Escherichia coli response regulator NarL controls transcription of genes involved in nitrate respiration during anaerobiosis. NarL consists of two domains joined by a linker that wraps around the interdomain interface. Phosphorylation of the NarL N-terminal receiver domain (RD) releases the, otherwise sequestered, C-terminal output domain (OD) that subsequently binds specific DNA promoter sites to repress or activate gene expression. The aim of this study is to investigate the extent to which the NarL OD and RD function independently to regulate transcription, and the affect of the linker on OD function.The frdA-lacZ or activate narG-lacZ reporter fusion genes. These in vivo expression assays revealed that the NarL OD, in the absence or presence of linker helix α6, constitutively repressed frdA-lacZ expression regardless of nitrate availability. However, the presence of the linker loop α5-α6 reversed this repression and also showed impaired DNA binding in vitro. The OD alone could not activate narG-lacZ expression; this activity required the presence of the NarL RD. A footprint assay demonstrated that the NarL OD only partially bound recognition sites at the narG promoter, and the binding affinity was increased by the presence of the phosphorylated RD. Analytical ultracentrifugation used to examine domain oligomerization showed that the NarL RD forms dimers in solution while the OD is monomeric.NarL OD constructs containing different linker segments were examined for their ability to repress The NarL RD operates as an on-off switch to occlude or release the OD in a nitrate-responsive manner, but has additional roles to directly stimulate transcription at promoters for which the OD lacks independent function. One such role of the RD is to enhance the DNA binding affinity of the OD to target promoter sites. The data also imply that NarL phosphorylation results in RD dimerization and in the separation of the entire linker region from the OD.The online version of this article (doi:10.1186/s12866-015-0502-9) contains supplementary material, which is available to authorized users. Bacteria, archaea, and lower eukaryotes rely on two-component signal transduction systems as a major strategy to monitor the extra- and intracellular environment for physical and biological changes . These mEscherichia coli, the availability of nitrate during anaerobiosis triggers the Nar two-component system . To. ToE. coths Fig.  , ranginior Fig. . AdditionarG promoter for the nitrate reductase operon, NarL-P acts as a transcriptional activator. Here, the consensus DNA binding sites are located upstream from the transcription start site and lack any 7-2-7 sites  and NarLC (131–216) conferred a slight elevation of activity in the absence of nitrate when compared to the vector control, but showed the same low level expression pattern. In contrast, wild-type NarL gave rise to a 20-fold increase in activity in response to the presence of nitrate. As expected, the NarLN control was unable to activate narG-lacZ gene expression. These results show that the RD is essential for NarL to activate the narG operon. Western blot analysis for these and the above transcription assays showed that all of the NarX and NarL proteins were expressed in vivo, and in relatively uniform concentrations (data not shown).At the tes Fig. . When tharL Fig. . NarLC . Binding patterns of NarLC (147–216), NarL, and NarL-P were compared. NarL-P clearly protected both the −89 and −195 regions  conferred a strong yet different protection pattern at the −89 region, and displayed much weaker protection at the −195 region as compared to NarL-P. This weak interaction primarily resulted from alterations in hypersensitive sites and not by typical zones of protection. Although NarL-P and NarLC (147–216) both bind the −89 site with the same relative affinity, their different binding pattern may be suggestive of improper occupancy by NarLC (147–216). At the −195 region, NarL-P binds more extensively and with a stronger affinity. The compromised binding by the OD at the −195 region signifies at least one reason for the inability of NarLC (147–216) to activate narG-lacZ expression. At this promoter region, the phosphorylated RD is required to impart enhanced binding affinity and full occupancy.One reason for the lack of 5 region , 29. Occe region , 31. To frdA-lacZ repression by NarLC constructs containing loop α5-α6 , which bestows full frdA-lacZ repression , which contains helix α6, bound DNA with equivalent binding affinity as the NarLC (147–216) control  bound DNA less tightly, supporting the hypothesis that loop α5-α6 exerts an inhibition on the NarL OD. The same results were observed by the NarLC truncation proteins when the (His)6-tag was placed at the C-terminal end (data not shown), suggesting that the inhibition by the α5-α6 loop is not due to the (His)6-tag. Furthermore, NarLC (131–216) and NarLC (126–216) are expected to have adopted an overall correct fold , 24.9 kDa , and 23.4 kDa  which were best fit by a monomer and dimer (Table N (NarLN-P) gave apparent molecular masses of 30.8 kDa, 30.2 kDa and 29.0 kDa, and were best fit by a population largely in the dimeric form. When examined at 160 μM, similar results were observed and with reasonably good residuals. Unphosphorylated NarLN had apparent molecular-masses of 25.9 kDa  and 23.0 kDa , while those for NarLN-P were 30.4 kDa  and 27.4 kDa . The slightly lower-than-expected measured molecular masses for NarLN-P (expected ~32 kDa) were somewhat surprising. NarLN-P autophosphorylates more efficiently with acetyl phosphate than full-length NarL  domain co-crystallizes with a DNA 7-2-7 site as a dimer [C (147–216) was determined by sedimentation to be 9.6 kDa at 60 μM and 22,000 rpm, which is identical to its sequence molecular mass. This domain, therefore, remains monomeric in solution (Table C (147–216) was also predominantly monomeric at 160 μM and 22,000 rpm. To test whether the linker region affects NarLC dimerization, the NarLC (126–216) construct, which contains the entire linker region, was also examined by sedimentation. The molecular mass of NarLC (126–216) was determined at 22,000 rpm and 60 μM to be 12.7 kDa (Table C (126–216) was also predominately monomeric. Residuals from the exponential fitting were small, indicating little molecular mass heterogeneity. For both NarLC constructs, additional data were taken at speeds of 15,000 rpm and 18,000 rpm, which gave similar results. Taken together, these sedimentation results demonstrate that NarLC, either alone or with the linker attached, is predominantly monomeric in solution.As mentioned, the NarL a dimer , 13; butDa Table  and 12.4in vivo and in vitro results indicate that the NarL RD performs different functions to regulate transcription, which depend on the requirements governing specific promoter regions. This idea is summarized in Fig. frdA and narG promoters. Transcriptional repression of frdA presumably involves occlusion of RNA polymerase and the transcriptional activator FNR at the transcription start site [Our art site , 33. AccC constructs that conferred strong constitutive repression of frdA-lacZ (NarLC (147–216 and NarLC (131–216)) were unable to activate narG-lacZ expression and their behavior was not constitutive. Based on our footprint experiment, the lack of narG activation by these constructs is at least attributed to their inability to properly bind the essential −195 region. Thus, release of the sequestered NarL OD alone is insufficient to induce transcription of the narG operon; rather, the presence of the NarL RD is also mandatory for transcriptional activation  but require phosphorylation to enhance binding and bestow full transcriptional activation [narG promoter is not as important as sequestering it from binding to the frdA promoter. Therefore, a response regulator having a sequestered OD does not necessarily indicate that the OD has intrinsic function at all times. Likewise, the OD of DevR (or DosR), a homologue of NarL, is also inhibited in the inactive state, but during hypoxia the DevR OD alone is unable to activate transcription of genes that require cooperativity [At the tivation –38. In orativity . CooperanapF verses yeaR promoters. NarLC alone was able to fully activate napF and only partially activate yeaR despite both having a 7–2–7 binding site upstream of the transcription start site (−40 to −49 for napF and −38 to −47 for yeaR). This also implies that the functions of the NarL RD depend more on promoter specificity than on the location of the NarL binding sites. Discrepant roles for the RD at different promoter regions have also been observed or implicated in other RRs. Unphosphorylated PhoP, which does not have a blocked OD, can bind DNA derived from the phoP promoter, but requires a phosphorylated RD to bind DNA derived from the msl3 promoter. The RD of FixJ was proposed to be dispensable at the nifA promoter [fixK and is expected to be vital at this promoter [Our results are also consistent with the aforementioned NarL studies by Lin and Stewart  that shopromoter .C (131–216), bestowed similar behavior to that seen in NarLC (147–216), which lacks this helix and is known to fold correctly from its crystal structure [C (131–216) and NarLC (147–216) showed constitutive repression of frdA-lacZ expression and similar DNA binding in vitro, this indicates that NarLC (131–216) also folded correctly. Supporting evidence that NarLC (131–216) (and NarLC (126–216)) folded correctly stems from the NMR structure of the Erwinia amylovora RcsB OD and linker segment, which shows structural homology to the corresponding region (residues 129–216) in full-length NarL [C (131–216) is predicted to form hydrophobic contacts with helix α10, which, in this form, blocks OD dimerization. These modest interactions, however, could render helix α6 flexible and account for its apparent movement that enabled NarLC (131–216) to function properly. Thus, our data support the expected relocation of helix α6 upon NarL phosphorylation that would enable OD dimerization on DNA via helix α10 [We investigated the affects of specific linker segments on NarL OD function and found discrepant activities between OD constructs containing linker helix α6 verses those containing linker loop α5-α6. The presence of helix α6, in NarLtructure . Since bgth NarL . Therefoelix α10 .C (126–216), compromised frdA-lacZ repression in vivo and weakened DNA binding to a high affinity 7–2–7 site in vitro. This may be explained by the hydrogen bond between M128 in linker loop α5-α6 and G170 in loop α7-α8 of the OD , may partially anchor the OD in this region and impart the relative intermediate inhibition observed. By this activation model, release of the OD upon NarL phosphorylation entails severing contacts between the OD and loop α5-α6. This activation mechanism also correlates with our previous EPR studies whereby phosphorylation was proposed to induce a hinge bending movement in the vicinity of the Gly126 [S. pneumonia where the presumed semi-activated form shows a conformational change of the same helix α6 and the loop which follows [In contrast, the addition of loop α5-α6, such as in NarLe Gly126 . Interese Gly126 . Helix α follows .Fig. 7HyBased on our results, we propose that phosphorylation of NarL has a dual purpose, to relieve OD inhibition and to enable RD homodimers. Liberation of the OD would entail severing contacts between the RD and OD as wells as between the linker and OD. Once separated, the domains play independent, yet concerted, roles in regulating gene expression. Liberation of the OD alone is sufficient to control some promoters, while control of other promoters requires additional events involving the receiver domain. Thus, the RD can act as a molecular switch or bestow additional complex functions, such as increasing the DNA binding affinity and forming other protein-protein interactions, which render it completely necessary for stimulating transcription.