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1
O’Sullivan, Audrey C., Gareth J. Sullivan, and Brian McStay. "UBF Binding In Vivo Is Not Restricted to Regulatory Sequences within the Vertebrate Ribosomal DNA Repeat." Molecular and Cellular Biology 22, no. 2 (January 15, 2002): 657–58. http://dx.doi.org/10.1128/mcb.22.2.657-668.2002.
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ABSTRACT The HMG box containing protein UBF binds to the promoter of vertebrate ribosomal repeats and is required for their transcription by RNA polymerase I in vitro. UBF can also bind in vitro to a variety of sequences found across the intergenic spacer in Xenopus and mammalian ribosomal DNA (rDNA) repeats. The high abundance of UBF, its colocalization with rDNA in vivo, and its DNA binding characteristics, suggest that it plays a more generalized structural role over the rDNA repeat. Until now this view has not been supported by any in vivo data. Here, we utilize chromatin immunoprecipitation from a highly enriched nucleolar chromatin fraction to show for the first time that UBF binding in vivo is not restricted to known regulatory sequences but extends across the entire intergenic spacer and transcribed region of Xenopus, human, and mouse rDNA repeats. These results are consistent with a structural role for UBF at active nucleolar organizer regions in addition to its recognized role in stable transcription complex formation at the promoter.
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Alvares, Lúcia E., Carlos Polanco, Olivier Brison, Luiz L. Coutinho, and Itamar R. G. Ruiz. "Molecular evolution of ribosomal intergenic spacers in Odontophrynus americanus 2n and 4n (Amphibia: Anura)." Genome 45, no. 1 (February 1, 2002): 71–81. http://dx.doi.org/10.1139/g01-134.
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Ribosomal intergenic spacers (IGSs) of Odontophrynus americanus 2n and 4n were cloned, restriction mapped, and partially sequenced. Three distinct regions, namely α, β, and δ, were identified in the IGSs. The α and β regions flanked the 28S and 18S rRNA genes, respectively, conserving an identical restriction pattern at each ploidy level. The δ region, located between α and β, was highly variable in size and restriction pattern, enclosing different BamHI subrepeats (B-SR), 87- to 530-bp-long. Sequence analysis showed that B-SRs were composed mainly of different arrangements of similar blocks of sequences. Another family of repetitive sequences was found in the δ region, clustered inside large BamHI fragments. These subrepeats are 189-bp-long and, although very similar in diploid and tetraploid IGSs, show a pattern of concerted evolution. A hypothetical functional role for the 189-bp repeats is discussed in view of their predicted secondary structure and presence of potential E2 binding sites inside diploid subrepeats. Although the same structural elements were present both in diploid and tetraploid IGSs, the higher level of repeatability of tetraploid IGSs suggests that common ancestor sequences have undergone several rounds of amplification after O. americanus polyploidy.Key words: Odontophrynus americanus, amphibian polyploidy, ribosomal DNA, intergenic spacer, IGS subrepeats.
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Pikaard, C. S., L. K. Pape, S. L. Henderson, K. Ryan, M. H. Paalman, M. A. Lopata, R. H. Reeder, and B. Sollner-Webb. "Enhancers for RNA polymerase I in mouse ribosomal DNA." Molecular and Cellular Biology 10, no. 9 (September 1990): 4816–25. http://dx.doi.org/10.1128/mcb.10.9.4816.
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The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is widespread in nature and has important functional consequences.
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Pikaard, C. S., L. K. Pape, S. L. Henderson, K. Ryan, M. H. Paalman, M. A. Lopata, R. H. Reeder, and B. Sollner-Webb. "Enhancers for RNA polymerase I in mouse ribosomal DNA." Molecular and Cellular Biology 10, no. 9 (September 1990): 4816–25. http://dx.doi.org/10.1128/mcb.10.9.4816-4825.1990.
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The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is widespread in nature and has important functional consequences.
