VEGFA promoter. Second, DNA-bound SerRS recruits the SIRT2 histone deacetylase to erase prior c-Myc-promoted histone acetylation. Thus, vertebrate SerRS and c-Myc is a pair of ‘Yin-Yang’ transcriptional regulator for proper development of a functional vasculature. Our results also discover an anti-angiogenic activity for SIRT2.Recent studies suggested an essential role for seryl-tRNA synthetase (SerRS) in vascular development. This role is specific to SerRS among all tRNA synthetases and is independent of its well-known aminoacylation function in protein synthesis. A unique nucleus-directing domain, added at the invertebrate-to-vertebrate transition, confers this novel non-translational activity of SerRS. Previous studies showed that SerRS, in some unknown way, controls VEGFA expression to prevent vascular over-expansion. Using in vitro, cell and animal experiments, we show here that SerRS intervenes by antagonizing c-Myc, the major transcription factor promoting VEGFA expression, through a tandem mechanism. First, by direct head-to-head competition, nuclear-localized SerRS blocks c-Myc from binding to the DOI:http://dx.doi.org/10.7554/eLife.02349.001 The network of blood vessels is one of the earliest structures to develop in a vertebrate embryo. A protein called Vascular Endothelial Growth Factor A (or VEGFA for short) is needed to promote the growth of these blood vessels, but too much VEGFA can cause blood vessels to grow too much and to grow abnormally.VEGFA gene, this unwrapping process starts when a protein called c-Myc adds chemical tags to the histones.Like most of the DNA in the nucleus, the gene for VEGFA is tightly wrapped around proteins called histones and must be unwrapped before it can be expressed as a protein. For the Recent research suggested that an enzyme called seryl-tRNA synthetase (or SerRS for short) also controls the expression of VEGFA. This came as a surprise because no other tRNA synthetase has a similar role during development. And although SerRS is known to enter the cell nucleus in vertebrates, researchers did not know what SerRS did in the nucleus to control the expression of VEGFA.Now, Shi et al. have discovered that SerRS controls blood vessel development in zebrafish embryos by counteracting the activity of c-Myc. It does this in two different ways: first, it directly blocks c-Myc from binding to and unpacking the DNA; and second, SerRS works with another enzyme to remove tags that are already on the histones. Shi et al. found that if the expression of this other enzyme  was reduced in zebrafish, the fish expressed more VEGFA and their blood vessels grew too much.Since blood vessel growth is important in the development of cancers, the findings of Shi et al. could also lead to a better understanding of how tumors develop, as well as how blood vessels develop normally.DOI:http://dx.doi.org/10.7554/eLife.02349.002 In fishagenesis  or ENU magenesis , caused Ser for protein synthesis in the cytoplasm. However, the role of SerRS in vascular development is independent of its enzymatic activity  transcript in the mutant fish embryos  protein, c-Myc functions through heterodimerization with the small bHLHZ partner MAX for binding to the Enhancer Box (E-box) DNA sequence (5′-CACGTG-3′) on its target genes  vs a control shRNA (sh-Con)  targeting the 3′ UTR of human SerRS mRNA was generated to knock down endogenous SerRS expression in human umbilical vein endothelial cells (HUVECs) and HEK 293 cells . For bot(sh-Con) . Conside(sh-Con) , or by e(sh-Con)  and HEK2(sh-Con) , respect(sh-Con) .10.7554/VEGFA gene, from 4 kb upstream (−4 kb) to 4 kb downstream (+4 kb) of the transcription start site , bound to DNA . Given tart site . We founpromoter .10.7554/To investigate whether the binding is repressive in nature, we performed a luciferase assay in which the majority of this promoter region (−1262 ∼ +46) was put in front of a luciferase reporter gene to test the transcriptional activity of SerRS. Strikingly, SerRS overexpression sharply reduced the elicitation of luciferase activity . In contIn further work, we showed that the inhibitory effect of SerRS persisted as the promoter region was shortened to −262 ∼ +46, suggesting that the SerRS-specific responsive element is within this 308-bp region . This DNd) of SerRS was 211.5 nM as measured by EMSA ((The direct interaction of SerRS with a slightly extended 27-bp DNA fragment (−62 ∼ −36) was confirmed by an electrophoretic mobility shift assay (EMSA) . The bin by EMSA  and 265 by EMSA  contains a nonclassical E-box sequence (−49CATGCG−44) that completely overlaps with the SerRS binding site  that would completely abolish c-Myc/Max binding . The N-terminal tRNA binding domain (TBD) of SerRS is used for recognizing the long variable arm of tRNAeraction . In facteraction , suggestTo further define the DNA binding sites on SerRS, we made additional deletion mutants of SerRS. Deletion of each of the two higher eukaryote-specific insertions in TBD and CD, respectively, which does not negatively impact tRNA binding , dramatiVEGFA promoter and their opposing roles on VEGFA expression, suggest that SerRS may compete with c-Myc for DNA binding and thus inhibit c-Myc-driven VEGFA expression. It is important to note that the binding affinity of the SerRS–DNA interaction  showed under-developed vasculature, which is accompanied with a reduced level of Vefga expression . In partA large scale protein–protein interaction study indicated a potential interaction between SerRS and sirtuin 2 (SIRT2) , a NAD+-To test this hypothesis, we firstly performed coimmunoprecipitation to confirm the interaction. The ectopically expressed SIRT2, but not SIRT1, effectively pulled down SerRS, and vice versa . The intVEGFA promoter, we knocked down the expression of SerRS in HEK 293 cells and detected the binding of SIRT2 to the VEGFA promoter. Knockdown of SerRS, but not GlyRS, significantly reduced the amount to SIRT2 bound to the VEGFA promoter  as with the injection of SerRS-MO   . Furtheranscript . TherefoVEGFA promoter; second, DNA-bound SerRS recruits the SIRT2 histone deacetylase to condense the chromatin at the VEGFA promoter, and thereby shut down the gene transcription. Importantly, in each aspect, these actions of SerRS directly compete with and thwart that of the VEGFA-promoting actions of c-Myc. While the opposing regulation of SerRS and c-Myc is applied on VEGFA expression, it is manifested at the organism level with respect to vascular development, making SerRS and c-Myc as a pair of ‘Yin-Yang’ regulators for proper development of a functional vasculature  can compete with c-Myc for binding to MAX and also recruit histone deacetylases to reverse the action of c-Myc-bound acetyltransferase to shut down the expression of c-Myc target genes . TherefoDrosphila, an invertebrate with a primitive ‘open’ circulatory system. The appearance of the MAX and Mad family genes is even earlier, and the genes were first identified in roundworms such as C. elegans , which is frequently affected by rearrangements or allelic loss in a variety of human malignancies . Anti-c-Myc, anti-SIRT2, anti-SIRT1, and anti-α-tubulin antibodies were purchased from Cell Signaling . Anti-V5 and anti-GlyRS antibodies were purchased from Invitrogen  and Abnova , respectively. Antibodies against histone H4 and acetylated H4 at Lys 16 (H4K16Ac) were purchased from Active Motif . Anti-SIRT2 antibody for chromatin immunoprecipitation was purchased from Thermo Fisher .Escherichia coli. The SerRS proteins were purified in tandem by Ni-NTA affinity , HiTrap Heparin High Performance , and HiLoad 16/600 Superdex 200 pg  columns. The GST-tagged SerRS constructs were subcloned into the pGEX-6P-1 vector  for expression in E. coli, and the proteins were affinity-purified using Glutathione-Sepharose 4B beads . The purities of the recombinant proteins were assessed by Coomassie blue staining following 4–12% Mini Gel  electrophoresis. Protein concentrations were determined using Bradford protein assay .For overexpressions in mammalian cells, human full-length, and NLS-deleted SerRS genes were cloned into the pFlag-CMV-2 vector , and human c-Myc, SIRT1, and SIRT2 genes into the pCDNA6-V5/His-C vector . For recombinant protein purification, human SerRS, c-Myc, MAX, and SIRT2 genes were subcloned into pET-20b(+) vector  to express with a C-terminal his-tag in g for 3 hr.HEK 293 cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and transfected with Lipofectamine 2000 . HUVEC cell were cultured in EGM complete medium  supplemented with 8% FCS in gelatin-coated dishes and transfected using lentivirus. DNA expressing a short-hairpin RNA (shRNA) designed against human SerRS (5′-GGCATAGGGACCCATCATTGA-3′), GlyRS (5′-GCATGGAGTATCTCACAAAGT-3′), SIRT1 (5′-GAAGTTGACCTCCTCATTGTT-3′) , or SIRTVEGFA; 5′-CGTCACCAACTGGGACGA-3′ and 5′-ATGGGGGAGGGCATACC-3′ for human β-ACTIN; 5′-GGCTCTCCTCCATCTGTCTGC-3′ and 5′-CAGTGGTTTTCTTTCTTTGCTTTG-3′ for zebrafish vegfa ; 5′-TCACCACCACAGCCGAAAGAG-3′ and 5′-GTCAGCAATGCCAGGGTACAT-3′ for zebrafish β-actin. The PCR reaction program started at 95°C for 10 min, followed by 45 cycles of 95°C for 20 s and 60°C for 1 min. Each experiment was carried out in triplicate. The VEGFA gene expression was normalized to that of β-ACTIN. Statistical analyses were performed with the software SigmaPlot (version 10.0). Student's t test was used to analyze the changes between different groups.Total RNA was isolated from cells by TRIzol Reagent . One milligram of the total RNA from each sample was reversely transcribed to cDNA by SuperScript II reverse transcriptase . All real-time PCR reactions were performed using the StepOnePlus Real-Time PCR system  with SYBR Select Master Mix . The primer pairs for the PCR reactions were: 5′-GAGGGCAGAATCATCACGAAG-3′ and 5′-TGTGCTGTAGGAAGCTCATCTCTC-3′ for human 4) were seeded on the gel and then cultured in EBM Basal Medium (without FCS) at 37°C and 5% CO2 for 24 hr to form tubes. Images of the endothelia cell tubular network were taken with a Leica DC350F CCD camera attached to an inverted Leica DMIL microscope. The length of the tubes was measured by ImageJ software.48 hr before the tube formation assay, HUVEC cells were infected with lentiviruses that produce different shRNAs as indicated. Pre-thawed matrigel basement membrane matrix (0.15 ml)  was transferred to 48-well plates and incubated at 37°C for 30 min to form a thin layer of gel. The infected HUVEC cells  for 10 min at room temperature. The reaction was stopped by adding 125 mM of glycine. ChIP assays were performed according to the protocol of ChIP-IT Express Enzymatic kit (Active Motif). After three washes, ChIPed DNA was analyzed on the StepOnePlus Real-Time PCR system using SYBR Select Master Mix (Applied Biosystems). A primer set (5′-GGGCGGATGGGTAATTTTCA-3′ and 5′-CTGCGGACGCCCAGTGAA-3′) targeting the eviously .VEGFA gene were PCR amplified and cloned into the pGL4.11[luc2P] vector (between Kpn I and Xho I sites) to create the pGL4-VEGFA firefly luciferase reporter plasmids. After 16 hr of incubation in 12-well plates, HEK 293 cells were transiently transfected with 100 ng of pGL4-VEGFA reporter plasmid and 500 ng of pFlag-SerRS, or pFlag-GlyRS or pFlag-CMV-2 empty vector as control. A Renilla luciferase control reporter plasmid pRL-SV40 (50 ng) was co-transfected for normalizing the transfection efficiency among different experiments.The luciferase activity was determined by using the Dual-Luciferase Reporter assay system . The promoter regions  of VEGFA