−araD139 Δ(argF-lac)U169 rpsLl50 relA1 flb-5301 deoCl ptsF25 rbsR [in vitro studies the narL and truncated narL genes were amplified from existing plasmid constructions using PCR technology. TaKaRa Extaq polymerase , was used for PCR and the amplified products were cloned into the BamHl and Hindlll restriction sites of the pQE9 plasmid. This construction adds an N-terminal histidine epitope to the NarL protein and also results in substitution of Met1 to Gly. NarL is in-frame beginning at Ser2 with the Hindlll restriction site introduced 54 bases downstream from the stop codon of the narL coding sequence. The N-terminal NarL derivative is in-frame beginning at Ser2 with a stop codon introduced at Leu142, immediately followed by a HindIII restriction site. This construct (2–141) is referred to as “NarLN.” The NarL C-terminal expression constructs (referred to as “NarLC”) are in-frame beginning at their indicated residue with the HindIII restriction site introduced 54 bases downstream from the stop codon of the narL coding sequence. NarLC truncation proteins were generated by PCR amplification. The amplified fragments were digested with BamHI and HindIII and then subcloned into the same sites of PQE9  to add a C-terminal histidine epitope for use in the EMSA experiment only). All restriction endonucleases, polymerases, and other enzymes were purchased from New England Biolabs except where noted.The bacterial strains, bacteriophages, and plasmids used in this study are listed in Table F25 rbsR . DesiredF25 rbsR . BacteriE. coli JM109 cells containing the recombinant over-expression vectors were cultured in 10 mL L Broth with 100 μg/mL ampicillin overnight at 37 °C, and then transferred to 500 mL L Broth with 100 μg/mL ampicillin and grown at 37 °C until the OD600 reached 0.7–1.0. IPTG  was added to a final concentration of 2 mM. After 3 hours, the cells were harvested by centrifugation at 4000 rpm at 4 °C and then resuspended in buffer , with the addition of DNase (5 mg/mL) and RNase A (10 mg/mL). Cells were broken by passage through a French Press at 12,000 psi. The cell lysate was spun at 16,000 g for 10 min and the supernatant was either incubated with Qiagen Ni-nitrilotriacetic acid resin before loading onto a column, or was loaded onto a 1 mL HiTrap™ chelating Sepharose™ HP column . (His)6-tagged NarL proteins were purified according to the manufacturer’s instructions and the proteins were eluted with the above (or related) buffer containing 0.5 M Imidazole. Collected protein fractions were dialyzed and concentrated against a storage buffer  using an Amicon Centricon 10, and stored at −80 °C. Protein concentrations were determined using a Bio-Rad protein assay kit with BSA as the protein standard.frdA-lacZ) or λPC51 (narG-lacZ) reporter fusions and is used in all in vivo β-galactosidase experiments as previously described [Strain RC11 was lysogenized with either λLK1 . Purified NarL or NarLC (147–216) (0–2000 nM) and labeled DNA template (1.4 nM) were incubated in 20 μL complex formation buffer  for 30 min at 22 °C. For assays using phosphorylated NarL, the protein was first phosphorylated for 20 min  and 25 mM acetyl phosphate was also added to the individual reaction mixtures. DNase I  was then added to each reaction mixture for 30 s. The digestion reaction was terminated with the addition of 15 μL stop solution . Samples were precipitated, washed with 70 % ethanol, and resusupended in 5 μL sample loading buffer  before separating on 8 M urea, 6 % polyacrylamide electrophoresis gels. The G sequencing reaction ladders were created using an alternate protocol to the Maxim-Gilbert sequencing reaction [DNase I footprinting assays were performed essentially as described . A 391 bg assays . This frreaction . Gel expEcoRI. The resulting 183 base-pair oligonucleotide contained the engineered, 7−2−7 palindromic narG −89/−89 site, 5’-TACCCCTAAAGGGGTA-3’ (heptamer sites underlined). The oligonucleotide was extracted from gels and labeled with 32P using the Klenow fragment of DNA polymerase (New England Biolabs) and purified using a Qiagen PCR clean-up kit. The EMSA was performed, as previously described [32P-labeled DNA probes with NarL protein constructs for 10 min at room temperature. The reaction mixtures were immediately run on a 6 % non-denaturing polyacrylamide gel electrophoresis (Invitrogen) and visualized by autoradiography.Products from the PCR reaction, using the pIS69  plasmid escribed , by incuN (NarLN-P) was prepared by incubating 246 μM protein with phosphorylation buffer  and acetyl phosphate (at a 1200:1 molar ratio of acetyl phosphate to protein) for 10 min at room temperature. Since NarLN and NarLN–P are indistinguishable by gel electrophoresis, our method of phosphorylating NarLN was validated using radioactive acetyl-phosphate, which showed that NarLN phosphorylates more efficiently than NarL  at 60 μM, however, was dialyzed in the buffer solution containing 150 mM NaCl or 500 mM (NH4)2SO4. Both samples were tested and showed that different salt concentrations produced no significant difference in their sedimented molecular masses. Similarly, dialyzing NarL–P in the buffer solution containing 100 mM NaCl or 500 mM NaCl did not significantly change the results.Phosphorylated NarL (NarL-P) or phosphorylated NarLC (147–216), NarLC (126–216), NarLN and NarLN-P. For low wavelength (240 nm) scans, a baseline was determined by pelleting the protein at 50,000 rpm. The data were initially fit with nonlinear, least-squares exponential for a single ideal species using the Beckman Origin-based software (Version 3.01) to give a weight-average molecular mass of all species in solution. When concentration and speed dependence of the molecular masses indicated association behavior, multiple runs  were analyzed using the “multifit” option of the Beckman global analysis software. The monomeric sequence molecular mass and various models  were tested to see which would give the best fit to the data. Partial specific volumes of 0.740 for NarL, 0.733 for NarLN, 0.734 for NarLC (147–216) and 0.739 for NarLC (126–216) calculated from the amino acid composition and corrected to 4 °C were used [Sedimentation equilibrium runs were performed at 4 °C in a Beckman Optima XL-A analytical ultracentrifuge using absorption optics. Samples were examined in 3 mm double sector, 12 mm double sector and 12 mm six sector cells at an appropriate wavelength  to ensure the absorbance was sufficient to give a good signal-to-noise ratio and the maximum absorbance was within the linear range of the instrument (less than 1.35 OD). Sedimentation equilibrium profiles were measured at 11,000, 15,000 and 18,000 rpm for NarL and NarL–P and 15000, 18,000 and 22,000 rpm for NarLere used , 50. The


Archaeon, Thermococcus kodakarensis, under different physiological conditions.Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic , and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile.Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcriptsT. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon.The results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The The online version of this article (doi:10.1186/1471-2164-15-684) contains supplementary material, which is available to authorized users. Archaea are prokaryotes, they resemble Bacteria in genome size, genome organization and the absence of a nuclear membrane, but their genetic information storage and expression components are generally more closely related to their eukaryotic than bacterial counterparts  was performed using the RNA-analysis pipeline READemption version 0.1.6 [segemehl version 0.1.4 [T. kodakarensis genome annotation [exp and C), generated with and without TEX digestion, and the remaining libraries were then manually checked to confirm these TSS and for additional TSS. As illustrated  and the ggplot2 package [DEseq[T. kodakarensis and microarray expression data for P. furiosus[heatmap.plus. The organization of genes into transcription units (operons) in the T. kodakarensis genome was taken from the DOOR2 database [The pooled sequence reads were de-multiplexed and the adapter sequences were removed. The reads in Fastq format were then quality trimmed using n 0.0.13 ) with a on 0.1.6  with defon 0.1.4 . An autoon 0.1.4 . The sofnotation  extendednotation  and the notation . TSS wer package . The bioge [DEseq was used furiosus taken frdatabase , and to MEME version 4.8.1 [MEME and potential RBS in previously unrecognized transcripts were sought by FIMO using standard settings [MEME generated position weight matrix (PWM) as input  < 0.06, and all potential homologues were then manually inspected. The number of identical nucleotides in sRNA alignments was divided by the total number of nucleotides in the query sRNA, and multiplied by 100 to obtain a percentage conservation value. Only conservation values ≥40% were retained for further analysis . The woon 2.1.g ). Orphann 2.2.28 ) with deT. kodakarensis dRNA-seq libraries. The Table lists the total number of sequenced cDNA reads considered in the analysis, the number of reads that were removed due to insufficient length (<12 nt) after poly(A)-tail trimming (before read mapping), the number of reads that were successfully mapped to the reference genome, the number of mappings, and the number of uniquely mapped reads. Percentage values  are also provide for the number of mapped reads and number of uniquely mapped reads. (XLSX 369 KB)Additional file 1: Table S1: Mapping