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McKee, B. D., L. Habera, and J. A. Vrana. "Evidence that intergenic spacer repeats of Drosophila melanogaster rRNA genes function as X-Y pairing sites in male meiosis, and a general model for achiasmatic pairing." Genetics 132, no. 2 (October 1, 1992): 529–44. http://dx.doi.org/10.1093/genetics/132.2.529.
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Abstract In Drosophila melanogaster males, X-Y meiotic chromosome pairing is mediated by the nucleolus organizers (NOs) which are located in the X heterochromatin (Xh) and near the Y centromere. Deficiencies for Xh disrupt X-Y meiotic pairing and cause high frequencies of X-Y nondisjunction. Insertion of cloned rRNA genes on an Xh- chromosome partially restores normal X-Y pairing and disjunction. To map the sequences within an inserted, X-linked rRNA gene responsible for stimulating X-Y pairing, partial deletions were generated by P element-mediated destabilization of the insert. Complete deletions of the rRNA transcription unit did not interfere with the ability to stimulate X-Y pairing as long as most of the intergenic spacer (IGS) remained. Within groups of deletions that lacked the entire transcription unit and differed only in length of residual IGS material, pairing ability was proportional to the dose of 240-bp intergenic spacer repeats. Deletions of the complete rRNA transcription unit or the 28S sequences alone blocked nucleolus formation, as determined by binding of an antinucleolar antibody, yet did not interfere with pairing ability, suggesting that X-Y pairing may not be mechanistically related to nucleolus formation. A model for achiasmatic pairing in Drosophila males based upon the combined action of topoisomerase I and a strand transferase is proposed.
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Robinett, C. C., A. O'Connor, and M. Dunaway. "The repeat organizer, a specialized insulator element within the intergenic spacer of the Xenopus rRNA genes." Molecular and Cellular Biology 17, no. 5 (May 1997): 2866–75. http://dx.doi.org/10.1128/mcb.17.5.2866.
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We have identified a novel activity for the region of the intergenic spacer of the Xenopus laevis rRNA genes that contains the 35- and 100-bp repeats. We devised a new assay for this region by constructing DNA plasmids containing a tandem repeat of rRNA reporter genes that were separated by the 35- and 100-bp repeat region and a rRNA gene enhancer. When the 35- and 100-bp repeat region is present in its normal position and orientation at the 3' end of the rRNA reporter genes, the enhancer activates the adjacent downstream promoter but not the upstream rRNA promoter on the same plasmid. Because this element can restrict the range of an enhancer's activity in the context of tandem genes, we have named it the repeat organizer (RO). The ability to restrict enhancer action is a feature of insulator elements, but unlike previously described insulator elements the RO does not block enhancer action in a simple enhancer-blocking assay. Instead, the activity of the RO requires that it be in its normal position and orientation with respect to the other sequence elements of the rRNA genes. The enhancer-binding transcription factor xUBF also binds to the repetitive sequences of the RO in vitro, but these sequences do not activate transcription in vivo. We propose that the RO is a specialized insulator element that organizes the tandem array of rRNA genes into single-gene expression units by promoting activation of a promoter by its proximal enhancers.
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Davidsen, Jeanne M., and Craig A. Townsend. "Identification and Characterization of NocR as a Positive Transcriptional Regulator of the β-Lactam Nocardicin A in Nocardia uniformis." Journal of Bacteriology 191, no. 3 (November 21, 2008): 1066–77. http://dx.doi.org/10.1128/jb.01833-07.