promoter from −262 to +46 bp was released from the pGL4-VEGFA plasmid by Kpn I and Xho I digestion. After purification by agarose gel electrophoresis, the 3′ end was radiolabeled using a standard Klenow fragment fill-in reaction with [α-32P]-dATP. The labeled DNA fragment was incubated with recombinant SerRS, c-Myc and MAX, or GlyRS in 20 µl binding buffer  for 1 hr at room temperature. DNase I  was then added to the mixture at a 2.5 U ml−1 final concentration and incubated for additional 40 min at room temperature. The reaction was stopped by adding 200 µl stop solution  to incubate for 30 min at 45°C. After extraction with phenol-chloroform and precipitation with ethanol, DNA fragments were resuspended in 80% formamide in 1x TE buffer and then denatured for 5 min at 95°C before separation by electrophoresis using 8% urea-polyacrylamide sequencing gels. Gels were dried and examined by autoradiography.The DNA of the VEGFA promoter and mutants were synthesized, annealed, and [32P]-labeled at the 5′ end by T4 DNA kinase (New England Biolabs) before purification using a sephadex G-25 spin column . The labeled oligonucleotides (0.08 pmol) were incubated with recombinant SerRS at indicated concentrations in binding buffer , 1 mM DTT ) for 30 min at room temperature. The samples were loaded to 5% native polyacrylamide gel (17.5 cm in length) and underwent electrophoresis at 250 V in running buffer . Afterwards, the gel was dried and examined by autoradiography.The 27-bp DNA oligonucleotide corresponding to SerRS binding site on the 3VO4, and protease inhibitor cocktail). Supernatants were incubated with indicated antibodies and protein-G-conjugated agarose beads (Invitrogen) for at least 2 hr. The beads were washed five times with wash buffer  and then subjected to SDS-PAGE and Western blotting analysis with indicated antibodies.HEK 293 cells were resuspended on ice with lysis buffer , 150 mM of NaCl, 0.5 mM of EDTA, 10% glycerol, 0.1% Triton X-100 and 1 mM of DTT. Equal amounts of GST or GST-SerRS fusion proteins were incubated with recombinant SIRT2 for two hours and pulled down by Glutathione-Sepharose 4B beads .+, 20 µM Fluoro-Substrate peptide, 1 µM recombinant SIRT2, and recombinant SerRS at different ratio indicated in The deacetylase activity of SIRT2 was measured by using CycLex SIRT2 Deacetylase Fluorometric Assay Kit . The reaction buffer contains 50 mM Tris-HCl (pH 8.8), 0.5 mM DTT, 0.25 mAU/ml Lysylendopeptidase, 1 µM Trichostatin A, 0.8 mM NADVEGFA promoter (5'-GGCGGG GCGGAGCCATGCGCCCCCCCCTTTATA-biotin-3′ and 5'-AAAGGGGGGGGCGCATGGCTCCGCCCCGCC-3′) were synthesized, annealed, and purified by electrophoresis on 8% native acrylamide gel. Binding kinetics was analyzed using a Biacore 3000 instrument . The DNA was immobilized through biotin–streptavidin interaction on a SA sensor chip, and the interaction reached to 300 Response Unit. A flow cell without immobilized DNA was used as a blank reference control. The immobilized DNA was stable over the course of the experiment. Baseline drift was less than 5 RU/h after the chip was washed with HBS-EP buffer  at 10 μl min−1 for 18 hr. SerRS proteins were injected using the KINJECT procedure for 300 s at 6.25, 12.5, 25, 50, 100, 200, 400, 800, and 1600 nM concentrations in HBS-EP buffer at 30 μl min−1. Dissociation was monitored by flowing HBS-EP buffer for 480 s at 30 μl min−1. The sensor chip was regenerated by a 60-s injection of 0.1% SDS, 10 mM NaOH to restore the original resonance signal of the surface. The injections were duplicated for each ligand concentration and were performed in random orders with buffer blanks injected periodically for double referencing. The variation between the replicates was less than 1%. Corrected response data were fitted with BiaevalTM 3.1 software and the apparent kinetic constants were calculated using data from the early parts of the association and dissociation phases. The fit was satisfactory for a simple 1:1 binding model.Biotin-labeled double-stranded DNA oligonucleotides corresponding to SerRS binding site on the Tg (Fli1a: EGFP) fish were maintained at 28.5°C under continuous water flow and filtration with automatic control for a 14:10 hr light/dark cycle. The night before injection, male and female fish were placed in a 1-L tank containing fish mating cage with an inner mesh and divider. Zebrafish embryos were obtained from natural spawning by removing the divider and stimulating with light. The embryos were kept at 28.5°C before and after microinjection. The antisense morpholinos (MOs) targeting SerRS or other genes were injected into the yolk of 1- to 2-cell stage embryos at the dosage of 4 ∼ 5 ng per embryo. The designs of SerRS-MO (5′-AGGAGAATGTGAACAAACCTGACAC-3′) (−1 tricaine (Sigma-Aldrich), mounted in 2% methylcellulose and photographed with a Nikon fluorescent microscope (AZ100) equipped with a Nikon CCD camera (Qimaging Retiga 2000R). All the experiments involving zebrafish had been conducted according to the guidelines established by the Institutional Animal Care and Use Committee (IACUC) at The Scripps Research Institute, IACUC approval number 09-0009. Statistical analyses were performed with the software SPSS Statistics 19. The effects of different morpholinos on ISV development were analyzed with χ2 test.Transgenic ACAC-3′)  and of SACAC-3′) . The Myc review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  functions in the nucleus to regulate VEGFA expression. The major findings are 1) SerRS binds directly to the VEGF promoter at a site that overlaps with a binding site for cMyc. 2) In vivo and in vitro experiments provide evidence that SerRS can compete for cMyc for binding at these sites. 3) The authors confirm a prior proteomic result suggesting interaction of SerRS with SIRT2. Evidence is presented that is consistent with SerRS acting to recruit SIRT2 to the VEGFA promoter where it actively represses VEGFA expression via histone deacetylation. Loss of function experiments in zebrafish that focus on vascular branching are supportive of mutually antagonistic roles of cMyc and SerRS in controlling vascular development, at least in part through regulation of the expression of VEGFA.One reviewer stated, 'Overall the findings are novel and interesting and suggest mutually antagonistic functions of SerRS and cMyc at the VEGF gene, and possibly other Myc target genes. SerRS could thus represent an alternative mechanism for negative regulation of Myc in addition to that provided by Mad repressors that form Mad/Max heterodimers'. The second reviewer stated, 'The findings are very novel, exciting, and important. The approach takes advantage of cell culture, in vitro, and zebrafish technologies, and is elegant, well-designed, and with appropriate controls.'The reviewers also raised the following major concerns that would need to be adequately addressed for the manuscript to be accepted:1) The evidence that SerRS binds to the VEGF promoter and antagonizes cMyc appears to be convincing. A major concern relates to the features of SerRS that enable it to bind to DNA in a sequence-specific manner. The authors make no mention of a conserved DNA binding domain in SerRS. Additional mapping studies are needed to define the amino acid sequences of SerRS required for sequence-specific DNA binding activity.2) At present there does appear to be sequence specificity, but perhaps too much so, in that all point mutations shown in 3) The shifted bands in the EMSA in 4) In  1) The evidence that SerRS binds to the VEGF promoter and antagonizes cMyc appears to be convincing. A major concern relates to the features of SerRS that enable it to bind to DNA in a sequence-specific manner. The authors make no mention of a conserved DNA binding domain in SerRS. Additional mapping studies are needed to define the amino acid sequences of SerRS required for sequence-specific DNA binding activity.The reviewers made an important point. To address this point, we performed additional experiments to identify DNA binding sites on SerRS through domain mapping and deletion mutagenesis. The results indicate that SerRS has an extensive DNA-binding site. All three domains of SerRS  are critical, but insufficient on their own, for DNA binding. To further define the DNA binding site on SerRS, we made 4 additional deletion mutants of SerRS at strategic positions. All 4 mutants lost the DNA binding activity. These results and the results on our investigation to address the second major concern of the reviewers  now constitute a new section of the manuscript entitled “Characterize the interaction between SerRS and DNA” and the new 2) At present there does appear to be sequence specificity, but perhaps too much so, in that all point mutations shown inabolished binding and the minimal binding site was rather long. Are there base pairs in the binding site that are not important for sequence specific recognition? Are there mutations that discriminate binding of cMyc and SerRS?The answer is ‘yes’ to both questions. We now tested a total of 11 single or double mutations in the 27 bp DNA of the SerRS binding site. Two double mutants of the E-box, including one that would completely abolish c-Myc/Max binding, did not affect the SerRS interaction; on the other hand, 5 different single mutations outside the E-box (on both the 5’ and the 3’ sides) that would not affect c-Myc/Max binding greatly weakened SerRS binding, indicating that SerRS and c-Myc/Max have distinct DNA binding specificities. These new data is now added to the manuscript and is part of the new 3) The shifted bands in the EMSA inshould be more precisely identified. For example, the band labeled as SerRS could be a “supershifted” complex containing SerRS as well as c-Myc-Max. Western blot analysis or antibody supershifts could clarify this issue. The authors should address why the Max/Max/DNA complex is not disrupted by SerRS.In parallel with the EMSA, we now performed the Western blot analysis as suggested to clarify this concern. Only SerRS was detected in the shifted band, indicating that SerRS and c-Myc/MAX cannot complex with the DNA simultaneously. This result is consistent with the result from the ChIP assay where SerRS and c-Myc were found to inhibit each other for binding to the VEGFA promoter. The result is now added to the manuscript as The reason for why SerRS cannot disrupt MAX/MAX-DNA complex is because that the affinity between MAX homodimer and DNA is significantly higher (Kd = 19.2∼48.7 nM) than that of the SerRS-DNA interaction (Kd = 211.5∼265 nm) and the c-Myc/MAX-DNA interaction (Kd = 90.5∼229 nM). This information is now added to the manuscript.4) Inthe authors show the interaction of SerRS with SIRT2 in an overexpression system only. The interaction between the endogenous proteins should be shown in cells, for example, by co-IP.We have performed the co-IP experiment to detect the interaction between the endogenous SerRS and SIRT2 as suggested. The experiment was successful and the new data are presented as


