing their dissociation constant. While these effects are likely of minor importance σ70-dependent transcription of biosynthetic operons We have then studied the effects of non-specific binding of core RNAPs and holoenzymes to DNA. Non-specific binding may in principle interfere with sigma factor competition if different holoenzymes and/or core RNAP have different non-specific dissociation constants by shifting the binding equilibrium such as to minimize the overall binding energy. If however, these dissociation constants are approximately the same, as it is likely the case under physiological ionic strength The last observation suggests that passive up-regulation of alternative sigma factor transcription can be expected during the stringent response, as proposed in vivo, a consistent set of all concentrations of the different holoenzymes under different conditions (e.g. different growth rates or different time points during a stress response or entry to stationary phase) would be invaluable. One may even imagine a partitioning of sigma factors and holoenzymes similar to a recent study for core RNAP We note that for a more detailed study of these passive effects B. subtilis has demonstrated interesting pulsing dynamics of a sigma factor, driven by cycles of auto-activation and sequestration Finally, sigma factors have been proposed as versatile components for synthetic gene circuits. Sequestration of sigma factors by anti-sigma factors and competition for core RNAP provide mechanisms for genetic switches Sigma-core binding is described by the equilibrium of the reactionAt equilibrium, they also fulfillIf two sigma factors compete for core RNAPs, we haveAt equilibrium, the dissociation constants Analytical expressions for the holoenzyme concentration can be found for some special cases see  but in gi.e. for whichWhile the onset of sigma factor competition is abrupt for very strong sigma-core binding, in general, there is a smooth transition. Thus, we define the onset of competition to be the point where the alternative sigma factors cause a 5% reduction of The results above can be extended to the scenario where more than two sigma factor species  with a promoter σ70-dependent promoter , hence For the case of a single sigma factor species with Dividing The binding of the holoenzyme to the cognate promoter Altσ-dependent genes) to the change of a control parameter The response coefficient Generally, the maximum of the response factor From the analytical solutions of the free binding case with strong core-sigma binding affinities  (stars)  . The ind(TIF)Click here for additional data file.Figure S2Effect of anti-sigma factors. (A) Formation of holoenzyme (TIF)Click here for additional data file.Table S1Dissociation constants of different holoenzyme species relative to , from reference  (second column) and according to our fit with  (third column).(PDF)Click here for additional data file.Text S1Analytical solutions.(PDF)Click here for additional data file.Text S2Values of the parameters used in the simulations.(PDF)Click here for additional data file.Text S3Estimate of association, dissociation and initiation rate from a Nσ-dependent promoter.(PDF)Click here for additional data file.


Tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the 5′ leader region of trp mRNAs, and downregulates expression of the operon by promoting transcription termination prior to the structural genes. Anti-TRAP (AT) is an antagonist that binds to tryptophan-activated TRAP and prevents TRAP from binding to RNA, thereby upregulating expression of the trp genes. AT forms trimers, and multiple trimers bind to a TRAP 11mer. It is not known how many trimers must bind to TRAP in order to interfere with RNA binding. Studies of isolated TRAP and AT showed that AT can prevent TRAP from binding to the trp leader RNA but cannot dissociate a pre-formed TRAP-RNA complex. Here, we show that AT can prevent TRAP-mediated termination of transcription by inducing dissociation of TRAP from the nascent RNA when it has bound to fewer than all 11 (G/U)AG repeats. The 5′-most region of the TRAP binding site in the nascent transcript is most susceptible to dissociation from TRAP. We also show that one AT trimer bound to TRAP 11mer reduces the affinity of TRAP for RNA and eliminates TRAP-mediated transcription termination in vitro.An 11-subunit protein called  Bacillus subtilis the trpRNA binding Attenuation Protein (TRAP) regulates transcription of the trpEDCFBA operon through an attenuation mechanism in response to changes in the intracellular concentration of tryptophan AG repeat interacts with Glu36 and Lys 37 from one subunit, and Lys 56 and Arg 58 from the adjacent subunit of TRAP  were stalled within the leader region such that some or all of the 11(G/U)AG repeats of the TRAP binding site were exposed on the nascent transcript. TRAP was then allowed to bind to the exposed RNA from the TEC, after which the ability of AT to prevent TRAP-mediated transcription termination was assessed.Binding studies using purified AT, TRAP and  to TRAP ,19. Thes to TRAP . Howeverp operon . Hence, trp attenuator after TRAP has bound the nascent RNA as long as it has not bound to all 11 (G/U)AG repeats. Under these circumstances, AT prevents TRAP-mediated termination by inducing TRAP to dissociate from the nascent trp mRNA. Our data also suggest that the 5′-most region of the TRAP binding site RNA is most susceptible to dissociation from TRAP. We also provide evidence that a complex of one AT trimer bound to a TRAP 11mer has significantly lower affinity for its target RNA than WT TRAP. Moreover, when examined using an in vitro transcription system, this AT-TRAP complex does not induce termination in the trp leader region.Our results indicate that AT can prevent TRAP-mediated termination at the trp operon in B. subtilis. Our findings add to the existing body of knowledge of gene regulation in bacteria. In addition, our studies may have broad implications in understanding how critical processes in both single-celled and multicellular organisms may be governed through interacting oligomeric proteins.In this report we have revealed an additional and/or alternate mechanism through which AT modulates TRAP-mediated attenuation control of the Escherichia coli K802. Templates for in vitro transcription were based on the plasmid pUCtrpL, which contains a 730-bp EcoRI-HindIII fragment (−411 to +318 relative to start of transcription) including the B. subtilis trp promoter, the regulatory leader region and the first 40 codons of the trpE gene (B. subtilis RNA polymerase was isolated from the strain MH5636 as described previously (Plasmids used in these studies were propagated in rpE gene . B. subteviously  with theWT AT as well as AT containing a Cys substitution at residue 7 (D7C AT) were purified as described by Chen and Gollnick . His-taget al.  starting at position +116 relative to the start of transcription  of the 11th repeat.The E111Q mutant EcoRI (EcoRI*) protein was purified as described by Potter et al. . This muet al. . EcoRI* et al. –25. We un Figure . This EctrpLEco116. Attempts to create these templates by placing the EcoRI recognition site upstream of +116 were unsuccessful because EcoRI* blocking the TEC at these sites was inefficient. The reason for this inefficient blocking is not known but it may involve the influence of the DNA sequences surrounding the GAATTC site on the affinity of EcoRI* binding (trp leader region at the 3′-end of the TRAP binding site between +76 and +92 (relative to the start of transcription) in the Eco116 template . When the TEC is blocked by EcoRI* on these templates, 9, 8 or 7 of the (G/U)AG repeats of the TRAP binding site are exposed on the nascent transcript respectively.Templates from which subsets of the 11 triplet repeats of the TRAP binding site are exposed on the nascent transcript when the TEC is blocked by EcoRI* were created based on pUC binding . Hence we Figure . These tin vitro transcription were created by polymerase chain reaction (PCR) using the plasmids described above. In each case, the primers used were the standard M13 Reverse primer and a primer corresponding to −104 to −83 of the trp leader region (relative to the start of transcription) (Integrated DNA Technologies). The PCR products were purified by phenol-chloroform extraction followed by ethanol precipitation. When transcription templates were created to bind to streptavidin-coated beads, the 5′-end of the M13 Reverse primer was biotinylated (Integrated DNA Technologies). The PCR products were purified as described above and the resulting 5′ biotinylated templates were coupled to streptavidin-coated paramagnetic beads (Promega) according to the manufacturer's instructions.Templates for 32P UTP (32P-labeled (GAGUU)11 RNA (∼5000 dpm) and 10 μg yeast RNA in filter binding buffer  containing 1 mM tryptophan. After incubation for 20 min at 37°C, 40 μl aliquots from each 50 μl reaction were filtered through a two membrane layer using a 96-well dot blot mini fold apparatus (Schleicher and Schuell). The membranes consisted of 0.45 μm nitrocellulose (Schleicher and Schuell), which retains protein and any RNA bound to the protein, on top of Hybond-N+ (Amersham), which retains unbound RNA. Each reaction was washed with 80 μl filter binding buffer. The membranes were dried and exposed to a phosphorstorage screen , which was scanned with a Storm Phosphorimager . Quantification of each assay was performed using ImageQuant Software . For each reaction, the amount of RNA on the nitrocellulose divided by total RNA on nitrocellulose and Hybond was used to calculate the fraction of RNA bound to TRAP. The data were analyzed using a non-linear single binding-site regression algorithm  to determine apparent dissociation constant (KD) values.A previously developed filter binding assay was used to measure RNA binding to TRAP or to AT-TRAP complexes . The 55 32P UTP . Increastrp attenuator was examined using an in vitro transcription attenuation assay based on the method described by Potter et al.  for 5 min at 37ºC prior to initiating transcription. Transcription was initiated by addition of RNA polymerase (RNAP) (50 μg/ml) together with 8 μM ATP and GTP as well as 2 μM UTP and 1 μCi [α-32P] UTP (3000 Ci/mmol), followed by incubation for 10 min at 37°C. Heparin (0.1 mg/ml) was then added to prevent re-initiation, and transcription was continued for 10 min at 37°C in the presence of 16 μM NTPs and 1 mM L-tryptophan either in the absence of TRAP, the presence of TRAP, or with TRAP that had been pre-incubated with AT.The effect of AT on TRAP-mediated termination at the r et al. . For reatrp transcript, AT and tryptophan-activated TRAP were combined prior to addition to the transcription reaction. To examine AT function after TRAP had bound the nascent trp transcript, AT was added after TRAP had been incubated in these reactions for 10 min at 37°C. For transcription reactions involving dissociation of EcoRI* from the DNA, 0.5 M KCl was added followed by an additional 10 min incubation at 37°C. All reactions were stopped by addition of an equal volume of stop solution . Samples were denatured at 95°C for 2 min and run on 6% polyacrylamide-8 M urea gels at 20 mA for 1 h. Gels were dried and exposed to a phosphorstorage screen, scanned and quantified as described in the previous section.To examine the ability of AT to interact with TRAP before it has bound to the nascent To allow comparisons of the effects of AT on TRAP when different DNA templates were used, we defined AT activity as the ability of AT to prevent TRAP-mediated termination. To assess AT activity, we first measured TRAP-mediated termination for each template as the increase in the percentage of TECs that terminate in leader region in the presence of TRAP above that observed in the absence of TRAP. AT activity was then calculated as the ratio of the decrease in TRAP-mediated termination in the presence of AT compared to TRAP-mediated termination in the absence of AT.in vitro transcription were constructed that expose either all 11 or subsets of the (G/U)AG repeats in the trp leader region as described above. In vitro transcription reactions were performed using these bead-bound templates as described above but in the absence of radiolabeled UTP, and the reactions were scaled up 8-fold. After transcription was allowed to elongate to the EcoRI* roadblock, TRAP was added followed by a 10 min incubation at 37°C. AT was added to TRAP either before or after it was allowed to bind to the nascent transcript, followed by a 10 min incubation. Bead-bound TECs were separated from the solution with a magnet and washed twice with transcription buffer containing 1 mM L-tryptophan to remove unbound TRAP. After washing, the beads were resuspended in transcription buffer with 0.1 mg/ml DNaseI and incubated for 15 min at 37°C to free the ternary complexes from the bead. These supernatants were filtered through a nitrocellulose membrane using a Minifold system (Schleicher and Schuell).Pull down assays were performed to examine association of TRAP with the nascent RNA in the absence or presence of AT. Biotinylated DNA templates for B. subtilis TRAP followed by goat anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (Bio-Rad). Detection was with Amersham ECL Plus using a Storm 860  at 450 nm. Images were quantified using ImageQuant software . For each template, the amount of background TRAP pulled down in the absence of EcoRI* was subtracted from the amount measured with the blocked TECs on each template. The amount of TRAP pulled down when the TEC was blocked with EcoRI* in the absence of AT was set to 100%.The amount of TRAP associated with the ternary complexes was determined by immunoblotting using polyclonal rabbit antibodies against Similar pull down assays were performed to examine the susceptibility of the 5′ or 3′ region of the RNA binding site to dissociate from TRAP. For these assays, instead of adding AT, DNA oligonucleotides were used to compete with TRAP for binding to the nascent RNA. 200 nM DNA oligonucleotides (oligo) were added to the transcription reaction after allowing TRAP to bind to the nascent transcript followed by 10 min incubation at 37°C. The following oligos were used: 5′ antisense, complementary to either 3 (G/U)AG triplet repeats of the 5′-most portion of the TRAP binding site (+32 to +52 relative to the start of transcription); 3′ antisense, complementary to 3 (G/U)AG repeats of the 3′-most portion of the TRAP binding site (+66 to +82 or +61); 5′ sense, containing the same sequence as the first three (G/U)AG repeats of the binding site (+32 to +52), and a non-specific oligo, containing 102 nt of random sequence with no similarity to the TRAP binding site.Bismaleimidoethane (BMOE) was used to crosslink AT and TRAP. BMOE is a homobifunctional crosslinker for covalent, irreversible crosslinking between sulfhydryl groups (Pierce). The distance between the functional groups in BMOE is 8 Å and therefore only cysteine (Cys) residues in close proximity are crosslinked. In the crystal structure of the AT-TRAP complex the side chains of Asn 20 of TRAP and Asp 7 of AT are at an appropriate distance and orientation to be crosslinked by BMOE . We intrPrior to the crosslinking reaction, N20C TRAP and D7C AT were passed through a G-25 Sephadex spin column equilibrated with crosslinking buffer (PBS pH 7.2 and 0.5 mM EDTA). N20C TRAP (0.02 mM) and D7C AT (0.08 mM) were mixed in the presence of 1 mM tryptophan in crosslinking buffer and BMOE (0.04 mM) followed by incubation at 37°C for 1 h. The reactions were quenched with 2 mM DTT at 37°C for 15 min. Crosslinking was analyzed on 15% SDS gels run at 60 mA for 1 h and then stained with Coomassie blue.1(WT)10, were purified on Ni agarose. Since only the N20C subunit can crosslink with D7C AT, only one AT trimer can crosslink to each (N20C)1(WT)10 TRAP