statistics of Additional file 2: Table S2: list of all identified TSS. The Table provides the positions and assigned classes of all identified TSS. When a TSS is assigned to more than one category, there is one row for each assignment with the associated gene. (XLSX 147 KB)T. kodakarensis and relates them to the dRNA-seq identified TSS. (XLSX 7 KB)Additional file 3: Table S3: Previously experimentally determined TSS. The Table lists all previously known or inferred TSS in Additional file 4: Table S4: List of likely mis-annotated genes. The Table lists positions of TSS and the corrected translational start sites of genes that were considered mis-annotated. (XLSX 10 KB)Additional file 5: Figure S1: Sequences upstream of sRK28 and TK1195. The sequences upstream of sRK28 and TK1195 and sRk28 are shown with the promoter elements , and the TSS (+1) documented by dRNA-seq, identified in bold text. The region encoding sRK28 is highlighted in red. The sequence encoding the 5′-UTR (grey) and the GTG translation initiating codon of TK1195 are identified. (PDF 23 KB)RNAfoldAdditional file 6: Figure S2: Secondary structure predictions of TEX resistant regions of transcripts from near the 3′-termini of genes. The sequences shown, transcribed from regions near the 3′-termini of the indicated genes, survived TEX digestion and so were prevalent in the dRNA-seq libraries. The secondary structures shown, and predicted stabilities, were generated by T. kodakarensis genome. A-E. The genomic locations of five TK HgcC transcripts (black boxes). The abundances of transcripts synthesized from these regions in T. kodakarensis cells growing exponentially  and in stationary phase  in sulfur medium, growing exponentially in pyruvate medium before  and 20 min after sulfur addition  are given by the peak heights. Data from the control library (C) not digested with TEX are shown in grey. As illustrated, an antisense RNA is also transcribed from the region encoding HgcC5. The relative abundance scales on the right of each panel allow direct comparisons of all data in that panel. The black scale bar in the top right corner of each panel is corresponds to 100 nt. (PDF 1 MB)Additional file 7: Figure S3: HgcC transcripts encoded in the T. kodakarensis genome. The Table lists all candidate snoRNAs identified in the T. kodakarensis genome. (XLSX 9 KB)Additional file 8: Table S5: snoRNAs in the T. kodakarensis genome. The Table lists all candidate sRNA identified in the T. kodakarensis genome. (XLSX 10 KB)Additional file 9: Table S6: Small non-coding RNAs in the DEseq derived expression data calculated as described in Methods. (XLSX 397 KB)Additional file 10: Table S7: Expression data for detected genes in all dRNA-seq libraries. The Table lists the average normalized read countings obtained by dRNA-seq and the T. kodakarensis. The Table lists all tRNAs, rRNAs and other well documented stable RNAs encoded in the T. kodakarensis genome and identifies their TSS. (XLSX 10 KB)Additional file 11: Table S8: tRNA, rRNA and other well documented non-coding RNAs in Ala-23S rRNA operon is cotranscribed with a snoRNA, designated Tko19. (B) The SRP RNA is transcribed from the stand opposite that designated in the genome annotation [exp; blue) and in stationary phase  in sulfur medium, growing exponentially in pyruvate medium before  and 20 min after sulfur addition  are given by the peak heights. Data from the control library (C) not digested with TEX are shown in grey. The relative abundance scales on the right of each panel allow direct comparisons of all data in that panel. The black scale bar in the top right corner of each panel is corresponds to 100 nt. (PDF 832 KB)Additional file 12: Figure S4: Transcription of the rRNA operon and SRP RNA. (A) As illustrated, the 16S-tRNAnotation . The abu2 database [exp and Sstat) and pyruvate (Pexp) libraries. (XLSX 8 KB)Additional file 13: Table S9: Read count percentage of transcriptional units. Genes were grouped into transcription units according to the DOORdatabase 41 and tdatabase , 41 withexp; blue) and in stationary phase  in sulfur medium, growing exponentially in pyruvate medium before  and 20 min after sulfur addition  are given by the peak heights. Data from the control library (C) not digested with TEX are shown in grey. The relative abundance scales on the right of each panel allow direct comparisons of all data in that panel. The black scale bar in the top right corner of each panel is corresponds to 100 nt. (PDF 309 KB)Additional file 14: Figure S5: Genome organization around TK2012 (encodes ferredoxin-3). As illustrated, a putative SurR binding site (red font) overlaps the BRE-TATA-box region of a promoter that directs transcription of an antisense RNA (black box) from the TK2012 region. The abundances of transcripts present in cells growing exponentially (SPositions weight matrix (PWM) used for the identification of RBS in the dRNA-seq data. Ribosome binding sites (RBS) were located in all annotated mRNAs by MEME[. A 10 nt window upstream of every start codon was extracted and scanned. The generated PWM was used as input for FIMO[Additional file 15: s by MEME. A 10 nt for FIMO. (ZIP 50


CDC123/CAMK1D type 2 diabetes association signal on chromosome 10 spans an intergenic region between CDC123 and CAMK1D and also overlaps the CDC123 3′UTR. To gain insight into the molecular mechanisms underlying the association signal, we used open chromatin, histone modifications and transcription factor ChIP-seq data sets from type 2 diabetes-relevant cell types to identify SNPs overlapping predicted regulatory regions. Two regions containing type 2 diabetes-associated variants were tested for enhancer activity using luciferase reporter assays. One SNP, rs11257655, displayed allelic differences in transcriptional enhancer activity in 832/13 and MIN6 insulinoma cells as well as in human HepG2 hepatocellular carcinoma cells. The rs11257655 risk allele T showed greater transcriptional activity than the non-risk allele C in all cell types tested. Using electromobility shift and supershift assays we demonstrated that the rs11257655 risk allele showed allele-specific binding to FOXA1 and FOXA2. We validated FOXA1 and FOXA2 enrichment at the rs11257655 risk allele using allele-specific ChIP in human islets. These results suggest that rs11257655 affects transcriptional activity through altered binding of a protein complex that includes FOXA1 and FOXA2, providing a potential molecular mechanism at this GWAS locus.Many of the type 2 diabetes loci identified through genome-wide association studies localize to non-protein-coding intronic and intergenic regions and likely contain variants that regulate gene transcription. The  CDC123/CAMK1D locus. We identify two T2D-associated variants that overlap predicted regulatory enhancer elements. We demonstrate that one variant, rs11257655, shows allele-specific transcriptional enhancer activity in mammalian cell lines relevant to type 2 diabetes. We also show differential protein-DNA binding suggesting that the rs11257655 type 2 diabetes- risk allele increased transcriptional activity through binding a protein complex that includes FOXA1 and FOXA2. This study demonstrates that genome-wide maps of regulatory elements are a useful resource to guide identification of variants differentially affecting transcriptional activity and provides insight into molecular mechanisms underlying a T2D susceptibility locus.GWAS have identified more than 1200 loci contributing to risk of disease, including more than 70 loci associated with type 2 diabetes. With a majority of associated variants localized to non-coding regions of the genome, focus has moved to identifying the functional variants explaining the association signals. One mechanism by which variants may act is to affect activity of enhancer elements regulating target gene expression. In this study, we take advantage of recent advances in genome-wide annotation of human regulatory elements to prioritize candidate functional variants at the  Type 2 diabetes is a complex metabolic disease with a substantial heritable component CDC123 (cell division cycle protein 123)/CAMK1D  locus on chromosome 10 contains common variants (MAF>.05) strongly associated with type 2 diabetes in Europeans  P = 1.5×10−8) P = 5.8×10−6) 2>.7) span an intergenic region of at least 45 kb between CDC123 and CAMK1D and overlap the 3′ end of CDC123CDC123/CAMK1D locus with reduced beta cell function, suggesting the beta cell as a candidate affected tissue 2 = 0.18 EUR, 1000G Phase 1) located 50 kb away from rs12779790 is associated with lung function The CDC123 is regulated by nutrient availability in yeast and is essential to the onset of mRNA translation and protein synthesis through assembly of the eukaryotic initiation factor 2 complex CDKAL1CAMK1D is a member of the Ca2+/calmodulin-dependent protein kinase family which transduces intracellular calcium signals to affect diverse cellular processes. Upon calcium influx in granulocyte cells and hippocampal neurons, CAMK1D activates CREB-dependent gene transcription CREB in beta cell survival, CAMK1D may have a role in beta cell insulin secretion. In cis-eQTL analyses, the rs11257655 type 2 diabetes risk allele was more strongly and directly associated with increased expression of CAMK1D than CDC123 in both blood and lung The transcript(s) targeted by risk variant activity at this locus remain unknown. CDC123/CAMK1D locus using genome-wide maps of open chromatin, chromatin state and transcription factor binding in pancreatic islets, hepatocytes, adipocytes and skeletal muscle myotubes. We measured transcriptional activity of variants in putative regulatory elements using luciferase reporter assays, and identified a candidate cis-acting SNP driving allele-specific enhancer activity in two mammalian beta cell-lines as well as hepatocellular carcinoma cells. We then evaluated DNA-protein binding in sequence surrounding this variant and identified allele-specific binding to key islet and hepatic transcription factors. Thus, our study provides strong evidence of a functional variant underlying the type 2 diabetes association signal at the CDC123/CAMK1D locus acting through altered regulation in type 2 diabetes-relevant cell types.In this study we aimed to identify the variant(s) underlying the association signal at the CDC123/CAMK1D locus, we considered variants in high LD  with GWAS index SNP rs12779790. To further prioritize variants for functional follow up, we used genome wide maps of chromatin state (2 = .74 with GWAS index SNP rs12779790), located 15 kb from the 3′ end of CDC123 and 84 kb from the 5′ end of CAMK1D, was a particularly plausible candidate overlapping islet, liver and HepG2 cell line DNase