For transcription initiation, σ54-RNA polymerase yields open promoter complexes through its remodelling by cognate AAA+ ATPase activators. Since activators can be bypassed in vitro, bypass transcription in vivo could be a source of emergent gene expression along evolutionary pathways yielding new control networks and transcription patterns. At a single test promoter in vivo bypass transcription was not observed. We now use genome-wide transcription profiling, genome-wide mutagenesis and gene over-expression strategies in Escherichia coli, to (i) scope the range of bypass transcription in vivo and (ii) identify genes which might alter bypass transcription in vivo. We find little evidence for pervasive bypass transcription in vivo with only a small subset of σ54 promoters functioning without activators. Results also suggest no one gene limits bypass transcription in vivo, arguing bypass transcription is strongly kept in check. Promoter sequences subject to repression by σ54 were evident, indicating loss of rpoN (encoding σ54) rather than creating rpoN bypass alleles would be one evolutionary route for new gene expression patterns. Finally, cold-shock promoters showed unusual σ54-dependence in vivo not readily correlated with conventional σ54 binding-sites.Enhancer-dependent transcription involving the promoter specificity factor σ This silenced state of RNAP may then have become a target for regulation. Intriguingly, to date no bacteria have been found where the gene encoding σ54 (rpoN) is present in the absence of any cognate AAA+ activator (54 functioning without activators are uncommon. Yet variants of σ54 bypassing activator requirements for making open promoter complexes exist and have been characterized extensively in vitro (54 can yield activator bypass forms. Quite why σ54 has seemingly not (or rarely) evolved to activator independence is rather unclear. Here we have investigated the in vivo global actions of wild-type σ54 in Escherichia coli bacteria and one bypass variant to explore potential restrictions on evolving bypass gene regulation systems.Amongst the strategies used to regulate transcription initiation in bacteria, the emergence of the enhancer-dependent system that uses the σnly seen ,2. Ratheomplexes ,4. Potenin vitro ,7. SinglE. coli increased activator bypass transcription suggesting multifactorial global constraints prevent unregulated transcription by the σ54 containing RNAP holoenzyme (σ54RNAP). Intriguingly, some genes with σ54 binding sites were found to be repressed in a σ54-dependent manner. In some cases the putative σ54 binding site overlapped that of a putative σ70 binding site suggesting a competition for promoter sites, which we confirmed by in vitro experiments. In other cases, σ54 and σ70 RNAP appear to act antagonistically through e.g. convergent transcription events.We found that only a very limited number of knock-outs (KOs) of non-essential genes in glnAp2 strains (Supplementary Table S1) P1vir transduction was used to insert rpoN208::Tn10 (K1471 donor strain) into the native chromosomal rpoN locus of BW25113 (recipient). Subsequently, transformation was used to transfer the pSB4A3 plasmid harbouring ΦPglnAp2-gfp  and pBAD18 cm . For the construction of the pspA strains (Supplementary Table S1) P1vir transduction was used to insert rpoN208::Tn10 (K1471 donor strain) into the native chromosomal rpoN locus of BW25113 (recipient). Next the ΦPpspA-lacZ  into the BW25113 rpoN208::Tn10 ΦPpspA-lacZ. The F3K3 plasmid containing the filamentous phage pIV secretin was also transformed into this strain. To transfer the single-gene KOs from the KEIO collection (vir lysates were produced from the pooled strains. These were then used as a donor for P1vir transduction of the KOs into the pspA-SABRS recipient strain (Supplementary Table S1).For the construction of the llection  and smalin vivo, strains were grown on LB indicator  plates and in minimal Gutnick liquid media supplemented with L-glutamine (54 function (since the nitrogen assimilation pathways in E. coli rely on σ54 for expressing appropriate levels of glutamine synthetase). The antibiotic concentrations used were 30 μg/ml kanamycin, 50 μg/ml chloramphenicol and 50–100 μg/ml ampicillin.For gene expression assays lutamine  as the npspA-lacZ in vivo, β-Galactosidase assays were carried out as described  and green fluorescence  were measured simultaneously using a BMG FLUOstar Omega microplate reader. Promoter activity was expressed as fluorescence emission at 520 nm per OD600 measured in triplicate.To measure activity of ΦPescribed . To deteNcoI, a 6-base cutter. Re-ligation of the resulting fragments into circular DNA was done in a volume of 100 μl. This DNA was used as a template for PCR, using kanamycin cassette-specific forward and reverse primers (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to align sequenced regions to the E. coli chromosome and to determine the identity of the deleted gene.Inverse polymerase chain reaction (PCR) was used to identify selected KO mutants (Supplementary Table S2). Chromosomal DNA from strains that were positive for β-galactosidase activity on indicator plates and in liquid culture (Miller assay) was purified and digested with  primers . Product® Genomics suite 6.6, including a QA/QC step to gauge the sequencing quality. Each sample yielded close to equivalent total reads with 100% of reads aligned to the E. coli K-12 reference genome (NC_000913) (Supplementary Table S4). For each gene, reads were normalized for both the number of reads and length of RNA and expressed as RPKM (reads per kilobase per million)  for further processing. Total RNA was isolated from the cell pellets using a bead mill and the mirVana RNA isolation kit (Ambion) including DNase treatment. Ribosomal RNA molecules were depleted from the total RNA preparations using the RiboZero rRNA Removal Kit (Bacteria) (Epicentre). The rRNA depleted RNAs were fragmented with RNase III. Then, the RNA fragments were poly(A)-tailed using poly(A) polymerase and the 5′ PPP structures were removed using RNA 5′ polyphosphatase (Epicentre). An RNA adapter was ligated to the 5′-phosphate of the RNA fragments. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). The cDNA pools were size fractionated in the size range of 200–450 bp using a differential clean-up with the Agencourt AMPure kit. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina and sequenced on a Illumina HiSeq 2000 system using 50 bp read length. Near equimolar amounts of cDNA were used for sequencing. The cDNA reads were analysed via the RNA-seq workflow within Parteke NC_0009  PEG 8000) at 37°C for 10 min. For the σ70/σ54 competition assays, 0.4 μM σ54 were either added before or with σ70 for binding competition. The reactions were quenched by formamide stop dye and run on a 20% sequencing gel. Signals were detected by Fuji PhosphorImager and quantified using the AIDA Image Analyzer software (raytest).Small primed RNAs (spRNAs) were synthesized in 10 μl volumes containing: 0.125 μM σ54 lacking the repressive Region I, σ54ΔRI, can bypass activator requirement for transcription in vitro  in single copy integrated into the att site of the E. coli chromosome we sought bypass activity from the σ54-dependent pspA promoter in vivo. Reporter strains lacked rpoN and were complemented with either the wild-type or the ΔRI form of σ54 expressed from pBAD18 cm . While rpoN and ΔRIrpoN were under control of the araBAD arabinose inducible promoter, leaky expression (without arabinose) yielded sufficient levels of σ54 to enable pIV-dependent transcription from the pspA promoter . Thus unless stated otherwise (Supplementary Table S2) the experiments were performed in the absence of arabinose. Under standard growth conditions, no clear bypass transcription was observable with the σ54ΔRI form  or on lactose as a carbon source for increased expression of PpspAlacZ. Screens were carried out at 37 and 42°C, and in the absence and presence of 0.001% arabinose (Supplementary Table S2). Colony PCR was used to confirm all necessary genetic elements  were intact in candidate upregulated clones. Coverage was three to four-fold of the chromosome assuming all KOs were represented in the library.To seek genetic determinants potentially restricting pspA promoter based on an increased β-galactosidase activity on indicator plates (Supplementary Table S2). Of these, six KOs  which were found in a screen at 37°C also show increased β-galactosidase activity in liquid culture. These KOs were directly identified by inverse PCR and sequencing. Five KOs were discounted as being direct restrictive determinants of in vivo levels of bypass transcription on the basis of (i) just causing changes to the cell's envelope which led to enhanced β-galactosidase activity, potentially a permeability issue with the ONPG substrate or (ii) reconstitution of the native chromosomal rpoN locus. The ttdR gene however encodes a nucleoid binding protein and its loss seems to genuinely cause increased bypass transcription .We considered that the amount of a gene product might be limiting, rather than inhibitory, for supporting bypass transcription by σ54 and compared outcomes to those from cells with wild-type σ54 and to cells lacking σ54. Cells were cultured at 42°C, which enhances bypass in vitro .We conducted RNAseq of cells containing the ΔRI bypass form of σin vitro , to favo54 compared to cells with σ54ΔRI or no σ54 revealed no major global bypass transcription  from class I and II for binding repression by σ54in vitro  using initiating dinucleotides (−1/+1 or +1/+2). The experiments were performed under two conditions. In the first, σ54 and σ70 were co-incubated with the RNAP prior to addition of promoter DNA probes. Here σ54 was in three-fold molar excess to σ70 and RNAP. This competition from excess σ54 reduced the amount of spRNA synthesised by 10–60% . Since the binding affinity and dissociation constant of σ54 and σ70 for RNAP are very similar  despite lacking any clear consensus −12/−24 sequences . However, in vitro transcription from cspA  has been shown to be essential for mounting a cold shock response through the transcription of σ54-dependent cold shock transcriptional regulators BkdR and YplP (cspA and cspH promoters contain putative heat shock sigma factor (σ32) binding motifs , and may influence DNA structure to normally repress bypass transcription.The σen cycle . Bioinfofunction . Recent proteins . Studieds of RNA ,29–33. Orcoiling ,32. The glnAp2 promoter) bypass transcription could be elevated by altering the TGCA sequence around the −12 GC element  correlation between low-level bypass transcription with a less conserved C −12 nucleotide and (ii) an apparent lack of in trans encoded repressors of bypass transcription.Earlier work from the Gralla lab established that with single promoter studies (the  element . Where wwith DNA ,35. We pin vitro ,21,36. O54 (54 can act to repress gene transcription by other forms of RNA polymerase through a binding competition for promoter sites. Our analysis of any repressive capacity is unlikely to be comprehensive, since a single growth condition was studied. Given more σ54 binding sites than conventional activator dependent σ54 promoters have been identified using a range of genomic methods, we propose that the repressive function is pervasive. Indeed, the σ54 factor is designed to inhibit RNAP by penetrating and blocking DNA entry routes (54 or some of its domains was to globally repress transcription. The unusual behaviours of the cold-shock promoters with respect to σ54 dependence is intriguing, and suggests some non-canonical functions of σ54 and or its holoenzyme remain to be discovered. These may involve factors outside of the well-recognized AAA+ activators which reorganize σ54 at its classical promoter sites.Overlapping promoters and competition between sigma factors are recognized as being important regulatory features of a range of genes –41, alth54 . Here, wy routes ,42,43, aSupplementary Data are available at NAR Online.