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ABSTRACT Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis, which shows moderate activity against a broad spectrum of gram-negative bacteria. Within the biosynthetic gene cluster of nocardicin A, nocR encodes a 583-amino-acid protein with high similarity to a class of transcriptional regulators known as streptomyces antibiotic regulatory proteins. Insertional inactivation of this gene resulted in a mutant showing morphology and growth characteristics similar to the wild type, but one that did not produce detectable levels of nocardicin A or the early precursor p-hydroxybenzoyl formate. Similar disruptions of nocD, nocE, and nocO yielded mutants that maintained production of nocardicin A at levels similar to the wild-type strain. In trans complementation of the nocR::apr mutant partially restored the wild-type phenotype. Transcriptional analysis of the nocR::apr mutant using reverse transcription-PCR found an absence of mRNA transcripts for the early-stage nocardicin A biosynthetic genes. In addition, transcription of the genes responsible for the biosynthesis of the nonproteinogenic p-hydroxyphenylglycine (pHPG) precursor was attenuated on the nocR disruption mutant. NocR was heterologously expressed and purified from Escherichia coli as an N-terminal maltose binding protein-tagged fusion protein. DNA binding assays demonstrated that NocR is a DNA binding protein, targeting the 126-bp intergenic region between nocF and nocA. Within this intergenic region is the likely binding motif, a direct hexameric repeat, TGATAA, with a 5-bp spacer. These experiments establish NocR as a positive transcriptional regulator of the nocardicin A biosynthetic pathway, coordinating the initial steps of nocardicin A biosynthesis to the production of its pHPG precursor.
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Putnam, C. D., and C. S. Pikaard. "Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers." Molecular and Cellular Biology 12, no. 11 (November 1992): 4970–80. http://dx.doi.org/10.1128/mcb.12.11.4970.
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Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.
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Putnam, C. D., and C. S. Pikaard. "Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers." Molecular and Cellular Biology 12, no. 11 (November 1992): 4970–80. http://dx.doi.org/10.1128/mcb.12.11.4970-4980.1992.
Full textAbstract:
Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.
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Milligan, Laura, Laurence Decourty, Cosmin Saveanu, Juri Rappsilber, Hugo Ceulemans, Alain Jacquier, and David Tollervey. "A Yeast Exosome Cofactor, Mpp6, Functions in RNA Surveillance and in the Degradation of Noncoding RNA Transcripts." Molecular and Cellular Biology 28, no. 17 (June 30, 2008): 5446–57. http://dx.doi.org/10.1128/mcb.00463-08.
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ABSTRACT A genome-wide screen for synthetic lethal (SL) interactions with loss of the nuclear exosome cofactors Rrp47/Lrp1 or Air1 identified 3′→5′ exonucleases, the THO complex required for mRNP assembly, and Ynr024w (Mpp6). SL interactions with mpp6Δ were confirmed for rrp47Δ and nuclear exosome component Rrp6. The results of bioinformatic analyses revealed homology between Mpp6 and a human exosome cofactor, underlining the high conservation of the RNA surveillance system. Mpp6 is an RNA binding protein that physically associates with the exosome and was localized throughout the nucleus. The results of functional analyses demonstrated roles for Mpp6 in the surveillance of both pre-rRNA and pre-mRNAs and in the degradation of “cryptic” noncoding RNAs (ncRNAs) derived from intergenic regions and the ribosomal DNA spacer heterochromatin. Strikingly, these ncRNAs are also targeted by other exosome cofactors, including Rrp47, the TRAMP complex (which includes Air1), and the Nrd1/Nab3 complex, and are degraded by both Rrp6 and the core exosome. Heterochromatic transcripts and other ncRNAs are characterized by very rapid degradation, and we predict that functional redundancy is an important feature of ncRNA metabolism.
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Ridley, Kristian A., Jonathan D. Rock, Ying Li, and Julian M. Ketley. "Heme Utilization in Campylobacter jejuni." Journal of Bacteriology 188, no. 22 (September 15, 2006): 7862–75. http://dx.doi.org/10.1128/jb.00994-06.