peaks, islet and liver FAIRE peaks, H3K4me1 and H3K9ac chromatin marks, and FOXA1 and FOXA2 ChIP-seq peaks in HepG2 cells (2 = .71 with rs12779790), overlapped a HepG2 DNase peak and displayed occupancy by FOXA1 and FOXA2 binding in HepG2 cells  showed differential allelic enhancer activity in both orientations in all three cell lines . Enhancer activity represents greater than a 1.4-fold  to 2.1-fold (832/13) increase in transcriptional activity relative to the non-risk allele in both the forward and reverse orientations. Compared to an empty vector control, enhancer activity was greatest in the islet cell lines .To evaluate transcriptional activity of the SNPs in predicted regulatory regions, 150–200 bp surrounding each SNP allele was cloned into a minimal promoter vector and luciferase activity was measured in two beta cell lines, 832/13 rat insulinoma and MIN6 mouse insulinoma cells, and in HepG2 liver hepatocellular carcinoma cells. Four to five independent clones for each allele were generated and enhancer activity was measured in duplicate for each clone. A 151-bp region including rs11257655  and no allele-specific activity in islet cells  or C (non-risk) allele were incubated with 832/13, MIN6 or HepG2 nuclear lysate and subjected to electrophoretic mobility shift assays (EMSA). Band shifts indicative of multiple DNA-protein complexes were observed for both rs11257655 alleles  and high (20 mM) concentrations of glucose for 16 hours, CAMK1D expression increased  in these tissues would be necessary to establish the mechanisms by which increased expression leads to diabetes risk.To determine whether atocytes . These dP = .22; . In 832/CDC123/CAMK1D locus. We prioritized candidate cis-regulatory variants and tested whether prioritized variants exhibited allele-specific transcriptional enhancer activity. We provide transcriptional reporter and protein-DNA binding evidence that rs11257655 is part of a cis-regulatory complex differentially affecting transcriptional activity. Additionally, we validate FOXA1 and FOXA2 as components of this regulatory complex in human islets.Integration of genome-wide regulatory annotation maps with disease-associated variants identified through GWAS has great potential for elucidation of gene-regulatory variants underlying association signals. In this study, we expand the lexicon of disease-associated functional regulatory variation by examining the type 2 diabetes-association signal at the CDC123/CAMK1D locus and evaluated the location of high LD variants  at the locus relative to known transcripts and to putative DNA regulatory elements. We identified two variants that overlapped putative islet and/or liver regulatory regions and none located in exons. We did not assess variants in lower LD (r2<.7), and additional functional SNPs may exist at this locus acting through alternate functional mechanisms untested in the current study.In recent years, progress has been made in following up mechanistic studies of GWAS type 2 diabetes-association signals CAMK1D or CDC123, may be associated with type 2 diabetes. Our subsequent analysis of protein binding revealed complexes that favored the rs11257655-T allele in 832/13, MIN6 and HepG2 cells. Consistent with predictions that the rs11257655-C allele may disrupt binding to the FOXA1 and FOXA2 transcription factors, we demonstrated that only the T allele of rs11257655 leads to FOXA1- and FOXA2-mediated supershifts. The ChIP enrichment of FOXA1 and FOXA2 in human islets from carriers of the T allele is concordant with EMSAs using nuclear extract from mouse and rat cell lines, further demonstrating the utility of rodent islet cell models to characterize human regulatory elements. Our results suggest that a cis-regulatory element surrounding rs11257655 may act in both islet and liver cells. Although we provide evidence that rs11257655 alleles differentially bind FOXA1 and FOXA2 in vivo, it is important to note that this enrichment was detected in isolated human islets. Future experiments will be needed to validate effects of rs11257655 within a whole organism environment. For example, recently zebrafish have been used to assay the regulatory potential of DNA sequences Based on our observation of type 2 diabetes-associated SNPs in regions of islet and liver open chromatin, we measured transcriptional activity in two mammalian islet cell models, rat 832/13 and mouse MIN6 insulinoma cells and in one hepatocyte cell model, human HepG2 hepatocellular carcinoma cells. In agreement with our previous observations FOXA1 and FOXA2 are members of the FOXA subclass of the forkhead box transcription factor family and are essential transcriptional activators in development of endodermally-derived tissues including liver and pancreas CAMK1D and CDC123 are candidate transcripts affected by variation at this locus. Cis-eQTLs in both blood and lung support an effect on CAMK1D but not CDC123. In blood, initial eQTL evidence for both genes were further analyzed by conditional analyses on the T2D lead SNP or rs11257655. The conditional analyses abolished the cis-eQTL signal for CAMK1D but not for CDC123, providing evidence that the T2D GWAS signal and the CAMK1D cis-eQTL signal are coincident CAMK1D with rs11257655 as a lead associated variant (P = 1.1×10−7); this and other T2D GWAS variants are the strongest cis-eQTLs for CAMK1D, while no significant eQTL is observed for CDC123CAMK1D transcript level, consistent with the direction of transcriptional activity we observed for this allele in islet and liver cells. Many eQTLs are predicted to be shared among tissues 2 = .74–.76) between beta cells and blood-derived lymphoblastoid cell lines, fat and skin CAMK1D eQTL may also exist in islets. Some eQTLs differ across tissues, and evidence of a consistent eQTL in islets would be valuable. Knockout mice provide further evidence supporting CAMK1D as a target gene. In FoxA1/FoxA2 beta cell-specific knockout mice, Camk1d expression was reported to be slightly reduced  CAMK1D is a more plausible target for differential regulation by rs11257655 alleles.Both CAMK1D may act in type 2 diabetes biology is unclear. CAMK1D is a serine threonine kinase that operates in the calcium-triggered CaMKK-CaMK1 signaling cascade CAMK1D activates CREB- (cAMP response element-binding protein) dependent gene transcription by phosphorylation CAMK1D also has been reported to regulate glucose in primary human hepatocytes CDC123 as a target for regulation by rs11257655.The mechanism by which CDC123/CAMK1D locus on chromosome 10. We identify rs11257655 as part of a cis regulatory complex in islet and liver cells that alters transcriptional activity through binding FOXA1 and FOXA2. These data demonstrate the utility of experimentally predicted chromatin state to identify regulatory variants for complex traits.In conclusion, we extend follow up studies of GWAS-identified type 2 diabetes-associated variants to the 2≥.7, EUR, with the GWAS index SNP rs12779790, 1000G Phase 1 release), two SNPs showed evidence of open chromatin Variants were prioritized for functional study based on linkage disequilibrium (LD) and evidence of being in an islet or liver regulatory element based on data from the ENCODE consortium 2. 832/13 cells were cultured in RPMI 1640 (Cellgro/Corning) supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES and 0.05 mM β-mercaptoethanol. MIN6 cells were cultured in DMEM (Sigma), supplemented with 10% FBS, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol. HepG2 hepatocellular carcinoma cells were cultured in MEM-alpha (Gibco) supplemented with 10% FBS, 1 mM sodium pyruvate and 2 mM L-glutamine.Two insulinoma cell lines, rat-derived 832/13 Fragments surrounding each of rs11257655 (151 bp) and rs34428576 (179 bp) were PCR-amplified  from DNA−5 cells per well were seeded in 24-well plates. At 80% confluency, cells were co-transfected with luciferase constructs and Renilla control reporter vector  at a ratio of 10∶1 using Lipofectamine 2000 (Invitrogen) for 832/13, and using FUGENE-6 for MIN6 and HepG2 cells . 48 h after transfection, cells were lysed with passive lysis buffer (Promega), and luciferase activity was measured using the Dual-luciferase assay system (Promega). To control for transfection efficiency, raw values for firefly luciferase activity were divided by raw Renilla luciferase activity values, and fold change was calculated as normalized luciferase values divided by pGL4.23 minimal promoter empty vector control values. Data are reported as the fold change in mean (± SD) relative luciferase activity per allele. A two-sided t-test was used to compare luciferase activity between alleles. All experiments were carried out on a second independent day and yielded comparable allele-specific results.Approximately 1×10C/T]TACTGGGCAT 3GGGCAAGTGTACACTTGCCC 3′ATGCCCAGTA[ . Double-stranded oligonucleotides for the risk and non risk alleles were generated by incubating 50 pmol complementary oligonucleotides at 95°C for 5 minutes followed by gradual cooling to room temperature. EMSA's were carried out using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific). Binding reactions were set up as follows: 1× binding buffer, 50 ng/µL poly (dI•dC), 3 µg nuclear extract, 200 fmol of labeled probe in a final volume of 20 µL. For competition reactions, 67-fold excess of unlabeled double-stranded oligonucleotides for either the risk or non-risk allele were included. Reactions were incubated at room temperature for 25 minutes. For supershift assays, 4 µg of polyclonal antibodies against FOXA1  or FOXA2  was added to the binding reaction and incubation proceeded for a further 25 minutes. Binding reactions were subjected to non-denaturing PAGE on DNA retardation gels in 0.5× TBE (Lonza), transferred to Biodyne nylon membranes (Thermo Scientific) and cross-linked on a UV-light cross linker (Stratagene). Biotin labeled DNA-protein complexes were detected by chemiluminescence. EMSAs were carried out on a second independent day and yielded comparable.Nuclear cell extracts were prepared from 832/13, MIN6, and HepG2 cells using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific) according to the manufacturer's instructions. Protein concentration was measured with a BCA protein assay (Thermo Scientific), and lysates were stored at −80°C until use. 21 bp oligonucleotides were designed to the sequence surrounding rs11257655 risk or non-risk alleles: Sense 5′ biotin- 20 to total 450 µL at room temperature for 30 minutes with rotation. 