VDR), which participates in proliferation, differentiation, cell death under both physiological and pathological conditions. On that account, active vitamin D, the ligand of VDR, is used as an adjuvant therapy to control infection, slow down progression of chronic kidney diseases, and cancer chemotherapy. Interestingly, vitamin D may not be able to augment VDR expression optimally in several instances where epigenetic contributes to down regulation of VDR; however, reversal of epigenetic corruption either by demethylating agents (DACs) or histone deacetylase (HDAC) inhibitors would be able to maximize expression of VDR in these instances.Illicit drug abuse is highly prevalent and serves as a powerful co-factor for HIV exacerbation. Epigenetic alterations in drug abuse and HIV infection determine expression of several critical genes such as vitamin D receptor ( Drug abuse is an important public health problem in the United States, affecting both the user and their families. Drug abuse and addiction not only cost the United States over six billion annually in health care but are also associated with loss of productivity due to associated factors . Illicit2D3) is the hormone synthesized in the skin following to sun exposure while vitamin D2 is a synthetic form and often found in fortified food. To become biologically active , the vitamin D undergoes a series of enzymatic conversions in the liver and kidneys   ; howeverure 2D3) . VDR heture 2D3) . VDR ligure 2D3) .VDR expression in unstimulated T cells is controversial, whereas activated T cells show robust expression . VDR alsVDR and other vitamin D target genes. Vit D induces VDR histone acetylation through histone acetyl transferase to enhance transcription of VDR  not only down regulated DNA methylation but also restored VDR mRNA expression (EZH2) may increase the H3K27 trimethylation at VDR promoter causing the suppression of target gene in colorectal cancer cells. Vit D is also demonstrated to cause attenuation of methylation on WNT genes , is the most studied epigenetic mechanism contributing to down regulation of gene transcription and maintains chromatin in its inactive state through Cytosine–Phosphate-Guanine (CpG) repetitive sequences . Vit D hpression . On the pression  or in copression . Similarpression . Interespression . Lin et NT genes , e-cadhecadherin , and PDZcadherin .VDR promoter , and HDAC1-3 on the NR4B promoter region. Ethanol has also been demonstrated to induce apoptosis in T cells through VDR down regulation  and its expression has been modulated by MeCP2 in mice ,” the tendency for addicted animals to prefer locations associated with drug administration. Since HDAC inhibition augmented morphine-induced CPP it suggests that HDAC activity contributes to the development of addiction . A recenasticity . Morphin in mice . In thespromoter . Currentpromoter  through promoter .fosB promoter (gene responsible for addiction). Inhibition of HDACs alters behavioral responses to drugs like cocaine. de novo DNA methylation (Cocaine induces substantial changes in brain gene expressions . Levine hylation .C-fos promoter (mPFC, OFC, and Nac (Mecp2, a gene essential for learning and memory. Vit D provided protection against the serotonin-depleting effect of amphetamine in the brain of animals in the setting of repeated dosage administration. Vit D deficient rats traveled farther in locomotion test after acute dosage administration (MeCP2, HDAC1, and HDAC2 in mediating MA-induced down regulation of GluA1, GluA2, and GluN1 transcription levels. They showed the MeCP2 and CoREST induced recruitment of HDAC2 onto the chromatin, resulting H4K5, K12, and K16 deacetylation and decreased H4K5ac, K12ac, and K16ac binding onto Glutamine11 and 2 DNA sequences. Histone H3 methylation at the Fobs promoter after repeated amphetamine exposure led to decreased transcription (Amphetamine is being used to treat many ailments . It can promoter  and DNA  and Nac . Deng etstration . Vit D tstration . MA has stration . In anotstration . Cadet acription . Consistcription .Repeated injections of marijuana also caused decreased H3K9me2 and increased H3K4me3 at sites flanking the proenkephalin of the rat .HIV and drugs provide an environment which is conducive to short term and long term epigenetic modifications leading to alterations in gene expression. Epigenetic alterations are also dependent on use of single or multiple drugs. Since both HIV and drugs such as morphine modulate the immune system, environment in these scenarios is likely to be complicated by ongoing opportunistic infections including, bacterial, viral, and fungal. These infections may themselves carry potential to modulate epigenetics. Therefore, epigenetics is a complex issue in drugs of abuse in general and specifically in the presence of HIV infection. However, epigenetic alterations are reversible and thus strategies can be developed to reverse them.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila, the transcription factors Pph13 and Orthodenticle (Otd) direct both aspects of differentiation: rhabdomeric opsin transcription and rhabdomere morphogenesis. We demonstrate that the orthologs of both proteins are expressed in the visual systems of the distantly related arthropod species Tribolium castaneum and Daphnia magna and that their functional roles are similar in these species. In particular, we establish that the Pph13 homologs have the ability to bind a subset of Rhodopsin core sequence I sites and that these sites are present in key phototransduction genes of both Tribolium and Daphnia. Furthermore, Pph13 and Otd orthologs are capable of executing deeply conserved functions of photoreceptor differentiation as evidenced by the ability to rescue their respective Drosophila mutant phenotypes. Pph13 homologs are equivalent in their ability to direct both rhabdomere morphogenesis and opsin expression within Drosophila, whereas Otd paralogs demonstrate differential abilities to regulate photoreceptor differentiation. Finally, loss-of-function analyses in Tribolium confirm the conserved requirement of Pph13 and Otd in regulating both rhabdomeric opsin transcription and rhabdomere morphogenesis. Taken together, our data identify components of a regulatory framework for rhabdomeric photoreceptor differentiation in Pancrustaceans, providing a foundation for defining ancestral regulatory modules of rhabdomeric photoreceptor differentiation.A hallmark of visual rhabdomeric photoreceptors is the expression of a rhabdomeric opsin and uniquely associated phototransduction molecules, which are incorporated into a specialized expanded apical membrane, the rhabdomere. Given the extensive utilization of rhabdomeric photoreceptors in the eyes of protostomes, here we address whether a common transcriptional mechanism exists for the differentiation of rhabdomeric photoreceptors. In  Drosophila (fruit fly) transcription factors, Pph13 and Orthodenticle, are expressed in photoreceptors of Pancrustaceans, Tribolium (red flour beetle) and Daphnia (water flea), and are capable of executing conserved functions of rhabdomeric photoreceptor differentiation. In particular, Tribolium and Daphnia orthologs are capable of substituting and rescuing the photoreceptor differentiation defects observed in their corresponding Drosophila mutants. Furthermore, loss of function analysis in Tribolium of both Pph13 and orthodenticle genes demonstrate they regulate opsin transcription and morphogenesis of the photoreceptor apical membrane. Our data illuminate a framework for rhabdomeric photoreceptor differentiation and provide the foundation for defining the ancestral regulatory modules for rhabdomeric differentiation and potential modifications that underlie the functional diversity observed in rhabdomeric photoreceptors.Visual systems are populated by one of two fundamental types of photoreceptors, ciliary and rhabdomeric. Each photoreceptor type is defined by the opsin molecule expressed and the final morphological form adapted to house the phototransduction machinery. Here we address whether a common transcriptional mechanisms exists for the differentiation of rhabdomeric photoreceptors. We demonstrate that orthologs of two  Drosophila (fruit fly) visual system (reviewed in Rhabdomeric (r) photoreceptors are one of two fundamental types of photoreceptors that have been described Drosophila, two homeodomain proteins have been identified that are critical for regulating r- photoreceptor differentiation. The first, Orthodenticle (Otd), is the Drosophila ortholog of a conserved family of Otd/Otx homeodomain transcription factors, which are essential for head and brain development across species Drosophila eyes, otd is required for both aspects of differentiation Drosophila retinas  sensitive r- opsin, and rh5, a blue (B) sensitive r- opsin in the two inner photoreceptors of Drosophila ommatidia rh6, the Drosophila ancestral long-wave (LW) opsin Drosophila ommatidium In Drosophila eye Drosophila, suggesting that the two proteins cooperate and have overlapping functions with respect to photoreceptor morphology rh6 and rh2otd mutants, phototransduction is abolished in Pph13 mutants due to the loss and reduced transcription of several key components of the phototransduction machinery Drosophila Rhodopsin promoters Drosophila Pph13 is necessary and sufficient for driving photoreceptor specific reporter gene expression from the artificial 3XP3 promoter Drosophila, the question we address here is whether Pph13 and Otd functions represent a common regulatory pathway of arthropod r-visual photoreceptor differentiation.The second critical transcription factor is PvuII-PstI homology 13 (Pph13), a paired-class homeobox protein that is similar to the vertebrate Aristaless-related homeodomain (Arx) proteins Tribolium castaneum (red flour beetle), a second insect, and Daphnia (water flea), a crustacean. Together, insects and crustaceans define the superclade Pancrustacea within the Arthropoda Daphnia, Tribolium, and Drosophila r- photoreceptor differentiation would indicate a pathway common to the ancestor that generated both lineages, at least 500 million years ago Tribolium and Daphnia and found only within LW r- opsins. Further, the Tribolium and Daphnia Pph13 homologs have retained similar DNA binding capabilities to their respective endogenous RCSI sites and we confirmed their functional equivalency to direct photoreceptor differentiation in Drosophila photoreceptors by transgenic rescue. The Otd paralogs of Tribolium and Daphnia are comparable in their ability to direct rhabdomere morphogenesis but exhibit differential abilities with respect to r- opsin regulation in Drosophila. Lastly, functional analyses in Tribolium reveal that both Pph13 and Otd homologs are essential for both aspects of photoreceptor differentiation, rhabdomere creation and r-opsin expression. In particular, Pph13 is a critical factor for LW r-opsin expression and Otd2 is necessary for the transcription of UV sensitive r-opsin. In summary, our data identify common components for rhabdomeric photoreceptor differentiation among Pancrustaceans, providing a foundation for defining the ancestral transcriptional mechanisms for rhabdomeric photoreceptor differentiation throughout Bilateria.To examine whether Pph13 and Otd could represent a common set of transcription factors required for r- visual photoreceptor