hetero-11mer. These TRAP hetero-11mers were mixed with 5-fold excess D7C AT in crosslinking buffer and crosslinked with BMOE as described above. Complexes containing D7C AT crosslinked to (N20C)1(WT)10 TRAP were purified on two successive immunoaffinity chromatography columns. The first contained antibodies against B. subtilis TRAP 1(WT)10 TRAP complex, which is referred to as TRAP-AT = 1:1.To generate specific complexes with one AT trimer bound to each TRAP 11mer, we created TRAP hetero-11mers composed of one N20C subunit and 10 WT subunits . To do slis TRAP  and the trp leader RNA but cannot dissociate RNA from TRAP after it has bound AG triplet repeats , and those that read through the attenuator and continue to the end of the template . Since 14 nt of the nascent transcript are within the blocked TEC AG repeats of the TRAP binding site are exposed on the nascent transcript from the stalled TEC. When TRAP binds to these transcripts, approximately 3 or 2 subunits of the TRAP 11mer should be unbound respectively. When AT was added after TRAP had bound to the nascent transcript, the AT activity correlates with the number of free TRAP subunits that are not predicted to be bound by RNA AG repeats are exposed on the nascent transcript when the TEC is blocked by EcoRI* Figure . This arAddition of either a non-specific oligo, or a sense oligo with the same sequence as the 5′-most (G/U)AG repeats of the TRAP binding site (5′ sense), did not affect the amount of TRAP associated with the TEC Figure . In contThe observed differences in the effects of oligos targeted to the 5′ or 3′ regions of the TRAP binding site could be due to the 3′ region of the RNA being less accessible as a result of close juxtaposition of TRAP and RNAP in the blocked TEC Figure . To testTogether these results indicate that within the TRAP-RNA-TEC complex, the 5′-most portion of the RNA of the TRAP binding site is more susceptible to dissociation from TRAP. These observations suggest that this portion of the RNA may be less stably bound to TRAP. If so, then AT may also initiate dissociation of TRAP from the 5′-most portion of the bound RNA.3) can bind to each TRAP 11mer 11 RNA. To do so, we used chemical crosslinking to create a complex with one AT trimer specifically bound to each TRAP 11mer. BMOE is a homobifunctional crosslinker specific for sulfhydryl groups. With 8 Å between the reactive maleimide groups, BMOE only crosslinks proteins with cysteine (Cys) residues in close proximity. WT TRAP does not contain any Cys residues  as compared to WT TRAP (KD = 0.55 nM)  Figure .3 bound per TRAP 11mer on its ability to induce transcription termination at the trp attenuator in vitro. Adding increasing amounts of tryptophan-activated WT TRAP increased the number of terminated transcripts accompanied by a decrease in the fraction of read-through transcripts, indicative of TRAP-induced termination at the trp attenuator .The explanation for the differences in the observed ability of AT to induce TRAP to dissociate from isolated RNA as compared to a nascent transcript is not yet clear. However, it seems likely that the difference relates to the absence or presence of RNAP. Several observations suggest that TRAP interacts directly with RNAP to induce transcription termination. First, we have found that TRAP can induce efficient termination within a modified rminator . SecondlInteraction between TRAP and RNAP may alter the manner in which TRAP binds to RNA so as to allow AT to induce dissociation of TRAP. Comparing the activity of AT on TRAP bound to the nascent transcripts from blocked TECs on the Δ10–11Eco116 and Δ10–11Eco116EX templates supports this proposal. The RNAs from these TECs expose nearly identical numbers of (G/U)AG repeats but on the former template the 3′-most repeat is immediately adjacent to RNAP whereas the Δ10–11Eco116EX template encodes 16 additional nucleotides between the last repeat and the EcoRI site on the DNA. Hence, TRAP is likely in close juxtaposition with RNAP on the Δ10–11Eco116 template Figure  but may trp attenuator region . Hence, this mutant protein may contain a substitution in the region of TRAP that interacts with RNAP to induce transcription termination. AT is approximately 3-fold less efficient at inducing dissociation of E60K than WT TRAP from the nascent transcript from TECs blocked on Δ9–11Eco116 template (data not shown). Together these observations are all consistent with the suggestion that TRAP interacts directly with RNAP and that this interaction is important for AT-mediated dissociation of TRAP from the nascent transcript.A recently identified TRAP mutant (E60K) has also provided evidence for a role of an interaction between TRAP and RNAP in influencing AT's ability to modulate TRAP function. E60K TRAP binds RNA and tryptophan with similar properties as the WT protein but fails to induce termination within the AT is approximately twice as effective at inducing dissociation of TRAP from stalled TECs that expose 7 or 8 (G/U)AG repeats as when 9 repeats are exposed Figure . The strtrp operon  AT trimers would bind to a subset of the available TRAP 11mers. Alternatively, it is possible that binding of as few as one AT trimer can interfere with the ability of a TRAP 11mer to bind RNA and/or cause transcription to terminate in the trp leader region. To test this hypothesis, we created a model complex with one AT trimer bound per TRAP 11mer using site-specific chemical crosslinking. This complex showed approximately 7-fold lower affinity for RNA containing 11 (GAGUU) repeats  also binds RNA with ∼7-fold lower affinity than WT TRAP, and this mutant protein shows ∼14-fold reduced regulation of a trpE‘-’lacZ fusion in vivo as compared to WT TRAP (3 bound to a TRAP 11mer may similarly reduce the ability of TRAP to regulate the trp operon.Several studies have shown that multiple AT trimers can bind to the TRAP 11mer ,31. Howecking AT . If mults Figure  as well o Figure . It is ntrp operon at low ratios of AT3 to TRAP 11mer may be that each AT3 binds to multiple TRAP 11mers. Each trimer of AT contains three identical binding sites for TRAP (3 to TRAP 11mer ratios using AU as well as small Angle X-Ray Scattering have provided further evidence for multiple TRAP 11mers that are linked by AT trimers at low ratios of AT3 to TRAP 11mer (An alternate explanation for the observed increase in expression of the for TRAP . The cryfor TRAP . Analytifor TRAP . More reAP 11mer .trp operon in B. subtilis. Interaction of AT with TRAP during transcription of the leader region may provide a second opportunity for AT to prevent TRAP-mediated termination of the trp operon. Modulation of TRAP-mediated termination by AT after TRAP has initiated binding to the regulatory leader region may increase the likelihood that transcription reads through the trp attenuator so that the trp genes are expressed. AT-mediated regulation of the trp operon during transcription of the leader region may provide more flexibility to fine-tune tryptophan synthesis.In this report, we have provided evidence for an additional mechanism through which AT modulates TRAP-mediated attenuation control of the Gene regulation is indispensable for maintaining normal cellular function and for versatility and adaptability of an organism to its environment. Abnormal gene regulation is linked to various diseases. Therefore, fine-tuning the level of gene expression is necessary to maintain cellular homeostasis. Akin to the fundamental processes in eukaryotic cells, tryptophan metabolism in bacteria is highly regulated via RNA-protein and protein–protein interaction. These molecular interactions allow the cell to adapt to the environment depending on the availability of the nutrient tryptophan. Our findings may be applicable in understanding how crucial processes are regulated through interacting oligomeric proteins in both lower and higher organisms, including humans.Supplementary Data are available at NAR Online.