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ABSTRACT A putative iron- and Fur-regulated hemin uptake gene cluster, composed of the transport genes chuABCD and a putative heme oxygenase gene (Cj1613c), has been identified in Campylobacter jejuni NCTC 11168. Mutation of chuA or Cj1613c leads to an inability to grow in the presence of hemin or hemoglobin as a sole source of iron. Mutation of chuB, -C, or -D only partially attenuates growth where hemin is the sole iron source, suggesting that an additional inner membrane (IM) ABC (ATP-binding cassette) transport system(s) for heme is present in C. jejuni. Genotyping experiments revealed that Cj1613c is highly conserved in 32 clinical isolates. One strain did not possess chuC, though it was still capable of using hemin/hemoglobin as a sole iron source, supporting the hypothesis that additional IM transport genes are present. In two other strains, sequence variations within the gene cluster were apparent and may account for an observed negative heme utilization phenotype. Analysis of promoter activity within the Cj1613c-chuA intergenic spacer region revealed chuABCD and Cj1613c are expressed from separate iron-repressed promoters and that this region also specifically binds purified recombinant FurCj in gel retardation studies. Absorbance spectroscopy of purified recombinant His6-Cj1613c revealed a 1:1 heme:His6-Cj1613c binding ratio. The complex was oxidatively degraded in the presence of ascorbic acid as the electron donor, indicating that the Cj1613c gene product functions as a heme oxygenase. In conclusion, we confirm the involvement of Cj1613c and ChuABCD in heme/hemoglobin utilization in C. jejuni.
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Havemann, Stephanie A., and Jamie S. Foster. "Comparative Characterization of the Microbial Diversities of an Artificial Microbialite Model and a Natural Stromatolite." Applied and Environmental Microbiology 74, no. 23 (October 3, 2008): 7410–21. http://dx.doi.org/10.1128/aem.01710-08.
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ABSTRACT Microbialites are organosedimentary structures that result from the trapping, binding, and lithification of sediments by microbial mat communities. In this study we developed a model artificial microbialite system derived from natural stromatolites, a type of microbialite, collected from Exuma Sound, Bahamas. We demonstrated that the morphology of the artificial microbialite was consistent with that of the natural system in that there was a multilayer community with a pronounced biofilm on the surface, a concentrated layer of filamentous cyanobacteria in the top 5 mm, and a lithified layer of fused oolitic sand grains in the subsurface. The fused grain layer was comprised predominantly of the calcium carbonate polymorph aragonite, which corresponded to the composition of the Bahamian stromatolites. The microbial diversity of the artificial microbialites and that of natural stromatolites were also compared using automated ribosomal intergenic spacer analysis (ARISA) and 16S rRNA gene sequencing. The ARISA profiling indicated that the Shannon indices of the two communities were comparable and that the overall diversity was not significantly lower in the artificial microbialite model. Bacterial clone libraries generated from each of the three artificial microbialite layers and natural stromatolites indicated that the cyanobacterial and crust layers most closely resembled the ecotypes detected in the natural stromatolites and were dominated by Proteobacteria and Cyanobacteria. We propose that such model artificial microbialites can serve as experimental analogues for natural stromatolites.
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Zhang, Yanmei, Xuelin Shen, Xiaoqin Sun, Jia Liu, Yifeng Xia, Xin Zou, and Yueyu Hang. "Molecular Distinguishment of Trapa natans L. Varieties in Taihu Lake Region of China and Development of a RAPD-SCAR Marker for Authentication of ‘Heshangling’." HortScience 54, no. 8 (August 2019): 1319–23. http://dx.doi.org/10.21273/hortsci14006-19.
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Water chestnut (Trapa natans L.) is a group of annual, floating-leaved aquatic plants that serves as food and medical resources in many countries. However, the molecular method for distinguishing different T. natans L. resources is lacking. In this study, we detected genetic diversity of several chloroplast and nuclear genic or intergenic sequences in four varieties of T. natans and one wild type of Trapa incisa Siebold & Zuccarini to evaluate their potential as molecular markers. Our data revealed that the three chloroplast fragments (rbcL, matK, and pbsA-trnH) show no sequence difference among all tested samples. Only one nucleotide substitution is detected for the nuclear ribosomal internal transcribed spacer (ITS) in the T. natans variety Shuihongling. Four nucleotide substitutions are detected for the nuclear carotenoid isomerase (CRTISO) gene in the variety Hongxiuxie. In contrast, a total of 29 polymorphic sites are detected for a Toll and interleukin-1 receptor-nucleotide binding site–leucine rich repeat (TNL) gene in the five samples, among which six are nucleotide substitutions and the rest are insertions/deletions. The five samples could be fully distinguished from each other based on the TNL gene. To specifically authenticate ‘Heshangling’, 33 randomly amplified polymorphic DNA (RAPD) markers were adopted to amplify genomic sequences from the five samples. A pair of sequence characterized amplified region (SCAR) primers were designed based on the results of RAPD markers, which could specifically amplify one target band from all eight individuals of ‘Heshangling’, but none from any individuals of other T. natans varieties or one T. incisa. Taken together, a TNL sequence was provided in this study to distinguish four T. natans varieties and one T. incisa. Furthermore, a RAPD-SCAR marker was developed for efficient authentication of ‘Heshangling’.
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Xavier, Ana Claudia C., Frederico Canato Martinho, Izabel C. G. Camões, and Lilian F. Freitas. "Investigation of virulence factors of Enterococcus faecalis strains isolated in secondary/ persistent infections." Brazilian Dental Science 17, no. 1 (January 24, 2014): 32. http://dx.doi.org/10.14295/bds.2014.v17i1.961.
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<p><strong>Objective</strong>: More virulent strains may result from the acquisition of genes by genetic exchange, pathogenicity islands in several species encoding toxins, adhesion factors and other factors associated with virulence. The aim of this study was to investigate the prevalence of E. faecalis strains in secondary endodontic/ persistent using endodontic infection by culture and PCR technqiues; and to investigate for the presence of virulence factor genes of gelatinase (gelE), cytolysin activator (Cyla), surface adhesin of Enterococcus (ESP) and collagen adhesin of Enterococcus (ACE). <strong>Material and methods</strong>: Microbial samples were obtained from 12 teeth with secondary/ persistent endodontic infection showing apical periodontitis. Culture techniques were used including serial dilution, plating, incubation, and biochemical identification. For PCR detection, samples were analyzed using a species-specific primer of the 16S rDNA and the downstream intergenic spacer region. <strong>Results</strong>: Culture and PCR detected the test species in 3/12 (25%) and 5/12 (41.6%) of teeth, respectively. A total of 38 Enterococcus faecalis strains were isolated and submitted to the virulence factor genes analysis. PCR products consistent with genes encoding surface adhesion (ESP), gelatinase (gelE) and collagen binding antigen (ACE) were found in 26/38 (68%), 31/38 (81%) and 38/38 (100%) of the isolates. The Cytolysin activator (Cyla) gene was not recovered from E. faecalis isolates. <strong>Conclusions</strong>: In conclusion, the present study revealed by culture and molecular methods revealed a high prevalence of E. faecalis in teeth with secondary/ persistent endodontic infection. Moreover, of a clinical relevance, we found different E. faecalis strains carrying different virulence determinants.</p><p><strong>KEYWORDS</strong> Bacteria; E. faecalis; Root canal, virulence. </p>
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Wang, Jun, Xin-Yi Dai, Xiao-Dong Xu, Zi-Yi Zhang, Dan-Na Yu, Kenneth B. Storey, and Jia-Yong Zhang. "The complete mitochondrial genomes of five longicorn beetles (Coleoptera: Cerambycidae) and phylogenetic relationships within Cerambycidae." PeerJ 7 (September 5, 2019): e7633. http://dx.doi.org/10.7717/peerj.7633.