differentiation, we chose to investigate their orthologs from two key nodal species, Drosophila r- visual photoreceptor differentiation was conserved, we investigated the conservation of orthologs in the genome sequences of the distantly related arthropod species, Tribolium castaneum and Daphnia pulexTribolium had been previously shown to possess two paralogs of Otd: Otd1 and Otd2 Parhyale hawaiensisDrosophila were previously considered unresolved due to the low level of sequence conservation outside the homeodomain; within Diptera there has been a reduction to only one otd paralog  was incorrect. Further examination of upstream genomic regions revealed that the DNA encoding the 5′ portion of the homeodomain was present. This conclusion was confirmed by RT-PCR (data not shown) and in the genome draft of a second related species: Daphnia magna. The complete Daphnia pulex Pph13 cDNA sequence thus included sequence from previously annotated loci JGI_V11_8835 and JGI_V11_313449. Sequence conservation between Daphnia, Tribolium, and Drosophila homologs was confined to the homeodomain  that may be of similar ancient origin based on conservation in non-arthropod invertebrates  studies of the development of the axonal photoreceptor connections with lamina neurons predict a model in which the photoreceptors begin to differentiate at the midline and move laterally as they mature Daphnia magna. In Daphnia pulex, there are 27 annotated r-opsin paralogs Drosophila, Daphnia pulex contain representatives of UV, LW and B- light sensitive r-opsins. This diversity includes 23 LW opsins that split between the LOPA and LOPB clades Daphnia magna but for our examination we assayed the expression of a pool of three putative representatives from LOPA  Tribolium contains two r- opsins, one of which belongs to the LW opsin subfamily and one of which belongs into the UV sensitive subfamily Tribolium homolog of Drosophila visual Gβ (Gβ76C), LOC662674 (beetlebase.org), we also detected a RCSI site  contained a potential RCSI site  Drosophila RCSI sites . These experiments revealed that SI sites . Interesin vivo expression patterns and in vitro binding assays provided strong evidence that Pph13 and Otd regulate r- visual photoreceptor cell differentiation in Drosophila, Tribolium and Daphnia. To test for functional equivalency among the orthologs and, more importantly, the ability to direct photoreceptor differentiation, we examined whether Daphnia and Tribolium orthologs were capable of rescuing the photoreceptor defects observed in Drosophila Pph13 and otd mutants.The Drosophila Pph13 mutants have two distinct characteristics. First, Pph13 is necessary for expression of opsin rh6  against each corresponding mRNA for injection into Tribolium larvae. None of the DsRNAs affected developmental timing, viability, or external morphological structures as compared to mock injections; scanning electron microscopy of the adult eye did not reveal any major effects on the external organization of the compound eyes  Daphnia Otd1 is orthologous to Drosophila Otd or Tribolium Otd1, it has the ability to rescue both rh6 repression and rh3 activation whereas Tribolium Otd1 the ortholog of Drosophila Otd rescued only the activation of rh3. Moreover, even though Tribolium Otd1 is expressed in all photoreceptors and has the ability to activate r- opsin transcription in Drosophila, this ortholog is apparently dispensable for r- opsin expression within Tribolium. Interestingly, the three vertebrate Otx homologs also exhibited differential rescuing activities of rh3, rh5 and rh6 regulation otd locus in insect and crustacean lineages contributed to different subfunctionalization trajectories. While the functional diversification that resulted from this appears bewildering, it implies continued evolutionary interchangeability, which may have been key to consolidating all Otd-related functions onto a single homolog during the loss of otd2 in the lineage to dipteran species.Consistent with the evidence of independent duplication events in the insect and crustacean lineages, we find that there is no simple correlation between the expression profiles of Drosophila eye is a good example of this paradigm. Interestingly, our functional analysis in Tribolium reveals differences in how Pph13 and Otd are employed in directing rhabdomere morphogenesis compared to Drosophila. First, within Tribolium, the reduction of either Otd1 or Otd2 generates non-overlapping defects in rhabdomere morphogenesis while the simultaneous knockdown of both genes leads to complete failure of rhabdomere formation. This outcome could result from incomplete subfunctionalization, leaving a limited degree of genetic redundancy in place. Alternatively, the two paralogs may have limited capacities to compensate for the downregulation of the sister paralog via expression level increase. Second, in Drosophila, both Pph13 and Otd are providing independent and overlapping functions to generate the rhabdomere Tribolium, the knockdown of either Pph13 alone or the knockdown of both otd paralogs is sufficient to eliminate rhabdomeres. Thus, there appears to be significantly less redundant control of rhabdomere formation in Tribolium in contrast to Drosophila. Assuming the general conservation of rhabdomere structure target genes, this finding implies evolutionary turnover of Otd and Pph13 dedicated target genes in the context of rhabdomere formation. Given the well defined binding properties of Pph13 and Otd and the conservation of the Pph13 binding sites between Drosophila and Tribolium, it should be feasible to elucidate the diverged distribution of Otd versus Pph13 targets in Tribolium versus Drosophila. Such studies will expand our understanding of the evolution of gene regulatory networks by specifically testing the proposal that downstream network components enjoy greater degrees of evolutionary freedom than intermediate upstream components The activation of structural gene batteries forms the endpoint in the gene regulatory network control of cell differentiation Daphnia Pph13 and otd2 have not been successful. Fortunately, with the advent of TALEN and CRISPR technology Pph13 relates to the diversification of ciliary and rhabdomeric photoreceptors during early metazoan evolution and whether Pph13 or Otd is required for the differentiation of non-visual rhabdomeric photoreceptors, as exemplified by intrinsic photosensitive retinal ganglion cells (ipRGC) Ever since Darwin pondered about the evolution of the eye Tribolium Pph13 and otd2 were constructed from RT-PCR reactions from total RNA isolated from beetle heads. The otd1 cDNA was a gift from Dr. G. Bucher. cDNAs representing Daphnia magna Pph13 and otd2 were constructed from RT-PCR reactions from total RNA isolated from whole adults. The Daphnia magna cDNA of otd1 was isolated by screening an embryonic cDNA library Pph13-GAL4 was generated by inserting the immediate upstream 1.6 kb of genomic DNA extending from the first coding Methionine of the Pph13 locus into pCHS-GAL4. Drosophila strains utilized: cn bw, hzycn bw Pph13uviotdy w; Sp/Cyo; UAS-Flag-otd/TM2uvi; otd-GAL4, Pwiz6/Cyo; TM2/TM6Botdotd rescue experiments females of uvi; otd-GAL4, Pwiz6/Cyo; TM2/TM6Botd were crossed to w; +; UAS – otd X transgenic lines and only non CyO;TM6B male progeny were examined. For Pph13 rescue experiments w; cn bw Pph13/CyO; Pph13-GAL4/TM6B homozygous flies were crossed to w; cn bw Pph13/CyO; UAS- Pph13 X/TM6B and only non CyO; TM6B progeny were examined.cDNAs representing All cDNAs were cloned into pCDNA 3.1(Invitrogen) and EMSAs were performed as described in pu11, wm26, or v late stage larvae; pu11 and m26 contain a 3XP3-GFP and a 3XP3-RFP reporter, respectively To generate RNAi knockdown animals, 1 ug/ul of total probe, DsRNA was injected into th) of RT reaction and samples were collected at 25 or 30 cycles. All reactions were repeated three times with two independent sets of total RNA. Equal amounts of PCR reactions were analyzed by gel electrophoresis. The list of PCR primers used can be found in Total RNA was isolated using Trizol and DNAase treated. First strand synthesis was accomplished with the Superscript III (Invitrogen) kit. Each reaction contained 2 ug of total RNA and oligo-dT as primers. PCR amplification was performed with 1 ul (1/20Tribolium release 3.0 gene models (http://www.Beetlebase.org/) using TopHat2 version 2.0.9 Tribolium, read counts were normalized across samples using the DESeq package (version 1.12) in R/Bioconductor Tribolium LW and UV opsin are listed in For each condition, duplicate sets of total RNA from entire animals (<12 hours old) were isolated. Stranded RNA sequencing libraries were constructed using the TruSeq Stranded mRNA Sample Preparation Kit  according to manufacturer's instructions. Libraries were quantified using the KAPA SYBR FAST Roche LightCycler 480 2X qPCR Master Mix , pooled in equal molar amounts, and sequenced on a HiSeq2000 instrument  using a 100 bp paired-end run. HISeq read sequences were cleaned using Trimmomatic version 0.30 Drosophila and Tribolium heads were prepared as previously described Tribolium Pph13 polyclonal antibody, 171, was created by injecting rats with a GST-fusion protein representing amino acids 104–220 of the protein. The Daphnia magna Otd2 polyclonal antibody was prepared in guinea pigs against a bacterially expressed C-terminal portion of the protein (amino acids 235–395). The Daphnia magna Otd1 polyclonal antibody was prepared in rats against a bacterially expressed portion of the protein (amino acids 220–351).The in situ hybridization in Tribolium and Daphnia was performed as previously described Tribolium Pph13 (1∶20), rat anti- Daphnia Otd1 , guinea pig anti- Daphnia Otd2  mouse anti-Rh3 , mouse anti-Rh5  and rabbit anti-Rh6 . For Otd2, in Daphnia, signals were amplified with the Tyramide Signal Amplification (TSA) system (PerkinElmer). FITC and Rhodamine conjugated secondary antibodies were utilized (Jackson ImmunoResearch). Immunofluoresence studies were performed as previously described Drosophila samples were from newly emerged adults and for each genotype at least three retinas from three different heads were examined.Whole mount RNA Many of the sequences utilized in this study can be found in Figure S1http://homeodb.zoo.ox.ac.uk/).Phylogenetic tree of Orthodenticle and related homeodomain transcription factors. Maximum likelihood tree estimated in MEGA 5.2.2 (TIF)Click here for additional data file.Figure S2http://homeodb.zoo.ox.ac.uk/).zPhylogenetic tree of Pph13/Aristaless and related homeodomain transcription factors. Maximum likelihood tree estimated in MEGA 5.2.2 (TIF)Click here for additional data file.Figure S3in situ hybridizations with sense probes in the developing retina of adult Tribolium. A–C. Dissected retinas 30–40 hrs APF of developing Tribolium castaneum adult visual system. None of the sense probes for Pph13 (A), otd1 (B), otd2 (C) demonstrate a specific expression pattern in the developing retina. At this developmental time point it is impossible to discern the developing retina field from surrounding tissue without a molecular marker for the photoreceptors.RNA (TIF)Click here for additional data file.Figure S4Daphnia pulex. Phylogeny of 27 Daphnia pulex opsin proteins inferred using the Neighbor-Joining method with bootstrap test (500 replicates). Scale bar represents genetic distance as number of amino acid substitutions per site. Tree modified from original data presented in Phylogenetic tree of r- opsins of (TIF)Click here for additional data file.Figure S5Daphnia magna. Mixed staged embryos were examined for expression of long wave (LW) A r-opsins , long wave B r-opsins , ultra violet (UV) r-opsin , otd2 , and Pph13 . Circles represent expression in the putative eye. None of the sense probes demonstrate a specific expression pattern in the developing retina.RNA in situ hybridizations with sense and antisense probes in the developing retina of (TIF)Click here for additional data file.Figure S6D. melanogaster, T. castaneum and three D. pulex homologs inferred from Neighbor-Joining method with bootstrap test (500 replicates). Scale bar represents genetic distance as number of amino acid substitutions per site. Loci in red indicate Gβ containing an RCSI site. Gβ76C-flybase.org, LOC662674-beetlebase.org.Phylogenetic tree of Gβ comparisons. Evolutionary relationships of Gβ proteins of (TIF)Click here for additional data file.Figure S7Daphnia magna Pph13. Electrophoretic mobility shift assays of Daphnia magna Pph13 protein on its endogenous Gβ RCSI site with or without 25 fold excess of cold competitor RCSI sites Click here for additional data file.Figure S8Daphnia magna Otd2 in an otd mutant background.  