100 µL (1 mg) of streptavidin-agarose dynabeads were added and the reaction incubated for a further 20 minutes. Beads were washed and DNA-bound proteins were eluted in 1× reducing sample buffer (Invitrogen). Proteins were separated on NuPAGE denaturing gels and protein bands stained with SYPRO-Ruby. Protein bands displaying differential binding between rs11257655 alleles were excised from the gel and subjected to matrix assisted laser desorption time-of-flight/time-of-flight tandem mass spectrometry (MS) and analysis at the University of North Carolina proteomics core facility. For peptide identification, all MS/MS spectra were searched against all entries, NCBI non-redundant (NR) database, using GPS Explorer Software Version 3.6 (ABI) and the Mascot (MatrixScience) search algorithm. Mass tolerances of 80 ppm for precursor ions and 0.6 Da for fragment ions were used. In addition, two missed cleavages were allowed and oxidation of methionine was a variable modification.DNA affinity capture was carried out as previously described 3). To reverse crosslinks, 5 M NaCl was added to each sample to a final concentration of 0.2 M, and incubated overnight at 65°C; to remove protein, samples were incubated with 10 uL 0.5 M EDTA, 20 uL 1 M Tris (pH 6.5) and 3 uL of Proteinase K (10 mg/mL) at 45°C for 3 hours. DNA was extracted with 25∶24∶1 phenol:choloform:isoamyl alcohol, precipitated with 100% ethanol with 1 µl glycogen as a carrier, and resuspended in TE (Sigma). qPCR was performed in triplicate using SYBR Green Master Mix. Primers were designed to amplify a 99-bp region surrounding rs112576555; 5′-CTACTGCTTCTCCGGACTCG ′3′ and 5′- TGGCCTCAAGAGG GAGATAA -3′. Primers for a 133-bp control region not overlapping open chromatin and located 27 kb away were 5′-GCACCCATGGTACTGAAACC -3′ and 5′- CTTTTCCCG AGGAAGGAACT -3′. Dissociation curves demonstrated a single PCR product in each case without primer dimers. Fold enrichment was calculated as FOXA1/FOXA2 enrichment divided by IgG control. A one-sided t-test was performed to compare enrichment based on the direction of binding observed using EMSA.Human islets from non-diabetic organ donors were provided by the National Disease Research Interchange (NDRI). Use of human tissues was approved by the University of North Carolina Institutional Review Board. Islet viability and purity were assessed by the NDRI. Islets were warmed to 37°C and washed with calcium- and magnesium-free Dulbecco's phosphate-buffered saline (Life Technologies) prior to crosslinking. For chromatin immunoprecipitation (ChIP) studies, approximately 2000 islet equivalents (IEQs) were crosslinked for 10 min in 1% formaldehyde (Sigma-Aldrich) at room temperature. Islets were lysed and chromatin was sheared on ice using a standard bioruptor  to a size of 200–1000 bp. IP dilution buffer  was added, 5% of the volume was removed and used as input, and the remainder was incubated overnight at 4°C on a nutating platform with FOXA1 or FOXA2 antibody or a species-matched IgG as control. Antibodies used for ChIP were the same as for EMSA; FOXA1 (Abcam) and FOXA2 (Santa Cruz). Protein A agarose beads (Santa Cruz) were added and incubated for 3 h at 4°C. Beads were then washed for 5 minutes at 4°C with gentle mixing, using the following solutions: Low Salt Buffer ; High Salt Buffer ; LiCl buffer , twice; and TE buffer (Sigma-Aldrich), twice. Chromatin was eluted from beads with two 15-minute washes at 65°C using freshly prepared Elution Buffer  were used  mRNA levels of mouse and rat cells. All PCR reactions were performed in triplicate in a 10-µl volume using a STEPOne Plus real-time PCR system (Life Technologies). Serial 3-fold dilutions of cDNA from pooled human tissues, 832/13 or MIN6 cells as appropriate were used as a reference for a standard curve. Statistical significance was determined by two-tailed t-tests.Total cytosolic RNA was isolated using the RNeasy Mini Kit (Qiagen). RNA concentrations were determined using a Nanodrop 1000 . For real-time reverse transcription (RT)–PCR, first-strand cDNA was synthesized using 8 ul of total RNA in a 20 µl reverse transcriptase reaction mixture . cDNA was diluted to contain equivalent to 20–55 ng/µl input RNA. To measure total human mRNA levels of ere used . TaqMan Figure S1Regulatory potential at rs11257655 and rs36062557. UCSC genome browser (hg18) diagram showing that rs11257655 and rs36062557 overlap regions of open chromatin, detected by DNase hypersensitivity and FAIRE, and histone modifications, including H3K4me1 and H3K9ac in islet, liver, and HepG2 cells. H3K27ac and H3K4me3 histone modifications are also shown. rs11257655 and rs36062557 are also located near to HepG2 ChIP-seq peaks for FOXA1 and FOXA2. DNA sequences amplified to evaluate transcriptional activity in dual-luciferase reporter assays and to evaluate enrichment of binding to FOXA1 and FOXA2 are indicated.(TIF)Click here for additional data file.Figure S2P values were calculated by a two-sided t-test.Transcriptional activity at rs34428576. Enhancer activity was measured in 832/13 cells (A) and HepG2 cells (B) for alleles of rs34428576. No difference was observed between alleles in 832/13 cells. In HepG2 cells, moderate allele-specific activity was observed only in the reverse orientation. Error bars represent standard deviation of 4–5 independent clones for each allele. Results are expressed as fold change compared to empty vector control. (TIF)Click here for additional data file.Figure S3Chromosome 10 region not overlapping open chromatin does not show binding to FOXA1 and FOXA2 in human islets. A negative control region 28 kb downstream of rs11257655 was not substantially enriched in FOXA1- (A) and FOXA2- (B) bound chromatin. Error bars represent standard error of two to three islets for each represented genotype.(TIF)Click here for additional data file.Figure S4CDC123 and CAMK1D expression and response to glucose.  Evidence that CAMK1D and CDC123 are expressed in various human tissues. cDNA from human islets, hepatocytes, blood and adipocytes was analyzed by real-time PCR using gene-specific primers for CAMK1D (A) and CDC123 and B2M (B). mRNA level was normalized to B2M.  Effect of glucose stimulus on CAMK1D and CDC123 expression level. 832/13 and MIN6 insulinoma cells were treated with low (3 mM) and high (15 mM) glucose for 16–18 hours. cDNA was analyzed by real-time PCR using TaqMan gene expression assays for CAMK1D (C) and CDC123 . mRNA level was normalized to RSP9. High glucose treatment resulted in a significant increase in CAMK1D mRNA level (C) but not CDC123 in MIN6 cells (D). High glucose treatment resulted in increased CDC123 mRNA level in 832/13 cells. Error bars represent the standard deviation of 4–5 samples for each treatment. P values were calculated by a two-sided t-test.(TIF)Click here for additional data file.Table S1DNA sequences amplified for luciferase activity assays.(DOCX)Click here for additional data file.Table S2PCR primers for quantitative real-time PCR in human tissues.(DOCX)Click here for additional data file.


Epstein-Barr virus (EBV) establishes latent infections as multicopy episomes with complex patterns of viral gene transcription and chromatin structure. The EBV origin of plasmid replication (OriP) has been implicated as a critical control element for viral transcription, as well as viral DNA replication and episome maintenance. Here, we examine cellular factors that bind OriP and regulate histone modification, transcription regulation, and episome maintenance. We found that OriP is enriched for histone H3 lysine 4 (H3K4) methylation in multiple cell types and latency types. Host cell factor 1 (HCF1), a component of the mixed-lineage leukemia (MLL) histone methyltransferase complex, and transcription factor OCT2 (octamer-binding transcription factor 2) bound cooperatively with EBNA1 (Epstein-Barr virus nuclear antigen 1) at OriP. Depletion of OCT2 or HCF1 deregulated latency transcription and histone modifications at OriP, as well as the OriP-regulated latency type-dependent C promoter (Cp) and Q promoter (Qp). HCF1 depletion led to a loss of histone H3K4me3 (trimethylation of histone H3 at lysine 4) and H3 acetylation at Cp in type III latency and Qp in type I latency, as well as an increase in heterochromatic H3K9me3 at these sites. HCF1 depletion resulted in the loss of EBV episomes from Burkitt's lymphoma cells with type I latency and reactivation from lymphoblastoid cells (LCLs) with type III latency. These findings indicate that HCF1 and OCT2 function at OriP to regulate viral transcription, histone modifications, and episome maintenance. As HCF1 is best known for its function in herpes simplex virus 1 (HSV-1) immediate early gene transcription, our findings suggest that EBV latency transcription shares unexpected features with HSV gene regulation.IMPORTANCE EBV latency is associated with several human cancers. Viral latent cycle gene expression is regulated by the epigenetic control of the OriP enhancer region. Here, we show that cellular factors OCT2 and HCF1 bind OriP in association with EBNA1 to maintain elevated histone H3K4me3 and transcriptional enhancer function. HCF1 is known as a transcriptional coactivator of herpes simplex virus (HSV) immediate early (IE) transcription, suggesting that OriP enhancer shares aspects of HSV IE transcription control. Epstein-Barr virus (EBV) is a human gammaherpesvirus that has been etiologically linked to diverse malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, and AIDS-associated non-Hodgkin's lymphomas , 2. LikeThe different patterns of EBV latent cycle gene expression have been referred to as latency types , 5. DuriIn proliferating tumor cells, EBV latency requires the consistent expression of EBNA1. EBNA1 is a sequence-specific DNA-binding protein that binds to multiple repeat elements in the viral origin of plasmid replication (OriP) . EBNA1 b9–EBNA1 is required to activate transcription of EBNA2 during the establishment of latent infection and immortalization of naive B lymphocytes . EBNA1 i18–O-glycosyltransferase OGT, ubiquitin hydrolase RNF2 (BAP-1), and the phosphatase PPA1  bind to three nonamer elements that flank EBNA1 binding sites . These Tase PPA1 . How hos2 environment. Bacterial expression plasmids for OCT2 and EBNA amino acids (aa) 428 to 619 were constructed by cloning into the BamHI (5′) and SalI (3′) sites of PET 28(-) His vector. Small hairpin RNAs (shRNAs) for OCT2 (shOCT2), host cell factor 1 (shHCF1) and the control (shControl) were obtained from the Sigma/TRC (The RNAi Consortium) collection of targeted shRNA plasmid library . The shEBNA1 was made by cloning small hairpin RNA into the same lentiviral vector as in the Sigma/TRC shRNA library. Lentivirus particles were generated in 293T-derived packaging cell lines. The plasmids containing FLAG-tagged EBNA1 (f-EBNA1) have been described previously  and a MutuI virus-derived lymphoblastoid cell line (LCL) were grown in RPMI 1640 medium (Gibco BRL) containing 15% fetal bovine serum and antibiotics penicillin and streptomycin (50 U/ml). HEK 293T cells were culture in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and antibiotics. All the cells were cultured at 37°C and 5% COeviously . T7 HCF1eviously   was expressed and purified as a His-SUMO  fusion protein. After cleavage with SUMO protease, untagged protein was further purified by conventional chromatography. Proteins were estimated to be >90% pure after gel filtration chromatography. Electrophoresis mobility shift assay (EMSA) with EBNA1 has been described previously (3+) cryptate donor. His-tagged protein was labeled with an allophycocyanin acceptor (XL665) according to the manufacturers' instructions (Cisbio Bioassays). Binding conditions were essentially identical for those described for EBNA1 EMSA reactions. After incubation of protein and DNA for 1 h, the reactions were read for fluorescence resonance energy transfer (FRET) using an Envision detector according to the manufacturers' instructions (PerkinElmer). Similar binding conditions were also employed for the ALPHA screen assay using streptavidin donor beads and nickel chelate acceptor beads according to the manufacturers' instructions (PerkinElmer and reference EBNA1 DNA binding domain (DBD) (aa 459 to 607) was expressed and purified from eviously . The homeviously . Double-Chromatin immunoprecipitation (ChIP) assays were performed as described previously . Rabbit 6 cells that were then incubated for 48 h at 50°C in lysis buffer . The agarose plugs were washed twice in TE buffer (10 mM Tris [pH 7.5] and 1 mM EDTA). Pulsed-field gel electrophoresis (PFGE) was performed for 23 h at 14°C with an initial switch time of 60 s and a final switch time of 120 s at 6 V/cm and an included angle of 120° as described previously (Bio-Rad CHEF Mapper) .MutuI, Raji, SavI, and KemI cells and LCLs were infected with lentivirus as described above. After puromycin selection, cells were resuspended in 1× phosphate-buffered saline (PBS) and an equal amount of 2% agarose to form agarose plugs containing 1 × 10 Mapper) . DNA was Mapper) . The DNA2, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 20 mM sodium glycerophosphate, 5% glycerol, 1% Triton X-100, 0.5% sodium dodecyl sulfate, 1× protease inhibitors [Sigma], 1× phosphatase inhibitors [Sigma], and 1 mM phenylmethylsulfonyl fluoride [PMSF]). After rotation for 60 min at 4°C, the lysate was centrifuged for 20 min at 16,000 × g, and the supernatant was recovered. The cleared extracts were used for immunoprecipitation with antibodies as indicated in the figures.Cells were extracted with lysis buffer  detection of viral DNA was carried out as previously described  at 1,000 rpm for 5 min. Fluorescent escribed . Images Reverse transcription-quantitative PCR (RT-qPCR) assay of viral gene expression was performed as previously described .To investigate enhancer-like epigenetic marks of OriP, we analyzed by chromatin immunoprecipitation coupled to detection by quantitative PCR (ChIP-qPCR) the occupancy of several histone modifications in either type I (MutuI) or type III (LCL) latently infected cell lines . ChIP DNWe also examined the histone modification patterns more specifically at the transcription start sites for Cp and Qp, promoters known to be differentially regulated in type III and I latency . As expeTo identify potential factors that may contribute to OriP enhancer function, we assayed by ChIP-qPCR several candidate cellular factors that have been implicated in FR binding, like sequence-specific DNA-binding proteins EBNA1 and OCT2 or non-DNA-binding chromatin modulators of histone H3K4 methylation, like the MLL complex components HCF1, ASH2L, and RBBP5 . As expeE. coli (in vitro DNA binding assays. We set out to determine whether OCT2 could bind directly to either a single family of repeat (1×FR) or to a tandem pair of repeats (2×FR) and whether this binding was influenced by the presence of EBNA1  experiments . We wereTo identify subdomains of HCF1 and EBNA1 important for their interaction, we assayed deletion mutations of each protein in coIP assays . We assaTo assess the functional contribution of OCT2 and HCF1 to EBV latent cycle gene expression, we used lentivirus-mediated shRNA knockdown in LCLs and MutuI cells . We founTo investigate the mechanism of HCF1 and OCT2 in regulating the OriP enhancer, we assayed the effects of OCT2 and HCF1 depletion on histone modifications at the OriP locus . DepletiWe also examined the effects of HCF1 and OCT2 depletion on the Cp and Qp promoter regions more specifically . In MutuWe next asked whether OCT2 or HCF1 had any effect on viral episome maintenance. We assayed EBV episome structure using PFGE and Southern blotting . We firsin vitro and in vivo. We found that EBNA1 can also interact with HCF1 in an OriP-dependent manner. This interaction was dependent on the EBNA1 DNA binding domain and a region previously implicated in HAUSP7 interaction  immediately upstream of FR (EBV coordinates 7421 to 8042). This peak is particularly pronounced in type I latency where EBER transcription is expressed at higher levels. The expansion of the H3K4me3 through OriP and into the Cp region is commonly observed in type III latency where Cp is active, while a more restricted peak of H3K4me3 is observed in MutuI cells where Cp is inactive. In an effort to identify factors that maintain this high level of H3K4me3 at OriP, we assayed various candidate proteins from the histone H3K4 methylase complexes, including HCF1. Of these candidates, we found that HCF1 was enriched at OriP and that it could interact with EBNA1 in the context of OriP. We also confirmed the previous observation that OCT2 can bind to the FR element cooperatively with EBNA1 , 25. We HCF1 has essential functions in host cell cycle chromatin and gene regulation. HCF1 is posttranslationally processed by proteolysis into amino- and carboxy-terminal peptides that have distinct functions . HCF1 doEBNA1 interaction with HCF1 required the EBNA1 DBD along with amino acids 400 to 440. This region of EBNA1 is known to interact with HAUSP7 , and it Finally, a role for HCF1 and OCT2 in EBNA1-dependent transcription regulation raises important parallels with HSV-1 control of immediate early gene transcription. HSV-1 immediate early gene activation is critically dependent on VP16 and HCF1 functioning together, and cytoplasmic-nuclear shuttling of HCF1 regulates reactivation from latency in neurons , 48. HCF


During transcription, the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout the budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5′-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes. R-loops (RNA-DNA hybrids) are potentially deleterious for gene expression and genome stability, but can be beneficial, for example, during immunoglobulin gene class-switch recombination. Here we made use of antibody S9.6, with specificity for RNA-DNA duplexes independently of their sequence. The genome-wide distribution of R-loops in wild-type yeast showed association with the highly transcribed ribosomal DNA, and protein-coding genes, particularly the second exon of spliced genes. On RNA polymerase III loci such as the highly transcribed transfer RNA genes (tRNAs), R-loop accumulation was strongly detected in the absence of both ribonucleases H1 and H2 (RNase H1 and H2), indicating that R-loops are inherently formed but rapidly cleared by RNase H. Importantly, stable R-loops lead to reduced synthesis of tRNA precursors in mutants lacking RNase H and DNA topoisomerase activities. RNA-DNA hybrids associated with TY1 cDNA retrotransposition intermediates were elevated in the absence of RNase H, and this was accompanied by increased retrotransposition, in particular to 5′-flanking regions of tRNAs. Our findings show that RNase H participates in silencing of TY1 life cycle. Surprisingly, R-loops associated with mitochondrial transcription units were suppressed specifically by RNase H1. These findings have potentially important implications for understanding human diseases caused by mutations in RNase H. During transcription, the RNA polymerase opens the DNA duplex, and in the process rotates the DNA double helix by approximately one turn per 10 bp. This generates positive torsional stress ahead, and negative torsional stress in the wake, of the transcribing polymerase Other features besides negative topological stress strongly influence R-loop formation R-loops are generally regarded as highly deleterious, since the single stranded DNA is susceptible to damage. Moreover, it is believed that the structure can block both transcription and DNA replication, creating replicative stress and potentially causing further DNA damage (for reviews see Aicardi-Goutières syndrome (AGS) R-loops can be resolved by RNase H1 and/or RNase H2 , either of which can cleave the RNA component in the RNA-DNA hybrid, albeit with different efficiencies  on the yeast ribosomal DNA (rDNA), we observed that R-loops are common at specific sites, in particular within the 5′-region of the 18S rDNA mitochondrial RNA polymerase (mtRNAP). Notably, R-loops accumulated unevenly across intron-containing genes with the highest peak over exon 2. We show that R-loop accumulation at tRNA genes leads to reduced pre-tRNA synthesis specifically in mutants lacking both RNase H and Top1, or also Top2 activities. We also show that integration of TY1 retrotransposons in 5′-flanking regions of tDNAs is favored in cells depleted of both Top1 and cellular RNase H activities. We present evidence that both RNase H1 and RNase H2 are involved in cleaving RNA-DNA hybrids associated with cDNAs of TY1 retrotransposons. Unexpectedly, we also show that only RNase H1 is involved in processing of co-transcriptional R-loops at mtDNA transcription units.Here we determined the genome-wide distribution of RNA-DNA hybrids in budding yeast using chromatin immunoprecipitation (ChIP) with antibody S9.6 We previously reported that R-loops can be identified robustly by ChIP-QPCR analyses using the S9.6 antibody rnh1Δ rnh201Δ and triple GAL::TOP1 rnh1PΔ rnh201Δ (rnh1Δ rnh201Δ and GAL::TOP1 rnh1PΔ rnh201Δ (depleted of Top1), were greatly enriched in the S9.6 ChIP-seq data over the input chromatin . In each case, R-loop accumulation was further increased by depletion of Top1, although this increase was relatively small in the case of tRNA genes . In vitro treatment of S9.6 ChIP samples with recombinant RNase H strongly reduced R-loops associated with Pol III genes  and three tRNA genes  revealedII genes . This coThe conclusion that R-loop accumulation is highly increased over tRNA genes in strains lacking RNase H activities was supported by ChIP-Seq data . The expFor