The constitutive expression of the high-risk HPV E6 and E7 viral oncogenes is the major cause of cervical cancer. To comprehensively explore the composition of HPV16 early transcripts and their genomic annotation, cervical squamous epithelial tissues from 40 HPV16-infected patients were collected for analysis of papillomavirus oncogene transcripts (APOT). We observed different transcription patterns of HPV16 oncogenes in progression of cervical lesions to cervical cancer and identified one novel transcript. Multiple-integration events in the tissues of cervical carcinoma (CxCa) are significantly more often than those of low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL). Moreover, most cellular genes within or near these integration sites are cancer-associated genes. Taken together, this study suggests that the multiple-integrations of HPV genome during persistent viral infection, which thereby alters the expression patterns of viral oncogenes and integration-related cellular genes, play a crucial role in progression of cervical lesions to cervix cancer. Cervical cancer is the second leading cause of cancer-related death in women worldwide. The persistent infection by high-risk human papillomavirus (HR-HPV), such as genotype 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 are essential for the progression of cervical lesions A viral early promoter lied upstream of the E6 ORF, such as P97 in HPV16 To date, a full transcription map of oncogenic HPV16 and HPV18 in HPV-infected cells and raft tissues have been constructed It's well known that the integration of HPV genomes is a key event in cervical carcinogenesis ∧E4) and several integrated HPV transcripts  in HPV16-infected tissues. However, transcriptional selection in response to environmental changes is a dynamic process to achieve optimal gene expression for cell survival and carcinogenesis Transcription patterns of HPV16 in the tissues of cervical cancer have been reported Tissue samples of primary uterine cervical lesions containing dysplastic epithelium/tumor cells were collected from the Second Affiliated Hospital of Wenzhou Medical University  from December 2010 to April 2012. The presence of HR-HPV was detected by HCII test, and the screening of HPV16 in HR-HPV-positive samples was done by HPV genotypes detection kit  Total RNA from biopsy samples described above was isolated using TRIzol reagent  according to the manufacturer's instructions. To remove the residual DNA contamination, the RNA preparation was treated with Rnase-free Dnase I  according to the manufacturer's protocol. Purified total RNA was dissolved in Rnase-free water and stored at −80°C. The concentration and purity of total RNA were quantified by the ultraviolet spectrophotometer at 260 nm and 280 nm and 1% agarose gel electrophoresis. Only RNA samples with an A260/A280 ratio of 1.8–2.0 and high integrity were used for the further experiment.17-primer coupled to a linker sequence RTp1-HPV16E7 specific primer (5′-CGGACAGAGCCCATTACAAT-3′) and linker p0 (5′-GACTCGAGTCGACATCG-3′) as the reverse primer; and the PCR amplification was carried out in a reaction volume of 50 µl. Different from previous reports, the PCR cycles was increased to 35, and all specimens only performed one-round PCR reaction. To verify the specificity of this procedure, the “minus-RT” control in which reverse transcriptase was omitted from the reactions was also performed parallel.APOT assay reported previously was based on nested PCR reactions The APOT amplification products were visualized by 2.5% agarose gel electrophoresis. PCR products of interest were excised from the gel and extracted using DNA agarose gel recovery kit . The corresponding amplimeres were cloned into cloning vector  and DNA sequence analysis was executed using an ABI 3730 XL Genetic Analyser  according to standard protocols. Sequencing results were analyzed using the BLASTn program provided by the National Center for Biotechnology Information, USA. Additionally, the chromosomal integration sites were ascertained using the National Center for Biotechnology Information (BLAST) and European Molecular Biology Laboratory (EBI). Moreover, the fragile sites and genes of integration sites were defined using the NCBI fragile site map viewer and the UCSC Blat tool.The principle of the APOT assay is a 3′ rapid amplification of cDNA ends (RACE) PCR assay that achieves amplification and cloning of the region between a single short sequence in a cDNA molecule and its unknown 3′- end To analyze the HPV16 oncogene transcripts, 40 HPV16-positive cervical specimens  with good quality RNA were selected among 63 collected samples in this study. Total 133 transcripts containing viral fragments were found. Among these transcripts, 64 fragments had HPV16 E7-E1* sequences at their 5′- ends and directly connected with poly A at their 3′- ends . Further* sequences at their 5′- ends and directly connected to host genome sequences. However, there were two different integration sites of E1 region (at nt880 and nt1107) in this type  at nt 729. These HPV16 oncogene integrated transcripts could be divided three different types . Among this type . The sitMoreover, HPV16 oncogenes showed significantly different transcription patterns in the tissues of LSIL, HSIL and CxCa . Among tTo identify the individual chromosomal locations, all 64 fusion-transcripts containing viral and cellular sequences were further analyzed by BLASTn comparisons to the whole genome database. Our data show that all chromosomes, except for Chr21 and X, were integrated with HPV16 genome, confirming the previous reports that no preferential HPV integration site was seen in selection of the human chromosome AMICA1, DAPK1, EBAG9, PIBF1 were affected twice, MRPS31 four times and PRDX5 even six times by the viral integration. At the same time, the nearest host genes to each integration site in the direction of transcription were also analyzed , tumor suppressor genes (TIMP3), or tumor-related genes (PIBF1and MRPS31).The cellular flanking sequences of viral-cellular fusion transcripts were further examined for known genes. Most of these fused transcripts had a cellular sequence from the coding orientation of known genes and thirty transcripts had the cellular sequence from an intron region, and 8 transcripts were fused with a sense exon sequence of the predicted genes . Among tanalyzed . Among tIntegration of HPV genome into host chromosomes represents an early clonal event to provide an additional selective advantage for the expansion of the neoplasm. Viral transcripts have been detected by the APOT assay HPV16 transcription patterns in LSIL, HSIL, and CxCa were significantly different. We found that the Type C transcript was only detected in the samples of CxCa and more random integration sites existed in our tissue samples. Similar to previous reports miR-34a, MSH2, WWOX and TIMP3, et al) or oncogenes . Interestingly, most of them were not reported in previous documents MiR-34a, an important tumor suppressor, is down-regulated in cervical cancer miR-34a by destabilization of p53 and resulted in cell proliferation miR-34a gene might further interpret the phenomenon of reduced expression of miR-34a in cervical cancer. MSH2 is a DNA mismatch repair