Utilizing the established paradigm of +white  tissue”.Expression of (TIF)Click here for additional data file.Figure S9In vivo rescue of Rh6 opsin repression in Drosophila orthodenticle mutant background. A–E. Rh6 opsin protein expression in adult Drosophila retinas. A. Wild-type retina. Rh6 protein accumulates in approximately 70% of the central R8 photoreceptors. B. otd mutant. Rh6 expression is detected in numerous photoreceptors in each ommatidium and accumulation is diffuse due to the disruption of rhabdomere morphology. C–G. Rescue of otd mutant with: C. Drosophila (Dmel) otd. D. Tribolium (Tcas) otd1. E. Tribolium (Tcas) otd2. F. Daphnia magna (Dmag) otd1. G. Daphnia magna otd2. All Otd orthologs, except Tcas Otd1, are capable of repressing rh6 transcription and limiting Rh6 expression in a subset of R8 photoreceptors. Insets represent a magnified view of a region of each panel.(TIF)Click here for additional data file.Figure S10In vivo rescue of Rh5 opsin expression in Drosophila orthodenticle mutation. A–E. Rh5 opsin protein expression in adult Drosophila retinas. A. Wild-type retina. Opsin protein accumulates in the rhabdomeres and thus appears as a tube like structure. B. otd mutant. The absence of Otd activity results in the absence of Rh5 transcription and thus no protein is detected. C–G. Rescue of otd mutant with: C. Drosophila (Dmel) otd. D. Tribolium (Tcas) otd1. E. Tribolium (Tcas) otd2. F. Daphnia magna (Dmag) otd1. G. Daphnia magna otd2. Only with Tcas otd2 did we see the expression of Rh5 in the absence of additional melted signaling (TIF)Click here for additional data file.Figure S11Tribolium otd2 affects rhabdomere biogenesis. Transmission electron microscopy analyses of Tribolium adult rhabdomeres. (A–C) Three additional samples of phenotypes observed with the knockdown of otd2. All samples were from newly emerged adults. Scale bar is 2 um. The removal of otd2 resulted in rhabdomere degeneration as observed by the large separations between microvilli and extension of microvilli into the cell body.Knockdown of (TIF)Click here for additional data file.Table S1Daphnia, Tribolium and Drosophila RCSI sites. Motif search: Motif search for TAATNNNATTA carried out using perl script “motifsearch.pl” on 1000 bp upstream regions of 27 Daphnia pulex opsin genes and confirmed by eye. Daphnia pulex genome release 1. Due to gaps in genome coverage, we could not determine whether an RCSI site exists in Daphnia r- opsins: LOPB12, LOPB13, LOPB14, and UNOP1.Identification and Sequence of (DOCX)Click here for additional data file.Table S2Quantification of RNAi injections scored for the absence or presence of 3XP3-RFP.(DOCX)Click here for additional data file.Table S3in situ hybridizations.Probes regions for RNAi and RNA (DOCX)Click here for additional data file.Table S4PCR primers for reverse transcriptase-polymerase chain reaction assays.(DOCX)Click here for additional data file.Table S5RNAseq data for Tcas LW and UV opsins.(DOCX)Click here for additional data file.Table S6RNAseq total read counts.(DOCX)Click here for additional data file.Table S7Gene sequences utilized in this study.(DOCX)Click here for additional data file.Movie S1Daphnia magna. The nuclei are counter stained with DAPI.Confocal stack of Otd2 expression (green) in the two visual clusters at 48 hrs after egg deposit (AED) in (MOV)Click here for additional data file.


NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD), we then used Cap Analysis of Gene Expression (CAGE) to identify thousands of their targets genome-wide (KD-CAGE). The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN), and ISL1, and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6, and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 50 kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN , NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1, and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6, and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting ChIP-seq datasets. (1) A large fraction of binding sites are at distal enhancer sites and cannot be directly associated to their targets, without chromatin conformation data. (2) Many peaks may be non-functional: even when there is a peak at a promoter, the expression of the gene may not be affected in the matching perturbation experiment.Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK) which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs) and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5), we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with  Regulation of gene expression by combinations of transcription factors (TFs) is a fundamental process that determines cellular identity and functions. TFs have the ability to recognize and bind short sequence motifs throughout the genome, and, either alone or in combination with other TFs, modulate mRNA levels in a cell until it acquires the predetermined phenotype  in their promoter regions have helped to identify direct targets  and gastrin , ghrelin (GHRL), and transthyretin , glucagon (GCG), and islet amyloid polypeptide (IAPP), the biomarkers for pancreatic delta, alpha, and gamma cells, respectively, were lowly expressed or absent in TC-YIK cells. We next examined the expression of genes described in the beta cell gene atlas  as likely important factors in pancreatic development.CAGE profiling of the mouse orthologs throughout pancreatic development  detected 33 of the 42 TFs in at least one stage with most changing expression levels over time  important than housekeeping TFs that are more broadly expressed. Logic would suggest that those TFs expressed in an enriched manner are more likely to be regulated by other enriched TFs, and that their targets are also more likely to be enriched. To test our assumption, we first carried out siRNA perturbation of a set of enriched and non-enriched (but expressed) TFs in TC-YIK cells and assessed their effect on expression of enriched and non-enriched targets by qRT-PCR.Multiple siRNAs were tested for each enriched factor and the one with the best efficiency was kept; siRNAs for 26 TFs reduced expression below 50%, a further 7 suboptimal siRNAs reduced expression to 51–77% of that of the scrambled control, while for the remaining TFs we were unable to find an efficient siRNA . In an analogous way, ISL1 was the main antagonist in the network, where its knock-down affected 11 targets, all of which were up-regulated . Other likely key TFs, RFX6, SHOX2, PAX6, MNX1, LMX1A, and PDX1 also strongly affected several targets.Looking at the number of affected targets for each TF knock-down (out degree) and the number of knock-downs that affected each TF  led to the highest increase in insulin transcript, which may indicate a shift in the balance between TATA dependent and TATA independent transcription.Of note, knock-down of 28 of the 33 TFs enriched in TC-YIK and 7 of the 8 non-enriched TFs affected insulin expression levels, with the enriched factors The above section focused on a limited and biased set of 52 target transcripts. We next applied CAGE , NEUROD1 , RFX6 , and PAX6 . Note to readers, the following antibodies were also tried but failed in ChIP-seq: .List of antibodies used in the ChIP-seq experiments: All experiments were carried out as biological duplicates. Immunoprecipitated and input chromatin samples were incorporated into 4-plex ChIP-seq libraries using the NEBnext kit (New England Biolabs). Libraries were labeled with a 6 bp barcode and then pooled to be sequenced on Illumina HiSeq2000.Sequencing results were mapped to the human genome (hg19) using BWA software  with the option “–mask” to filter out bindings on repeats. The target sequences are the regions under the peaks and the background regions are randomly sampled sequences from the genome (Hg19) with similar GC content as the target sequences.We used HOMER software for de-novo motif discovery ]. All sequencing data used in this study has been deposited to DDBJ Read Archive (http://www.ddbj.nig.ac.jp/) with accession number DRA002420 (CAGE data) and DRA002468 (ChIP-seq data). CAGE expression profiles and enrichment of TFs for TC-YIK cell line are part of the FANTOM5 main data set. siRNA perturbations, CAGE-KD, and ChIP-seq experiments were generated separately for this study. Additional material can be found at the following URL .This work is part of the FANTOM5 project. Data download, genomic tools and co-published manuscripts have been summarized at AF designed the study and wrote the manuscript; ML carried out all bioinformatics analyses and wrote the manuscript; YI carried out the siRNA perturbations, qRT-PCR and chromatin immuno-precipitation experiments with help from AK; MI provided the CAGE libraries; TL and AH mapped the CAGE data; YN provided the TC-YIK cell line; JS helped with visualization in ZENBU; HK contributed to the ChIP-seq analysis and provided the set of CAGE peaks; HS, HK, PC, YH, and AF supervised the project.FANTOM5 was made possible by the following grants: Research Grant for RIKEN Omics Science Center from MEXT to Yoshihide Hayashizaki; Grant of the Innovative Cell Biology by Innovative Technology (Cell Innovation Program) from the MEXT, Japan to Yoshihide Hayashizaki; Research Grant from MEXT to the RIKEN Center for Life Science Technologies; Research Grant to RIKEN Preventive Medicine and Diagnosis Innovation Program from MEXT to YH. We thank Michiel de Hoon for proofreading the manuscript. We would also like to thank RIKEN BRC for providing the TC-YIK cell line samples and thank GeNAS for data production. ARRF is supported by a Senior Cancer Research Fellowship from the Cancer Research Trust and funds raised by the MACA Ride to Conquer Cancer.