tRNA heatmaps in The substantial increase in R-loops on tRNA genes seen in strains lacking RNase H activity presumably reflects their high transcription rates, whereas low levels of R-loops in wild-type strains apparently shows that these are normally cleared rapidly by RNase H. However, the ChIP-QPCR data  revealedThe presence of R-loops is expected to impede transcription elongation, increasing the time required for pre-tRNA synthesis. In contrast, the accumulation of negative supercoiling behind the polymerase in strains with reduced topoisomerase activity can increase transcription initiation rates via promoter opening.GAL-TOP1P), or of both Top1 and Top2 . Pre-tRNA synthesis was slightly affected under conditions of partial induction of GAL-TOP1P, specifically at 0 h depletion in medium containing galactose plus sucrose (which provides the cells with limited amounts of glucose), in both the triple and quadruple mutant strains, relative to the strains with functional RNase H  flanking the TYA and TYB open reading frames , whereas TYB encodes the protease, the integrase and the reverse-transcriptase/RNase H (RT/RNase H). ChIP-QPCR analyses revealed only low levels of RNA-DNA hybrids over Ty1 retrotransposons in the wild-type strain, but notable accumulation was seen in the double mutant rnh1Δ rnh201Δ, and even more in the triple mutant GAL-TOP1 rnh1PΔ rnh201Δ following depletion of Top1 for 6 h   construct inserted into a chromosomal Ty1 element  his3(AI) gene does not produce functional HIS3 mRNA, however, an intact HIS3 gene can be regenerated by splicing, cDNA synthesis and retrotransposition. Ty1 mobility can therefore be quantified by measuring the rate of His+ prototroph formation. In strains carrying double rnh1Δ rnh201Δ or single top1Δ mutations, the rates of TY1his3AI transposition were ∼12.5 and ∼5 fold higher, respectively, than the isogenic wild-type . Integration of Ty1 at tRNAGLY was strongly suppressed in strains lacking only Dbr1 or both Dbr1 and RNase H, relative to the loss of only RNase H  sequences were enriched with RNA-DNA hybrids . Notably, the no antibody controls (−Ab bars) showed no enrichment above background in any strain tested. The recovery of R-loops that mapped to the mtDNA was substantially reduced by treatment of ChIP samples from the WT and double rnh1Δ rnh201Δ mutant strains with recombinant RNase HI in vitro  relative to no-antibody control (−Ab black bars)  the distribution of S9.6 ChIP-seq reads was distinctly different over exon and intron sequences  (RPL28 (At equences . The deni-genes) . This wa) (RPL28 . The maj) (RPL28 . The asy) (RPL28 . This pa) (RPL28 . The dis) (RPL28 . Moreove)  transcription units, specifically in strains lacking RNase H1. The ∼80 Kb S. cerevisiae mt chromosome comprises relatively long transcription units such as the genes COX1/Q0045 and COB/Q0105, which expression and polycystronic structure (with multiple exons and introns) are extremely complex We observed the accumulation of R-loops over the The yeast mt DNA encodes proteins and RNAs with key roles in mitochondrial function In yeast the mtDNA is not essential for viability on glucose-containing medium and many mutations cause instability of the mtDNA, leading to a complete loss (rho(0) petites) or truncations (rho(-) petites) of this genome S. cerevisiae is expected to be more similar to the fungus C. albicans, which is mediated mainly by recombination-driven replication RNA-DNA hybrids are extensively formed in the circular 16.5 Kb mt chromosome of mammalian cells where they are believed to play important roles during DNA replication As for RNA Pol I, Pol III and mtRNAP genes, the actively transcribed mRNA genes were also associated with R-loops, albeit at much lower rates. Thus there appear to be a general link between transcriptional activity and R-loop formation in budding yeast, as recently reported in Competition between RNA packaging and R-loop formation is a normal feature of mRNA synthesis. R-loop formation at mRNA genes could be dictated by factors including increased residence in proximity to the DNA template of poorly packaged transcripts and the G.C content of the sequence (reviewed in splice sites (SS). Notably, depletion of the splicing factor ASF/SF2  in mammalian cells can lead to increased R-loop formation and genome instability H.sapiens, predicted that R-loops will generally be less stable around the 5′ and 3′ SS, due to differences in the helical stability of (pre-mRNA)/DNA and DNA/DNA duplexes Most intron-containing genes in yeast, particularly ribosomal protein genes, have a short exon 1, and this correlates with higher levels of expression relative to genes with longer exon 1 see . The cloS.cerevisiae were by standard techniques. For Top1 and Top2 depletion, cells (GAL-TOP1P and GAL-TOP1/2P strains) were grown at 30°C to OD600 ∼0.3–0.4 in complete Kaiser synthetic SGS minimal medium  then transferred to the pre-warmed complete SD minimal medium (2% glucose). Growth was continued for several hours and maintained in exponential phase by dilution with pre-warmed SD medium.Yeast strains and plasmids used in this study are listed in 600 ∼0.6, 50 OD600/ChIP sample) with formaldehyde (1%) was for 25 min at room temperature. Pellets were resuspended with 400 µL of FA-1 lysis buffer , mixed with 500 µL of glass beads , and vortexed  for 45 min at full speed at 4°C. Glass beads were removed and cross-linked chromatin was recovered by centrifugation at full speed for 10 min at 4°C (supernatant discarded). Eight-hundred microliters of FA-1 buffer were added on the top of the pellet. Sonication of chromatin was performed for 2 min  to yield an average DNA fragment size of ∼500 bp. Sonicated chromatin were spun for 15 min at full speed at 4°C and glycerol 5% was added to supernatants. Sonicated chromatin were mixed with sepharose Cl-4B beads (Sigma CL4B200) and cleared for 1 h at 4°C. Twenty microliters were kept for control Input chromatin. Immunoprecipitations were performed by mixing ‘cleared-sonicated chromatin’ with 35–40 µg of IgG2a of antibody S9.6 together with 100 µl bed of Protein A sepharose CL-4B beads  on a rotating wheel overnight at 4°C. To assess contribution of background, a ‘beads-only’ internal control was prepared in parallel to immunoprecipitated samples but without addition of any antibody. Beads were recovered  and washed successively with FA-1 buffer (plus CPI-EDTA 1×), FA-2 buffer , FA-3 buffer , and TE 1×  at 4°C. Cross-link reversal of sonicated- chromatin from samples ‘input chromatin’, ‘beads-only’ and ‘S9.6 immunoprecipitate’ were performed by incubating the washed beads overnight at 65°C in 250 µL of TE buffer containing 1% SDS and 1 mg/mL proteinase K. DNA was purified using Qiagen PCR purification kit and eluted with 55 µL of buffer EB containing RNase A (0.5 µg/mL). 10–20 ng/µl DNA were recovered from ‘S9.6 immunoprecipitates’ from wild-type cells as measured by Qubit dsDNA HS Assay Kit . Quantitative PCRs (qPCRs) were performed in triplicate in a MX3005P real-time PCR machine (Agilent Technologies) in 10 µl reaction containing: 5 µl of 2× TaKara SYBR premix Ex Taq II Tli Rnase H Plus (Clontech RR820L), 1 µl DNA (but 10% of input chromatin), 0.4 µl of 10 µM primers (see −(Ct ‘beads only’ - Ct ‘input chromatin’) and ΔΔCt “S9.6 immunoprecipitate” = 2−(Ct ‘S9.6 immunoprecipitate’ - Ct ‘input chromatin’). The ‘S9.6 immunoprecipitate’ and ‘input chromatin’ were further processed for ChIP-seq as described below.Immunoglobulins IgG2a of monoclonal antibody S9.6 mers see , 0.04 µlhigh fidelity (HF) DNA polymerase , 1× HF buffer , 0.2 µM ‘primer 1.2’, 0.2 µM ‘primer 2.2’ and 0.2 mM dNTP mix (for primer sequences see 200 ng of DNA (‘S9.6 immunoprecipitate’ and ‘input chromatin’) were linker-ligated as described mainly in the Illumina's hand book with some modifications. Step1 “Repair DNA ends”: 100 µl reactions contained, DNA, 1× T4 DNA ligase buffer , 0.4 mM dNTP mix, 15 units T4 DNA polymerase , 50 units T4 Polynucleotide Kinase  and 5 units DNA Polymerase I- Large (Klenow) Fragment . Reactions were incubated at 20°C for 30 min. DNA was purified using QIAquick PCR Purification Kit . DNA columns were centrifuged several times up to 10 min to ensure that no residual traces of ethanol are left in the column. DNA was eluted with 32 µl EB buffer  which was pre-heated to 55°C. Step2 “Add A”: 50 µl reactions contained DNA from step 1, 1× NEB buffer 2 , 0.2 mM dATP and 15 units Klenow Fragment 3′→5′ exo minus . Reactions were incubated for 30 min at 37°C. DNA was purified using MinElute spin column  and eluted with 10 µl EB buffer as described in step 1. Step 3 “Ligation with standard pair-end (PE) adapters”: 30 µl reactions contained DNA from step 2, 1× Quick DNA ligase buffer , 3.33 nM PE adapter mix and 1600 units Quick T4 DNA ligase   were washed successively with FA-1 buffer (plus CPI-EDTA 1×), TE 1× buffer (plus CPI-EDTA 1×) and 10 mM Tris-HCl pH 8 . Washed beads were re-suspended in 300 µl of reaction buffer containing RNAse H buffer 1× , 4% glycerol and 20 µg/ml BSA. Beads were incubated for 2.5 h at 37°C in absence or presence of 15 µl of recombinant E. coli RNase HI , with shaking at 1000 rpm (Eppendorf Thermomixer). RNase H reactions were stopped by adding 10 mM EDTA. Beads were washed successively with buffers FA-2, FA-3, and TE 1×, sonicated-chromatin was reverse-crosslinked, and DNA was recovered and analysed by q-PCR as described for the standard ChIP protocol.One-hundred microliters of bed of Protein A beads incubated with sonicated-crosslinked-chromatin and antibody S9.6  were resolved on standard 8% polyacrylamide-8.3M urea gels for low molecular weight RNAs. Northern hybridizations for tRNAs were performed overnight at 37°C in ULTRAHyb-oligo buffer  and washes done at 37°C in SSC 6×. Northern signals were generated by a Fuji FLA-5100 PhosphorImager and quantified with AIDA software (Raytest). For primer sequences see TY1his3AI-[Δ1]-3114) HIS3 gene was inserted in the TYB sequence of a Ty1 element in the antisense orientation  re-diluted to OD600 ∼0.01 in the same medium and aliquoted in 5 cultures of 10 ml each, and, 4) grown at 18°C until saturation (4–6 d). Cells were harvested, washed and re-suspended in 5 ml sterile water. For total number of colonies, aliquots of each culture (dilution 1∶106) were plated on SD Drop-Out -Leu minimal medium and incubated at 30°C. For HIS+ colonies, aliquots of each culture (dilution 1∶2) were plated on SD Drop-Out -Leu-His plates and incubated at 30°C. The rate of Ty1his3AI transposition is the number of HIS+ colonies divided by the total number of colonies . A measure of enrichment for each window was given as the ratio of the windows mean depth vs the intergenic mean (i.e. 50 bp window score = “average read depth in surrounding 100 bp”/“intergenic mean depth”). This method of normalization was mainly adapted from 50 bp reads were sequenced by an Illumina MiSeq Benchtop Sequencer, quality trimmed and filtered with Trimmomatic and aligned to the The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus Figure S1WT) and double mutant rnh1Δ rnh201Δ, and in triple mutant GAL-TOP1 rnh1PΔ rnh201Δ depleted of Top1 for 6 h at 30°C. Also shown is the input chromatin profile of the wild-type strain. Raw read counts were not corrected by the number of rDNA repeats. G+C content of the DNA sequence was calculated for 100 bp windows and is depicted as a blue intensity. Shown below the profiles is a diagram of one rDNA repeat which comprises the 35S rDNA gene, transcribed by Pol I in to the 35S pre-rRNA which is processed to 18S, 5.8S, and 25S rRNAs. 