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Cerambycidae is one of the most diversified groups within Coleoptera and includes nearly 35,000 known species. The relationships at the subfamily level within Cerambycidae have not been convincingly demonstrated and the gene rearrangement of mitochondrial genomes in Cerambycidae remains unclear due to the low numbers of sequenced mitogenomes. In the present study, we determined five complete mitogenomes of Cerambycidae and investigated the phylogenetic relationship among the subfamilies of Cerambycidae based on mitogenomes. The mitogenomic arrangement of all five species was identical to the ancestral Cerambycidae type without gene rearrangement. Remarkably, however, two large intergenic spacers were detected in the mitogenome of Pterolophia sp. ZJY-2019. The origins of these intergenic spacers could be explained by the slipped-strand mispairing and duplication/random loss models. A conserved motif was found between trnS2 and nad1 gene, which was proposed to be a binding site of a transcription termination peptide. Also, tandem repeat units were identified in the A + T-rich region of all five mitogenomes. The monophyly of Lamiinae and Prioninae was strongly supported by both MrBayes and RAxML analyses based on nucleotide datasets, whereas the Cerambycinae and Lepturinae were recovered as non-monophyletic.
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Zhu, Dan-Tong, Chi Zou, Fei-Xue Ban, Hua-Ling Wang, Xiao-Wei Wang, and Yin-Quan Liu. "Conservation of transcriptional elements in the obligate symbiont of the whitefly Bemisia tabaci." PeerJ 7 (August 16, 2019): e7477. http://dx.doi.org/10.7717/peerj.7477.
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Background Bacterial symbiosis is widespread in arthropods, especially in insects. Some of the symbionts undergo a long-term co-evolution with the host, resulting in massive genome decay. One particular consequence of genome decay is thought to be the elimination of transcriptional elements within both the coding region and intergenic sequences. In the whitefly Bemisia tabaci species complex, the obligate symbiont Candidatus Portiera aleyrodidarum is of vital importance in nutrient provision, and yet little is known about the regulatory capacities of it. Methods Portiera genomes of two whitefly species in China were sequenced and assembled. Gene content of these two Portiera genomes was predicted, and then subjected to Kyoto Encyclopedia of Genes and Genomes pathway analysis. Together with two other Portiera genomes from whitefly species available previously, four Portiera genomes were utilized to investigate regulatory capacities of Portiera, focusing on transcriptional elements, including genes related with transcription and functional elements within the intergenic spacers. Results Comparative analyses of the four Portiera genomes of whitefly B. tabaci indicate that the obligate symbionts Portiera is similar in different species of whiteflies, in terms of general genome features and possible functions in the biosynthesis of essential amino acids. The screening of transcriptional factors suggests compromised ability of Portiera to regulate the essential amino acid biosynthesis pathways. Meanwhile, thermal tolerance ability of Portiera is indicated with the detection of a σ32 factor, as well as two predicted σ32 binding sites. Within intergenic spacers, functional elements are predicted, including 37 Shine-Dalgarno sequences and 34 putative small RNAs.
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Gómez, Gustavo, and Vicente Pallás. "A Long-Distance Translocatable Phloem Protein from Cucumber Forms a Ribonucleoprotein Complex In Vivo with Hop Stunt Viroid RNA." Journal of Virology 78, no. 18 (September 15, 2004): 10104–10. http://dx.doi.org/10.1128/jvi.78.18.10104-10110.2004.
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ABSTRACT Viroids are highly structured plant pathogenic RNAs that do not code for any protein, and thus, their long-distance movement within the plant must be mediated by direct interaction with cellular factors, the nature of which is presently unknown. In addition to this type of RNAs, recent evidence indicates that endogenous RNAs move through the phloem acting as macromolecular signals involved in plant defense and development. The form in which these RNA molecules are transported to distal parts of the plant is unclear. Viroids can be a good model system to try to identify translocatable proteins that could assist the vascular movement of RNA molecules. Here, we demonstrate by use of immunoprecipitation experiments, that the phloem protein 2 from cucumber (CsPP2) is able to interact in vivo with a viroid RNA. Intergeneric graft assays revealed that both the CsPP2 and the Hop stunt viroid RNA were translocated to the scion. The translocated viroid is symptomatic in the nonhost scion, indicating that the translocated RNA is functional. The CsPP2 gene was cloned and sequenced. The analysis of its primary structure revealed the existence of a potential double-spaced-RNA-binding motif, previously identified in a set of proteins that bind to highly structured RNAs, which could explain its RNA-binding properties. The possible involvement of this phloem protein in assisting the long-distance movement of the viroid RNA within the plant is discussed.