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sepsis is a global burden and the primary cause of death in intensive care units worldwide. The pathophysiological changes induced by the host’s systemic inflammatory response to infection are not yet fully understood. During sepsis, the immune system is confronted with a variety of factors, which are integrated within the individual cells and result in changes of their basal state of responsiveness. Epigenetic mechanisms like histone modifications are known to participate in the control of immune reactions, but so far the situation during sepsis is unknown.In a pilot approach, we performed combined chromatin immunoprecipitation followed by high-throughput sequencing to assess the genome-wide distribution of the chromatin modifications histone 3 lysine 4 and 27 trimethylation and lysine 9 acetylation in monocytes isolated from healthy donors (n = 4) and patients with sepsis (n = 2). Despite different underlying causes for sepsis, a comparison over promoter regions shows a high correlation between the patients for all chromatin marks. These findings hold true also when comparing patients to healthy controls. Despite the global similarity, differential analysis reveals a set of distinct promoters with significant enrichment or depletion of histone marks. Further analysis of overrepresented GO terms show an enrichment of genes involved in immune function. To the most prominent ones belong different members of the HLA family located within the MHC cluster together with the gene coding for the major regulator of this locus—CIITA.We are able to show for the first time that sepsis in humans induces selective and precise changes of chromatin modifications in distinct promoter regions of immunologically relevant genes, shedding light on basal regulatory mechanisms that might be contributing to the functional changes occurring in monocytes. Sepsis is a global burden and the primary cause of death on ICUs all over the world –3. DurinHistones can be posttranslationally modified by the enzyme-catalyzed addition of chemical groups to their N-terminal tails, e.g. acetylation, phosphorylation or methylation. The specific presence or absence of these histone modifications in promoter regions is functionally correlated with the expression of the associated genes in defined genomic regions . TrimethBy influencing gene expression, histone modifications seem to be indirectly associated with the regulation of different kinds of cell functions. Therefore, also the regulation of histone modifying enzymes like histone deacetylases (HDACs) seems to play a key role in inflammatory gene expression. It has been demonstrated that HDAC3-deficient murine macrophages lack the ability to express inflammatory genes after LPS (lipopolysaccharide) stimulation, which is attributable to a secondary effect by the loss of LPS-induced IFN-β (interferone-β) expression . MoreoveBesides manipulation of acetylation through histone deacetylases, also changes in methylation have been found to be important during immune reactions. The histone demethylase Jmjd3 (jumonji domain containing 3) has been identified to regulate immune response in murine macrophages after induction by the transcription factor NF-kB . It remoThis is in line with the finding, that continuous interleukin-4 stimulation induces an increase in Jmjd3 expression and decreases H3K27 methylation at the promoter regions of alternative macrophage marker genes followed by an increased transcriptional activation of these genes .Animal experiments using the cecal ligation and puncture (CLP) model, mimicking the clinical condition of polymicrobial peritonitis, showed both a decreased H4ac and H3K4me3 with reduced recruitment of RNA polymerase II and associated with decreased cytokine gene expression in lung macrophages. These findings correlated with the induction of Interleukin-1 receptor-associated kinase-M, a negatively regulating protein of the TLR signaling pathways. Interestingly, only few genes showed a transiently reduced expression in response to LPS during sepsis .Despite all evidence from animal experiments, the global pathophysiological mechanisms are still unknown and the mounting evidence that genomic responses in mouse models only poorly mimic the situation during human inflammatory diseases raises the ultimate need for primary cell analysis from patients suffering from sepsis .++ CD16−) seems to play an important part in the initial pro-inflammatory defence mechanism [Monocytes are key players in the innate immune system and their cellular functions are of high importance during early immune reactions. During sepsis, the classical monocyte subpopulation CD14++ CD16− echanism . Studiesechanism .Despite all efforts in immune and sepsis research the exact mechanisms leading to the pathological monocyte dysfunction are still unknown. An analysis of epigenetic modifications of human immune cells may help to understand the genomic alterations occurring and therefore unravel novel pathophysiological mechanisms in patients suffering from sepsis.In order to set the stage for epigenomic studies in the future, we conducted genome-wide histone modification analysis of human CD14++ CD16- monocytes from two patients suffering from sepsis. Our study demonstrates for the first time that sepsis in humans does not result in an uncontrolled remodeling of the chromatin landscape, but in selective and precise changes of chromatin modifications in distinct promoter regions of immunologically relevant genes.To assess the genome-wide distribution of the chromatin modifications H3K4me3, H3K27me3 and H3K9ac in CD14++ CD16- monocytes from patients suffering from sepsis  of their promoter regions for at least one of the examined posttranslational modifications . EmployiMonocytes belong to the group of antigen-presenting cells. Antigen-presentation is a crucial process during early immune responses required for full activation of the host response. Upon uptake, microorganisms are digested within the phagolysosomes and peptides are embedded into MHC class II complexes and presented on the surface of the cell for recognition by CD4+ T helper cells. During late sepsis a diminished expression of monocytic HLA-DR, which is a member of the MHC class II molecules, is a well known characteristic and closely correlates to the global loss of monocyte function, ultimately leading into an immuneparalytic state of the host . The genOverall, our results indicate changes in chromatin modifications, which seem to appear in a very specialized manner. As an example of high interest in sepsis, we found alterations in the MHC class II locus and its corresponding master regulator CIITA. In line with results of earlier studies in monocytes from septic patients, which show a decreased expression of MHC II genes as well as the gene encoding CIITA , our finAnother finding with potential implications in the late pathophysiology of the disease is the alterations of the promoter regions of IL-1β as well as the IL1 Receptor 2 (IL1R2). While the promoter of IL-1β is depleted of active marks , both marks are accumulating at the IL1R2 promoter . RemarkaSepsis might lead to changes in the expression of certain important transcriptional regulatory factors, resulting in changes in the modification pattern of downstream promoters. Interfering with these factors might be a promising therapeutic approach, for which already experimental evidence exists. By applying the synthetic histone I-BET, which mimics acetylated histones, pro-inflammatory gene expression is abolished during systemic inflammation . In addiOur first results might be promising, but it is a long way to step out of the dark of applying treatment in a blackbox scenario towards the light of guided, surgical precise modulation of the epigenome. Before this might happen, we need to conduct further studies with larger sample sizes, and eventually including more histone modifications and especially more cell types—both hematopoetic and somatic. Moreover, as sepsis is a dynamic syndrome, we need to understand the spatiotemporal kinetic of epigenomic patterns and their functional consequences as well as differences depending on the underlying cause or focus of infection. Nevertheless, we believe that this bottom-up approach “from promoter to target” will raise new therapeutic options for the treatment of sepsis and other inflammatory diseases.All together this study provides the first comprehensive ressource of the epigenomic changes occurring in CD14 ++ CD16- monocytes of patients suffering from sepsis. The genome-wide maps of three histone modifications indicate that a defined set of immunologically relevant genes undergo specific alterations of their associated histone modification patterns in human sepsis.Ethics Statement: All experiments were conducted according to the principles expressed in the Declaration of Helsinki, with approval of the ethics committee of the medical faculty of the Justus-Liebig-University of Giessen, Klinikstrasse 32, D-35385 Giessen, Germany . Subjects provided written, informed consent on forms approved by the Institutional Review Board.Cell separation: For monocyte separation 30 ml blood sample was taken and immediately processed from 2 patients suffering from sepsis and 4 healthy donors. Ficoll-based density gradient centrifugation was used to isolate peripheral blood mononuclear cells. To isolate CD14++ CD16- monocytes, magnetic cell sorting  was conducted using CD16 MicroBeads (Miltenyi Biotec) to deplete CD16++ monocytes and CD14 MicroBeads (Miltenyi Biotec) to finally isolate CD14++ CD16- monocytes.Purity of CD14++ CD16- monocytes was measured using flow cytometry after incubation with FITC anti-human CD14 Antibody (BioLegend).6 cells per 200 μl lysis buffer) sonication was performed using Branson sonifier (fragment size 150–600 base pairs) under constant cooling. Preparation of immunoprecipiation was conducted after seperation of 10% input using agarose A/G-beads . Chromatin was mixed with antibodies for H3K4me3, H3K27me3 and H3K9ac at 4°C in a rotator for 3 hours. Different concentrations of salt wash buffer were to clear chromatin bead complex, before reverse Cross-linking (RNase A followed by 10% SDS/Proteinase K mix). Illustra GFX PCR DNA and Gel Band Purification kit  was used to purify DNA. DNA was eluted in pure H2O and quantitation of ChIP-DNA determined by Qubit Assay .ChIP protocol: ChIP was performed as described before . CD14++ Library amplification: We used MicroPlex Library Preparation kit (Diagenode) for library amplification with the presence of SYBR Green in a real-time PCR machine as previously described . AMPure Sequencing: Illumina HiSeq was used for sequencing of 4 barcoded libraries  for each patient and healthy donor on a single lane. Cluster generation was performed using the Illumina cluster station, sequencing on the Illumina HiSeq 2500 followed a standard protocol. The fluorescent images were processed to sequences using the Genome Analyzer Pipeline Analysis software 1.8 (Illumina). All sequencing data was deposited at NCBI’s gene expression omnibus (GEO) under the accession number GSE62908.http://www.bioconductor.org).Processing of ChIP-seq reads: ChIP-Seq reads were converted to fastq format and aligned to a precompiled hg19 reference index with BOWTIE . Sequenchclust function using the "complete" method.Differential promoter binding analysis: Read counts were determined for the promoters of all annotated hg19 RefSeq transcripts. The RefSeq transcriptional start sites (TSSs) were used to identify promoter intervals ranging from −1 to +1 kb around the TSS. For each promoter the number of reads mapping to the respective interval were extracted for patient and control samples. DESeq  was usedVisualization of binding profiles: After extension of reads, continuous coverage vectors were calculated and normalized per million reads to account for differential library sizes. Additonally the coverage vectors were smoothed in a 1000 bp window, quantile-normalized and subsequently converted into bigwig format for visualization in genome browsers. The coverage vectors were used to collect data in windows of different sizes spanning transcriptional start sites using the bigWigSummary tool  applicatIdentification of enriched GO terms: Clusters identified by hierarchical clustering as described above were used to identifiy over-reprasented GO-terms using DAVID .Statistics were calculated by the Mann-Whitney signed Rank test or paired t-test using R.S1 FigBoxplots shows z-scaled read counts of MHC genes for individual patients (#P1 and #P2: red boxes) and controls (#C1-#C4: blue boxes) for three modifications.