protein, and associated with DNA repair pathway ROCK2, an important signaling molecule, can promote cervical cancer metastasis by upregulating and activating the expression and function of moesin protein through RhoA/ROCK2 pathway FANCM which is a DNA translocase and highly related to DNA replication regulates checkpoint signaling and replication fork progression COX6B1 is related to cell apoptosis ESRRA also have been reported associated with cervical cancer PRDX5, EBAG9 and CD28, etc. These integrations were generally deemed of no interest. However, their sense sequences were associated with DNA restoration or tumor development and might affect both host and viral gene expression during the development of cervical cancer. The most integration in the antisense orientation was the gene encoding peroxiredoxin 5 (PRDX5), a protective emzyme against oxidative stress EBAG9 expression has been observed in several malignant tumors CD28 which maintains immune homeostasis plays a role in increasing susceptibility to cervical cancer The integration not only changes the transcription pattern relevant for the dysregulated expression of the viral oncogenes, but also affects the expression of the host gene with virus genome integration. The integration alters the expression of host genes in integration sites, even if this occurs within the intron sequences In conclusion, changes of the transcription patterns of HPV 16 early genes go along with the progression from cervical intraepithelial neoplasia to cervix carcinoma and viral genome integration into host chromosome. The change or selection of transcription patterns and the integration on the expression of host genes in the integration sites and flanking cellular sequence regions might all take part in oncogenesis of HPV16-induced cancers.Figure S1The types of viral sequences connected with poly A at their 3′- ends. The type of Class I shows E1 sequences directly ended with poly A; the type of Class II shows E1 spliced to E4 and then to L1 and also ended with poly A sequences. ▴, there are several truncation sites in E1 (data shown in (TIF)Click here for additional data file.Figure S2Different truncation sites in E1 region in the type of Class I. There are four truncated sites in E1, 880, 949, 1054 and 1234, respectively.(TIF)Click here for additional data file.Figure S3Several spliced donor sites in E1. In Type A there are two integration sites in E1, 880, and 1107, respectively. The solid boxes mean cellular sequences.(TIF)Click here for additional data file.


Trim33; the mutants displayed increased expression of the reporter transgene. ChIP-seq of Trim33 in testis revealed 9,109 peaks, mostly at promoters. This is the first report of ChIP-seq for Trim33 in any tissue. Comparison with ENCODE datasets showed that regions of high read density for Trim33 had high read density for histone marks associated with transcriptional activity and mapping to TE consensus sequences revealed Trim33 enrichment at RLTR10B, the LTR of one of the youngest retrotransposons in the mouse genome, MMERVK10C. We identified consensus sequences from the 266 regions at which Trim33 ChIP-seq peaks overlapped RLTR10B elements and found a match to the A-Myb DNA-binding site. We found that TRIM33 has E3 ubiquitin ligase activity for A-MYB and regulates its abundance. RNA-seq revealed that mice haploinsufficient for Trim33 had altered expression of a small group of genes in the testis and the gene with the most significant increase was found to be transcribed from an upstream RLTR10B. These studies provide the first evidence that A-Myb has a role in the actions of Trim33 and suggest a role for both A-Myb and Trim33 in the arms race between the transposon and the host. This the first report of any factor specifically regulating RLTR10B and adds to the current literature on the silencing of MMERVK10C retrotransposons. This is also the first report that A-Myb has a role in the transcription of any retrotransposon.Transposable elements (TEs) have been active in the mammalian genome for millions of years and the silencing of these elements in the germline is important for the survival of the host. Mice carrying reporter transgenes can be used to model transcriptional silencing. A mutagenesis screen for modifiers of epigenetic gene silencing produced a line with a mutation in  Almost half of the genomes of humans and mice are made up of transposable elements. During host evolution, subsets of these elements have periods of transpositional activity during which they spread throughout the genome. This is dependent on the transcriptional activity of these elements in the cells that contribute to the germline. Hosts have evolved pathways to silence their expression. A number of Trim family proteins have been found to have a role in silencing transposable elements, and it was previously shown that Trim33 shared this function in liver. However, the function of Trim33 in other tissues is poorly understood. Here we report a role for Trim33 in silencing a specific subset of retrotransposons that contain RLTR10B LTRs, in the germline. We also show the transcription factor, A-Myb, is responsible for activating transcription of these elements and it is likely that a subset of RLTR10Bs have recently evolved Myb DNA binding sites to capitalise on the critical role that the A-Myb transcription factor has in germ cells. Suppression of A-Myb activity by Trim33 provides a plausible mechanism by which the host keeps transposons in check. Approximately half of the genomes of humans and mice are made up of transposable elements (TEs) and numerous pathways, both genetic and epigenetic, have evolved to repress their expression. Each TE has had a period of transpositional activity during which it spreads through the genome and this is dependent on its transcriptional activity in the germline. It has been suggested that during periods of epigenetic reprogramming, such as occurs in the germline, when DNA methylation is low, other mechanisms are used to suppress the expression of recently transposed retrotransposons . BecauseModifiers of murine metastable epialleles Dominant (MommeDs) [MommeD lines have been identified and reported; MommeD1,2,4,5,8–10,12–14,16–19,23,27,28,30–40,42 [MommeD6,8 [MommeD44 and the consequences of this mutation on the transcriptome in the mouse.Unbiased genetic screens for modifiers of epigenetic gene silencing have been carried out in a number of model organisms, including the mouse, and have provided a valuable tool in the identification of the proteins responsible for mediating transcriptional silencing of inserted reporter transgenes , 3. The MommeDs) . The undMommeD44 heterozygous mutants showed an increase in the expression of the reporter transgene compared to that of their wildtype littermates. The underlying N-ethyl-N-nitrosourea-induced mutation was found to have produced a null allele of the gene Trim33, Tripartite motif containing 33, also known as Tif1γ, which codes for a protein that contains a tripartite motif with ubiquitin ligase activity and two chromatin binding domains; a bromodomain and a PHD (plant homodomain) finger. The function of Trim33 remains poorly understood. In cell culture, Trim33 can act as transcriptional coregulator via the TGFβ pathway [Trim33 in the liver of mice null for Trim24 results in increased expression of the VL30 class of retrotransposons [ pathway  and mice pathway . While fnsposons  and hepansposons . Unlike nsposons , the mecMommeD44 mice, demonstrates a role for Trim33 as a dominant transcriptional repressor of RLTR10B elements in the testis and a combination of bioinformatics and biochemical studies suggests that this repression involves the A-Myb transcription factor. This is the first report of ChIP-seq for Trim33 and the first report of a function for Trim33 in the testis.Trim33 has been shown to be highly expressed in the spermatogonia and primary spermatocytes of the testis  but its MommeD44 founder was identified because it showed an increase in the proportion of red blood cells expressing the GFP reporter , using the general method described previously[The experimental pipeline for the screen has been described previously . The mutreporter . As suchTrim33 gene, which is located in the linked interval. The A → T mutation changes a lysine to a premature stop codon  was used to perform ChIP in adult testis followed by high throughput sequencing of Trim33-bound DNA. Approximately 40 million reads were generated for both Trim33 chromatin immunoprecipitated DNA and an Input control and approximately 30 million of these uniquely aligned to the genome in both cases . Peaks wUsing the ENCODE testis dataset, we found that regions of high read density for Trim33 had high read density for H3K4me3 and H3K27ac . These hTrim33 was identified in a screen for genes involved in silencing of a GFP transgene, a locus that had been inserted into the genome. This raised the possibility that it might have a role in regulating other elements recently integrated into the genome, including LINE-elements and LTR-containing retrotransposons. Using the Trim33 ChIP-seq data, we mapped reads to consensus sequences for each repeat annotation from the rodent Repbase repository, which groups elements by sequence, e.g. IAPEZI, IAPLTR1_Mm, IAPLTR2b etc. Of the approximately 1600 repeat consensus sequences, we considered only the 309 that had a RPKM (reads per kilobase per million) >10 in testis  mice and carried out RNA-seq. Using the R package DESeq and Ensembl transcript annotations (NCBI build 37), we identified 39 genes that were significantly differentially expressed   showing that transcription initiates in the repeat element (MommeD44 heterozygotes compared to that of wildtype mice (We were keen to identify loci that might be sensitive to haploinsufficiency of Trim33 in the testis and that overlapped with Trim33 enrichment. We purified RNA from the testis of wildtype (n = 3) and heterozygous xpressed . Trim33 9 of 11) . Howevery RTqPCR  has an uy RTqPCR . In the  element . Clonal ype mice , despiteNmnat3.We used Cufflinks to establish transcript annotations guided by both the raw RNA-seq data and existing annotations for the mouse mm9 genome to identify other upstream RLTR10Bs that initiate transcription of an adjacent gene. This set was used to identify transcripts that were significantly differentially expressed and linked to a RLTR10B or RLTR10B2. Ten were found , includiMommeD44 mice and overlapping with an RLTR10B element, the Vmn1r181 gene  screen carried out in a human cell line to find proteins required for transgene silencing [Trim33, Bonus, was identified as a suppressor of variegation (i.e. reduced levels of Bonus result in an increased proportion of cells expressing the reporter transgene) when tested for its ability to alter transcription of a variegating reporter locus [Trim33 in a mutagenesis screen for modifiers of transcriptional silencing in the mouse. In this screen it also behaves as a suppressor of variegation.ilencing . The flyer locus . Here weTrimMommed44 allele are consistent with this report.Trim33 was identified in a zebrafish screen for genes involved in the development of the haematopoietic system , 31. It Trim33 is not thought to bind DNA directly and available evidence suggests that its transcriptional effects occur via DNA-binding cofactors. For example, the role of Trim33 in TGF-beta signalling in cell lines has been shown to involve the Smad transcription factors that bind DNA in a sequence-specific manner , 34. It. The modin vivo, found that most Trim33 peaks in testis overlap with peaks for H3K4me3 and H3K27ac and presumably not for H3K4me0. Others have suggested that the PHD and Bromodomain of Trim33 are required to activate its ubiquitin ligase activity [The role of the chromatin binding domains (PHD and Bromodomain) of Trim33 remains unclear. Previous studies, following overexpression of the chromatin binding domains in somatic cell lines, mapped the binding specificity to H3K4me0 and acetylated lysine residues , 21. HowMultiple mouse models of cancer have demonstrated a role for Trim33 as a tumour suppressor , 35, 36.Taken together, these findings indicate Trim33 can be considered a corepressor of transcription that functions by ubiquitinating DNA-binding cofactors and that it has important roles in development and cancer.Trim33 is expressed an order of magnitude higher in the testis than in any other adult mouse tissue . It is eth out of 546 elements analysed [Our study is the first report of ChIP-seq for Trim33 in any tissue. We show that Trim33 binds many active gene promoters in the testis (i.e. those promoters marked by H3K4me3 and H3K27ac). Trim33 also shows enrichment at RLTR10B elements, a subgroup of ERVK LTR retrotransposons, consistent with our hypothesis. RLTR10Bs are among the chronologically youngest retrotransposable elements in the mouse genome, ranked 5analysed . The onlanalysed . In geneKnowing that haploinsufficiency for Trim33 was sufficiently disruptive to increase expression of the reporter transgene in our mutagenesis screen, we were keen to test whether it would alter the expression of other genes in the testis. RNA-seq suggests no dramatic effect across the genome but confirms a role for Trim33 in silencing RLTR10B. The fact that the transgene and the RLTR10B elements, but not other Trim33-bound loci, are sensitive to Trim33 dosage is not understood. It might be that other Trim family members (and other factors) can compensate for reduced levels of Trim33 at most loci. It is likely that in the complete absence of Trim33, many of the Trim33 bound loci would be affected but the early demise of the homozygous embryo precludes testing.Nmnat3. This tandem arrangement has previously been shown to increase the affinity for Myb binding to DNA [We have identified a role for Trim33 in binding and silencing the RLTR10B-containing class of retrotransposon in testis. Many other groups of retrotransposons, including active IAP elements, are enriched for the same histone marks found on RLTR10B elements  but are g to DNA  A-Myb isg to DNA  but has Momme screen reporter transgene contains an AACTGTCT element in the HS40 alpha globin hypersensitive site and this fits the MYB binding consensus site–AACTG(C/T)C(A/T). A-MYB has been shown to act as a transcriptional activator in peripheral blood cells [The od cells . It is rMyb family members are subject to several types of post-translational modifications, including phosphorylation, acetylation, and