35S genes are flanked by the intergenic spacers IGS1 and IGS2 and the 5S rDNA gene, transcribed by Pol III. The direction of transcription is indicated by a tailed arrow. Chr = chromosome. Prominent R-loop peaks discussed in the text are highlighted by stars. ETS = external transcribed spacer. ITS = internal transcribed spacer. Profiles were generated using Integrative Genomics ViewerR-loops over the rDNA repeats are detected by S9.6 ChIP-seq. A: Analysis of R-loops by ChIP-seq using antibody S9.6 in the wild-type strain BY4741 ((TIF)Click here for additional data file.Figure S2WT (BY4741) and double mutant rnh1Δ rnh201Δ, and in triple mutant GAL-TOP1 rnh1PΔ rnh201Δ depleted of Top1 for 6 h at 30°C. Median profiles of control input chromatin from WT are also shown. The y-axis represents the relative enrichment of reads where values >1 are above the background level of sequencing Click here for additional data file.Figure S3TY1B hybridization probe (filled black rectangle) and relevant PvuII cleavage sites. Probe TY1B detects an ∼2 Kb PvuII DNA fragment of unintegrated Ty1 cDNA and variably sized PvuII DNA fragments >2 Kb containing the junction of Ty1 elements with chromosomal DNA at different locations in the genome. B: Southern analyses of Ty1 cDNA from strain WT (BY4741) and double mutant rnh1Δ rnh201Δ, grown at 22°C in YEPD medium (glucose 2%) until saturation, in the absence or presence of 600 µg/ml of phosphonoformic acid (PFA), which is an inhibitor of TY1 RT WT (BY4741) and mutant strains double rnh1Δ rnh201Δ, single GAL-TOP1P, triple GAL-TOP1 rnh1PΔ rnh201Δ, single dbr1Δ, triple rnh1Δ rnh201Δ dbr1Δ, double GAL-TOP1 dbr1ΔP, and quadruple GAL-TOP1 rnh1Δ rnh201Δ dbr1ΔP. Cultures were grown at 30°C in medium containing galactose and sucrose  and shifted for 6 h to medium containing glucose . TY1 cDNA band is indicated by a tailed arrow. Indicated to the left of the gel the migration position of a 2 Kb band from a DNA size ladder.TY1 cDNAs accumulate in mutants lacking cellular RNase H or also depleted of Top1. A: Diagrams of unintegrated Ty1 cDNA and a genomic Ty1 element, indicating the location of the (TIF)Click here for additional data file.Figure S4rnh1Δ rnh201Δ was grown in YEPD medium (glucose 2%) at 22°C in the absence or presence of 600 µg/ml of phosphonoformic acid (PFA), which is an inhibitor of TY1 reverse transcriptase (RT) COX1 and 21S rDNA), Pol III gene tRNA SUF2, mRNA gene RPL28 and CEN16 were analyzed by Q-PCR as described in Reduced accumulation of RNA/DNA hybrids at Ty1 in the absence of RT activity. Double mutant (TIF)Click here for additional data file.Figure S5WT (BY4741) and mutant strains double rnh1Δ rnh201Δ, triple GAL-TOP1 rnh1PΔ rnh201Δ and quadruple GAL-TOP1 rnh1Δ rnh201Δ dbr1ΔP. Yeast cultures grown at 30°C were shifted from galactose- and sucrose- containing-medium to glucose medium and harvested at 6 h depletion of Top1. Panel I. Blot probed with antibody anti-Ty1 Gag. The Gag-p49 and processed-Gag-p45 bands each appear as doublets. Panel II. Blot probed with antibody anti-beta-actin.Gag proteins are slightly increased in mutants lacking both Top1 and cellular RNase H. Immunoblots of cellular homogenates from strain (TIF)Click here for additional data file.Figure S6GLYtRNA in mutants lacking RNase H and/or Dbr1. Four independent isolates for each strain, WT (BY4741) and mutant strains single dbr1Δ, double rnh1Δ rnh201Δ, triple rnh1Δ rnh201Δ dbr1Δ, triple GAL-TOP1 rnh1PΔ rnh201Δ, and quadruple GAL-TOP1 rnh1Δ rnh201Δ dbr1ΔP, were grown until saturation at 18°C in medium containing both galactose and sucrose (permissive for GAL-TOP1P expression). Panel I. See legend in Panel II. Representative examples of SYBR-stained gels are shown, revealing integration of Ty1 cDNA upstream of the 16 GLYtRNA gene loci. Shown to the right of the gels DNA ladders with lengths in base-pairs (bp).PCR analyses of integration of Ty1 at (TIF)Click here for additional data file.Figure S7Model: Pol III-associated R-loops facilitate targeting of TY1 at 5′ flanking regions of tRNA genes. Ty1 integration upstream of tRNA genes is specifically targeted to the H2A/H2B interface of nucleosomal DNA in a ∼1 kb window (TIF)Click here for additional data file.Figure S8WT (BY4741) double mutant rnh1Δ rnh201Δ and from mutants triple GAL-TOP1 rnh1PΔ rnh201Δ and quadruple GAL-TOP1 rnh1Δ rnh201Δ dbr1ΔP depleted of Top1 for 6 h at 30°C. CEN16 and four different regions of COX1 gene were analysed by Q-PCR as described in ChIP-QPCR of R-loops at mtDNA in mutants lacking both Top1 and cellular RNase H. ChIP samples using antibody S9.6 (same as in (TIF)Click here for additional data file.Figure S9Formaldehyde-crosslinked R-loops associated with mRNA genes are slightly cleaved in vitro by recombinant RNase HI. ChIP samples are from strains WT (BY4741) and double mutant rnh1Δ rnh201Δ , and the telomeric region Tel01L, were analyzed by Q-PCR as described in (TIF)Click here for additional data file.Figure S10I. Raw transcriptome sequencing (RNA-seq) reads of exponentially growing wild-type strain BY4741 Ribosomal protein genes (RPG) and non-ribosomal protein genes (NRPG) are represented by a green or black vertical line, respectively. The plot region featuring the 500 highly expressed mRNA genes of group C4 including all the RPGs is magnified in panel II. n = number of genes. B: Plot of yeast mRNA intron-genes (i-genes) grouped according to the length of their first exon (exon 1) (panel I). The region of the plot featuring the i-genes with Exon 1 <300 bp is magnified in panel II: 87 RPGs (out of 89 i-genes) and 144 NRPGs (out of 181 i-genes) have an Exon 1 <300 bp. C: Boxplots of RNA-seq and Net-seq data from wild-type strain BY4741 YDR535C, YLR202C and YOR318C were excluded from our analysis in Expression of spliced genes with a “short-exon1” is generally higher than those with a “long-exon1.” A: Plot of RNA expression of yeast mRNA genes. Panel (TIF)Click here for additional data file.Figure S11ribosomal protein genes (RPG) (panel A) and the non-ribosomal-protein genes (NRPG) (panel B), and further segregated in to four sub-categories according to the length of their first exon (Exon 1): (0–50 bp), (50–100 bp), (100–150 bp) and (>300 bp). The y-axis represents the relative enrichment of reads where values>1 are above the background level of sequencing  in the wild-type strain (BY4741), grown at 30°C in YEPD medium (glucose 2%). Averaged reads were plotted on sequences encompassing Exon1-intron-Exon2 regions as described in (TIF)Click here for additional data file.Figure S12RPL28 (A), ACT1 (B) and EFB1 (E) and intronless genes SEH1 (A), FET5 and YPT1 (B), DUF1, MHF1 and ADH1 (C), LEU1 and PMA1 (D), SSA1 and VPS8 (E), and IBA57, RPS5 and ENT3 (F). The y-axis represents the relative enrichment of reads where values >1 are above the background level of sequencing  grown at 30°C in YEPD medium (glucose 2%). Shown for spliced genes (TIF)Click here for additional data file.Figure S13Comparison of R-loop distribution across Exon 2 of spliced-genes and across non-spliced ribosomal protein genes. Box plots of mean sequence read distribution of R-loops per gene across the second exon of the i-genes NRPG (I) and RPG (II), and the entire length of the e-genes RPG (III). Each box plot represents the log2 fold change of mean S9.6 ChIP-seq relative to input chromatin in the wild-type (BY4741), grown at 30°C in YEPD medium (glucose 2%). Box-plot representation shows median values (black line) +/−25% quartiles in the box and minimum/maximum distribution of the values (excluding outliers) in the whiskers Click here for additional data file.Figure S14non-ribosomal-protein genes (NRPG) (A) and the ribosomal protein genes (RPG) (B). The thermodynamic helical stability of polynucleotide sequences /DNA duplexes) and the concentrations of G.C nucleotides were calculated for non-overlapping windows of 9 bases as described in 9 and [G+C] nucleotides for the i-genes in each group were plotted on sequences encompassing Exon1-intron-Exon2 regions as described in Thermodynamic helical stability of DNA/DNA and (pre-mRNA)/DNA duplexes of yeast mRNA intron-genes. The mRNA intron-genes (i-genes) were split in to the (TIF)Click here for additional data file.Figure S15rnh1Δ mutants than in isogenic wild-type W303a. Strains wild-type W303a and mutant rnh1Δ were grown at 30°C in YEP medium containing 3% glycerol. After four days cells were washed with sterile water, diluted appropriately, plated onto YEP medium containing 2% glucose and incubated at 30°C. One feature of ‘petite’ −ade2 cells is to have a small size and the inability to convert an intermediate in the adenine biosynthesis pathway (AIR) into a red pigment due to impaired respiratory functions Click here for additional data file.Figure S16ΔG9 values of DNA/DNA and (pre-mRNA)/DNA duplexes and [G+C] nucleotides see ΔG9 and [G+C] for the e-genes in each group were plotted on sequences encompassing the entire length of the gene (0–100%). n = number of genes.Thermodynamic helical stability of DNA/DNA and (pre-mRNA)/DNA duplexes of yeast mRNA intronless genes. The mRNA intronless genes (e-genes) were split in to four groups according to their mRNA expression: panel A , panel B , panel C  and panel D . For (TIF)Click here for additional data file.Table S1List of strains and plasmids. A: Strains. B: Plasmids.(DOC)Click here for additional data file.Table S2Oligonucleotides used in Northern and Southern analysis. A: Oligonucleotides for Northern analysis of tRNAs and rRNAs. B: Oligonucleotides for Southern analysis of Ty1 cDNAs.(DOC)Click here for additional data file.Table S3Oligonucleotides used in ChIP-QPCR, PCR and ChIP-seq analysis. A: ChIP-QPCR primers. B: Standard PCR primers. C: ChIP-seq primers.(DOC)Click here for additional data file.Protocol S1Bioinformatic analysis.(DOC)Click here for additional data file.