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableSummary statistics describing differential association of histone modifications comparing patients and controls over 743 gene promoters with significant changes for at least one histone modification.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in Leishmania major and Trypanosoma brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription termination and increased expression of downstream genes. Thus, J regulation of RNAP II transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates termination and gene expression at specific sites within polycistronic gene clusters.Base J, β-d-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA polymerase (RNAP) II initiation and termination. Reduction of J in  Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, which cause African sleeping sickness, Chagas’ disease and leishmaniasis, respectively. Kinetoplastids are early diverged protozoa with unique genome arrangements where genes are organized into long clusters, or polycistronic transcription units (PTUs), which are transcribed by RNA polymerase (RNAP) II  II . RNAP IIRNAP) II . TranscrRNAP) II . Pre-mesnylation . In othenylation . In kineent gene , preventWhile little is known regarding the regulation of gene expression in kinetoplastids, the unique genome arrangement and polycistronic transcription of functionally unrelated genes has led to the assumption that transcription is an unregulated process in these organisms and that most gene regulation occurs post-transcriptionally ,17. ReceT. brucei, T. cruzi and L. major  for activity (T. brucei or growth in the presence of DMOG results in cells devoid of base J (J null)  ,34; howerganisms ,35.T. cruzi, following the deletion of either JBP1 or JBP2, resulted in the formation of more active chromatin at transcription initiation sites, increased RNAP II recruitment and PTU transcription rate, and global changes in gene expression  detection of the nascent transcript confirmed that we are measuring J-dependent defects in transcriptional termination, rather than RNAP II reinitiation events. Complete loss of J in T. brucei failed to indicate any defect in termination within cSSRs or HT sites. However, we localized base J at sites prior to the end of a PTU where the loss of J led to upregulated expression of the downstream genes within the same PTU. For one of these sites we show that the gene expression changes occurred at the level of transcription. Therefore, while base J regulates RNAP II termination in both L. major and T. brucei, it does so to different degrees and at different genomic locations. In L. major, J regulates termination at the end of each PTU to prevent the generation of antisense RNAs genome-wide. In contrast, while termination occurs at the end of each PTU in T. brucei in a J-independent manner, J-dependent termination within a PTU allows developmentally regulated expression of downstream genes.To explore the conservation of J function among kinetoplastids and avoid indirect effects associated with the use of BrdU and genetic deletions, we utilized DMOG to examine the role of J in regulating RNAP II termination in All restriction enzymes were purchased from New England Biolabs. Prime-It II random primer labeling kit was purchased from Stratagene. ECL (enhanced chemiluminescence) and Hybond-N+ were from Amersham. Goat anti-rabbit HRP (horseradish peroxidase) was purchased from Southern Biotec Inc. All other chemicals were purchased from Sigma Aldrich.T. brucei cell line 221a of strain 427 was cultured in HMI-9 medium as described previously  as described  was performed as described  according to the manufacturer's instructions with the following exception: the PCR amplification was performed for 12–16 cycles using the KAPA HiFi DNA polymerase (Kapabiosystems). The PCR product was purified and concentrated using AMPure XP beads (Beckman Coulter). Quality and concentration of all libraries was determined using a Bioanalyzer 2100 (Agilent) and high-throughput sequencing was performed on a HiSeq2000 (Illumina).The two full-length RNA-seq libraries were constructed from ∼1 μg of total RNA by Vertis Biotechnology AG. Briefly, total RNA was polyA-enriched using oligo(dT) chromatography and fragmented by ultrasound. Next, first strand cDNA was synthesized using N6 random primers followed by a strand-specific ligation of sequencing adapter to the 3′ and 5′ ends of the first stranded cDNA and PCR amplification of 10–20 cycles depending on the amount of starting material. High-throughput sequencing was performed on a HiSeq2000 (Illumina).Sequencing reads were mapped to the respective reference genomes (see Supplementary Table S2) using bowtie-2 with default ‘local-sensitive’ mode  and furtT. brucei cultures were grown in the presence of DMSO or 1-mM DMOG for 5 days. Nuclear run on was then performed using 1 × 109 cells as described  per the manufacturer's instructions. Quantification of selected genes was performed on an iCycler with an iQ5 multicolor real-time PCR detection system . Reaction conditions and thermocycling parameters used were the same as those described above. All transcripts were normalized using 40s rRNA. Briefly, triplicate cDNAs were diluted 1:10 and the 40s cDNA starting quantity (in nanograms) was determined using a standard curve (described above). The ratio of WT:WT DMOG-treated 40s cDNA starting quantity was used to normalize all subsequent transcripts analyzed by RT-qPCR. Primer sequences used in the analysis are available upon request.L. major grown in medium containing 5-mM DMOG for 10 days resulted in ∼32-fold reduction in the total level of base J , as reflected in small RNA degradation products. As expected, RNAP II transcription generally terminated at a defined location within the cSSR in wild-type cells. As described in L. tarentolae  that normally lacks J  site that lacked a detectable readthrough defect by small RNA-seq. A readthrough product was also not observed at a site (35.6) where DMOG failed to significantly reduce J and no readthrough defect was detected by small RNA-seq . We conclude that the small RNA-seq analysis accurately measures termination defects where base J regulates RNAP II termination at the end of PTUs to prevent transcriptional readthrough in L. major. Furthermore, consistent with a direct relationship between the strength of RNAP II termination and level of base J, we detected increased amounts of readthrough product with increased concentrations of DMOG (1–5 mM)  Figure . We haveL. major . OverallLeishmania spp. prompted us to examine T. brucei for similar transcription termination defects upon the loss of base J. Base J is not essential in T. brucei, as both JBP enzymes can be knocked out without obvious phenotypic effects  are located downstream of a J peak within a gene cluster, suggesting that base J may facilitate termination prior to the end of a gene cluster and attenuate the transcription of downstream genes. Consistent with RNAP II termination prior to the end of a PTU, H3V is also enriched upstream of many of the upregulated genes . An exam7% are loL. major, as was observed in L. tarentolae. Whereas previous analyses utilized a JBP2 KO treated with BrdU, the use of DMOG as an alternative method to reduce J levels in wild-type cells strongly suggests the epigenetic modification itself regulates RNAP II termination and that termination defects were not due to the loss of JBP2 protein or an indirect effect of BrdU or DMOG. We also demonstrate that J functions to regulate termination at HT sites in L. major in addition to cSSRs. Finally, we confirmed the termination defects identified by small RNA-seq as readthrough transcription using strand-specific RT-PCR. These findings suggest antisense RNAs were produced as a result of readthrough transcription, though we cannot exclude the possibility that some RNAP II reinitiation contributed to the production of additional antisense RNAs.We have clearly demonstrated an important function for base J in the regulation of RNAP II termination in L. tarentolae JBP2 KO cells led to the conclusion that readthrough transcription and subsequent generation of antisense RNAs were responsible for cell death .In contrast to cumented ,54. T. b pathway , though T. brucei failed to detect any termination defect, total RNA-seq analysis identified several sites where J appeared to promote transcription termination prior to the end of a polycistronic gene cluster. The loss of base J within a PTU resulted in increased transcript abundance of genes downstream of the J peak. The majority of these sites are devoid of chromatin modifications associated with transcription initiation, such as histone variants H2AZ, H2BV or histone acetylation, but are enriched for histone variants H3V and H4V, which are associated with transcription termination in T. brucei . Our nuclear run-on analysis supports the conclusion that increases in transcript abundance were due to increased transcription; therefore, it is possible that small RNA-seq analysis does not provide a complete picture of the primary transcriptome in T. brucei. It is plausible that the presence of the RNAi machinery affects the pool of small RNAs detectable by the deep sequencing approach used here, which requires a 5′-P and a 3′-OH. Consistent with this possibility, in Leishmania braziliensis, which contains a functional RNAi pathway, small RNAs indicative of readthrough are not observed at the single cSSR that lacks base J, in contrast to other RNAi negative Leishmania spp. [(Based on a model in which base J attenuates RNAP II elongation in ia spp. [ and demoT. brucei is consistent with the developmentally regulated synthesis of base J and absence of the modification in the procyclic stage of the parasite (Leishmania spp., where base J is found in all but one cSSR (T. brucei (Leishmania spp., but in T. brucei J has a more specialized function attenuating transcription and regulating gene expression at specific genomic loci. Additional experiments are underway to explore the mechanism through which J promotes RNAP II termination, specifically investigating whether the modification directly inhibits RNAP II elongation.The non-essential role of base J in parasite . It is pone cSSR , J is no. brucei . Thus, bAll sequencing data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE57621.Supplementary Data are available at NAR Online.