ubiquitination. Furthermore, the ubiquitination of B-Myb and C-Myb has been shown to inhibit the transcriptional activation functions of these two factors , 43. TriNmnat3 [Trim33 overlap with those that express high levels of A-Myb [Publically available datasets show that reduction in A-Myb in mouse testis results in a decrease in expression of Nmnat3 . We haveof A-Myb . Given tTranscriptional activity at retrotransposons is species specific, consistent with rapid evolution of retroviral subtypes . It is lLine3, a transgenic line that is homozygous for the GFP transgene and on an FVB/NJ inbred background, as previously described [MommeD44 allele was carried out on the Line3 background. The Line3C was used for linkage studies and was produced by crossing Line3 to C57BL/6J for 10 generations and selecting for homozygosity of the GFP transgene. All breeding crosses and experimental procedures using the MommeD44 allele were carried out using mice at least 5 generations from the founder mouse. Procedures were approved by the Animal Ethics Committee of LaTrobe University, under approval numbers AEC 12–74 and AEC 12–75. All mice sacrificed in the study were anesthetized using isoflurane and euthanised by cervical dislocation.The ENU screen was carried out in escribed . MaintenThe expression vector encoding A-MYB-FLAG was purchased from OriGene . TRIM33-GFP was obtained from DR Kyle Miller (Addgene plasmid #65399). Knockdown shRNA for Trim33 were obtained from Dr Joan Massague (Addgene plasmid # 15728).PCR was carried out using DNA from tail tips using the following conditions: 94 degrees Celsius 6 minutes, 30x cycles with 94 degrees Celsius 30 seconds, 60 degrees Celsius 45 seconds 72 degrees Celsius 45 seconds, followed by a final annealing step of 72 degrees Celsius 6 minutes. All primers used in the study are described in Flow cytometry of blood from 3 week old mice collected in FACSFlow Sheath Fluid (BD Biosciences) was carried out and analysed on a Guava easyCyte HT  and with the Guava InCyte software, respectively. Erythrocyte green fluorescence (525nm) was recorded and a GFP-positive gate was set to exclude 99% of wildtype erythrocytes.MommeD44 heterozygous mice were backcrossed twice to Line3C (see above) and phenotyped for GFP expression by flow cytometry. DNA from tail tissue collected during flow cytometry procedures was used to perform linkage analysis. The Illumina GoldenGate genotyping assay (Mouse Medium Density Linkage Panel) was used with 10 wildtype and 13 heterozygous mice. MommeD44 wildtype samples should only have heterozygous C57BL/6J SNPs surrounding the causative mutation and MommeD44 mutants should have FVB and C57BL/6J SNPs at this interval. The Mouse Medium Density Linkage panel contains 766 measurable SNPs between C57BL/6J and FVB/NJ. Samples were genotyped following the Illumina protocol and genotype calls were made using the Genotyping module of the GenomeStudio v1.1 software. Only samples with a call rate >95 were accepted. The linked interval was identified based on a peak in the LOD score. Fine mapping was carried out using primers amplifying C57BL/6J or FVB SNP loci that could be cut with restriction enzymes to determine genotype. Of the 95 F2 backcrosses, 8 mice had SNP profiles that were inconsistent with the mapping and were excluded. This small (<10%) error rate in phenotyping is commonly encountered in this screen [Exome capture was performed using the Roche NimbleGen reagents  as per the Illumina optimized Roche NimbleGen SeqCap User’s Guide (version 1.0) and using a Bioruptor  for DNA fragmentation. Libraries were sequenced using the illumina GAIIx platform and reads were aligned to the mouse  genome using bwa aln and bwa sampe programs . For furMommeD44 ENU mutations, other MommeD lines previously identified in the screen and described elsewhere were used [MommeD44 mutant mice. Sanger Sequencing or restriction enzyme digest with the enzyme Mse1 (New England Biolabs) that specifically cuts at the ENU mutation site in the Trim33MommeD44 allele was used.As control sequence, that lacked ere used . VarscanTimed matings between heterozygous mutant females and heterozygous mutant males were set up and the detection of a vaginal plug was counted as 0.5 dpc. Genotyping was carried out using DNA extracted from embryos.Bisulphite conversion was carried out on 1ug of DNA extracted from whole testis using the EpiTect Bisulphite Kit  according to the manufacturer’s instructions. The bisulphite conversion rate was at least 99% and sequences were analysed using the BiQ Analyser software. Oligonucleotides are provided in HEK293T and mouse embryonic fibroblasts were cultured in DMEM supplemented with 10% fetal calf serum  at 37ᴼC in a humidified 10% CO2 incubator. For transient expression of cDNA or RNAi vectors, HEK293 or MEF cells were transfected with expression plasmids using the Fugene 6 reagent  following the manufacturer’s instructions. For A-MYB survival assays, cells were transfected with indicated plasmids for 48 hours and then treated with cycloheximide to a final concentration of 10 ug/ml. Cells were lysed at indicated time points and A-MYB protein levels were assessed by Western blotting.2 1.5mM, EGTA 1mM, Triton X-100 1%, Glycerol 10%) and incubated overnight with anti-FLAG antibody at 4ᴼC. Following incubation, antibodies were washed in ONYX buffer and proteins were eluted in 8M urea lysis buffer. For protein co-immunoprecipitations the Nuclear Complex Co-IP Kit  was used as per manufacturer’s instructions. BCA  was used to quantify protein and total lysates were separated according to size on polyacrylamide gels .Whole-cell extract was prepared from testis of adult (12 week old) mice in 8M urea lysis reducing buffer . For ubiquitination assays, HEK293T cells were lysed in RIPA buffer , diluted 1:10 in ONYX buffer , rabbit polyclonal anti-A-Myb , mouse monoclonal anti-GFP , mouse monoclonal anti-HA , mouse monoclonal anti-Gapdh  mouse monoclonal anti-HSP70  and rabbit polyclonal anti-γ-Tubulin . Anti-Flag was a gift from Dr Lorraine O'Reilly at the Walter and Eliza Hall Institute of Medical Research. Antibodies used for immunoprecipitation were bead-conjugated anti-FLAG  and anti-IgG .t test.Total RNA was extracted from snap frozen tissues or cells using TRIzole reagent  according to manufacturer instructions. cDNA synthesis was carried out from total RNA using the QuantiTect Reverse Transcription Kit  and RTqPCR was performed with the QuantiTect SYBR Green reagent  with primers designed to span exon junctions in mRNA . SamplesRNA sequencing was carried out from total RNA submitted to the Australia Genome Research Facility . At least 20 million 100bp single end reads were generated on an Illumina HiSeq platform for each sample, using libraries generated using the illumina TruSeq RNA Sample Preparation kit . Initial QC was performed by AGRF. Reads were aligned to the mouse genome  using the program Tophat (version 2.0.11)  with theTestis tissue from three 12 week old wildtype mice were snap-frozen and sent to Active Motif for ChIP, library preparation, sequencing and initial data analysis. The rabbit polyclonal anti-Trim33  was used for ChIP. Sequencing was carried out for 75mer read lengths on the NextSeq 500 platform . Reads were aligned using the BWA program  with defTo estimate enrichment of repeat elements for ChIP-seq and RNA-seq datasets reads were mapped to a repeat assembly file containing a single FASTA entry for each repeat type defined in the rodent repeat sequences RepBase database  Click here for additional data file.S2 FigMommeD44 colony (n = 113) were measured for their percentage of GFP expressing erythrocytes and grouped by genotype for the Trim33 mutation.Mice from the (PDF)Click here for additional data file.S3 Fig(A) The Trim33 antibody specificity at a 1:10000 dilution was tested using protein extract from three independent wildtype samples. The lysate was separated on a polyacrylamide gel. (B) Wildtype (n = 7) and heterozygote (n = 6) testis extracts incubated with the Trim33 antibody and an antibody against γ Tubulin. (C) Densitometry analysis of B shows Trim33 is significantly reduced in heterozygotes compared to wildtypes , error bars indicate SEM. (D) Heterozygote and homozygote protein extracts, from pooled E9.5 embryos (N = 4 per sample), incubated with the Trim33 antibody and antibodies directed against the housekeepers γ Tubulin and Gapdh.(PDF)Click here for additional data file.S4 FigMommeD44 show no obvious differences in histological sections after staining with haematoxylin, compared to MommeD44 wildtype mice.Mice heterozygous for (PDF)Click here for additional data file.S5 Fig(A) Screenshots of Trim33 ChIP-seq (and Input) read density for four sites that were identified as Trim33 binding peaks. Shown are the 5kb loci centred on each of the peaks and labelled according to their distance from the nearest gene transcription start site: an intronic locus located within the Fgf2 gene, two gene promoter loci (Ccdc28a + Pdzd8) and an intergenic Trim33 binding site 5Kb upstream of the gene Heyl. The intronic locus within Fgf2 gene is located at an RLTR10B retrotransposon. (B) ChIP qPCR for six loci, two representing negative controls (Untr6 + Untr17) with primers located in regions not enriched for Trim33 and the four Trim33 enriched regions from A. Error bars indicate S.D. from 3 technical replicates.(PDF)Click here for additional data file.S6 FigSeveral motifs were significantly enriched in highly significant  Trim33 ChIP-seq peaks (N = 2338) that were significantly homologous to known DNA binding consensus sequences from the JASPAR CORE 2014 database(PDF)Click here for additional data file.S7 FigA) Heat plot of ChIP-seq read density, clustered by similarity, for publically available ChIP-seq data for the A-Myb (plus input)–GEO accession GSE44690 and Trim33 and input in testis, at all RLTR10B and RLTR10B2 elements. Of the two clusters, only cluster 1 is enriched in the Jaspar Core Database Myb consensus binding sequence using the MEME program. (B) ChIP-seq data from another study [Click here for additional data file.S8 FigThe MEME suite program MAST was used to search for Jaspar Core Database Myb consensus binding sites (shown in green) in the 447 ERV2 elements in the rodent RepBase database. Those elements with an E value of less than 10 are shown. The E value for each element is equal to the combined p-value of the sequence times the number of sequences searched.(PDF)Click here for additional data file.S9 FigHprt and Gapdh. RLTR10B expression increased when A-MYB was overexpressed and the effect is reversed when ubiquitin was also overexpressed. Error bars indicate SEM, * p-value > 0.05. The expression levels of Flag-A-MYB in MEFs and HSP70 are shown (bottom panel).Immortalized MEF cells were transfected with FLAG-tagged A-MYB or FLAG-tagged A-MYB and HA-tagged ubiquitin for 36h. RLTR10B retrotransposon expression was measured and normalized to the expression of housekeeper genes (PDF)Click here for additional data file.S10 FigHprt and Rps2, the number of samples for each group was at least 4, * p value ≤ 0.05, error bars = SEM. Ptgds and Serpina3n did not validate as significantly different between wildtypes and heterozygotes.The expression for genes predicted to be significantly differentially regulated by RNA-seq  was test(PDF)Click here for additional data file.S11 FigNmnat3 locus begins at the RLTR10B element upstream.Splice junction tracks generated by the IGV genome browser program are shown. Each of the tracks represents a mRNA sequence built by the IGV browser program from the split reads mapped by the testis RNA-seq data. The thickness of the bands indicates the relative coverage of split reads supporting usage of a particular splice site. The majority of mRNA across the (PDF)Click here for additional data file.S12 FigNmnat3 gene in testis. Bisulfite primer design inside the locus is indicated. The methylation state of individual animals (two per genotype) is shown as columns of cloned PCR products. Filled in circles represent methylated CpGs and white circles represent unmethylated CpGs. Each mouse is represented by eight or greater clones and averages for each sample and the two groups are indicated. The bisulfite conversion rate of the non CpG cytosines is indicated as 99%.DNA methylation was measured inside the RLTR10B that functions as an upstream promoter of the (PDF)Click here for additional data file.S13 FigA) Expression of RLTR10B elements in testis was measured using primer pairs that either included the Myb binding sites (F2 –R1) or not (F1 –R1). Increased expression of RLTR10B elements containing the Myb binding sites was seen in MommeD44 heterozygotes. Expression was normalized to two housekeeping genes, Hprt and Rps2, and then normalised to the average expression levels from wildtype mice. The number of samples for each group was at least 3, * p value ≤ 0.05, error bars = SEM. (B) Expression of an ectopic transcript, originating at an RLTR10B upstream of the Vmn1r181 gene, was measured in MommeD44 heterozygous and wildtype testis. Primer annealing locations are shown. Increased expression was detected in heterozygous tissue, normalized to the housekeeping genes Hprt and Rps2, * p value ≤ 0.05, error bars = SEM. (C) Expression of RLTR10B elements was measured in E9.5 embryos from the same litter; wildtype, heterozygous or homozygous for the MommeD44 mutation. No change in expression was detected across genotypes, normalized to the housekeeping gene Gapdh.((PDF)Click here for additional data file.S14 FigRNA-seq data from another study , accessi(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableBowtie2 was used to map raw data from Trim33 ChIP-seq to consensus sequences from the rodent RepBase repository. RPKM values were used to normalize for the size of each element and the library size(XLSX)Click here for additional data file.S3 TableBowtie2 was used to map raw data from Trim33 ChIP-seq to consensus sequences from the rodent RepBase repository. RPKM values were used to normalize for the size of each element and the library size(XLSX)Click here for additional data file.S4 TableRNA-seq data was mapped back to the Ensembl annotated (NCBI build 37) genome and DEseq was used to identify differentially expressed genes.(XLSX)Click here for additional data file.S5 TableA set of transcript annotations was created using Cufflinks and the mapped RNA-seq data. 10 overlaps were identified.(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.