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Woo, Andrew J., Jonghwan Kim, Jian Xu, Hui Huang, and Alan Cantor. "Role of the Krüppel-Type Zinc Finger Transcription Factor ZBP-89 In Human Globin Gene Regulation and Erythroid Development." Blood 116, no. 21 (November 19, 2010): 2067. http://dx.doi.org/10.1182/blood.v116.21.2067.2067.
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Abstract Abstract 2067 The molecular mechanisms underlying developmental globin gene regulation remain incompletely understood. Prior studies have identified key cis-regulatory elements within the beta globin locus that contain core regions of closely spaced functional binding sites for GATA, NF-E2p45/maf and GT/GC box binding transcription factors. We recently identified the GT/GC-box binding transcription factor ZBP-89 as a novel GATA-1 interacting partner, and showed that it is involved in erythroid development in mice (Woo et al. 2008. Mol. Cell Bio. 28:2675-2689). Brand et al. independently isolated ZBP-89 in NF-E2p45/mafk complexes from induced mouse erythroid leukemia (MEL) cells (Brand et al. 2004. Nat. Struct. Mol Biol. 11:73-80). In the current study, we show that ZBP-89 protein levels increase during in vitro erythroid differentiation of human bone marrow derived CD34+ cells. This correlates with the onset of alpha and beta globin gene transcription. ChIP-chip studies using ENCODE v2.0 arrays demonstrate that ZBP-89 occupies key cis-regulatory elements within both the beta globin (locus control regions HS3, HS2; delta and beta proximal promoters; and an intergenic region between gamma1 and delta globin) and alpha globin (HS-48, HS-40, HS-10 and alpha globin proximal promoters) loci in primary human erythroid precursors. Comparative analysis across the entire ENCODE array reveals a strong positive correlation between ZBP-89 occupancy, RNA polymerase II occupancy, and the activating histone marks acetylated histone 3 (AcH3) and trimethylated histone 3 lysine 4 (H3K4me3); and a negative correlation with the repressive mark trimethylated histone 3 lysine 27 (H3K27me3). Motif analysis under the ZBP-89 occupancy peaks indicates a preference for GGGG(G/A)NGGGG in vivo binding sites. Lentiviral shRNA mediated knock down of ZBP-89 in the in vitro differentiated CD34+ cells results in 30–50% reduction of alpha-, gamma-, and beta-globin gene expression, as well as modestly decreased expression of a number of additional erythroid-specific genes. Co-immunoprecipitation experiments demonstrate physical association between ZBP-89 and the GCN5/Trapp histone acetyltransferase complex. Based on these findings, we propose that ZBP-89 participates with GATA-1 and NF-E2 in the final epigenetic changes required for high-level expression of globin and other erythroid genes in terminally differentiating human erythroid cells. Disclosures: No relevant conflicts of interest to declare.
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Panigrahi, Sunil K., Gagan Deep Jhingan, Indrani Som, Alok Bhattacharya, William A. Petri, and Sudha Bhattacharya. "Promoter Analysis of Palindromic Transcription Units in the Ribosomal DNA Circle of Entamoeba histolytica." Eukaryotic Cell 8, no. 1 (October 31, 2008): 69–76. http://dx.doi.org/10.1128/ec.00254-08.
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ABSTRACT rRNA genes of Entamoeba histolytica are organized as palindromic ribosomal DNA (rDNA) units (I and II) in a 24.5-kb circle. Although the two rDNAs are identical in sequence, their upstream spacers are completely different. Since the intergenic sequences (IGS) of all rDNA copies in other organisms are conserved and contain transcription regulatory sequences, the lack of sequence conservation in the IGS prompted the question of whether both rDNAs are indeed transcriptionally active. We mapped the transcriptional start points (tsp's) and promoters of the two rDNAs. A 51-bp sequence immediately upstream of the tsp's was highly conserved in both units. In addition, both units had an A+T-rich stretch upstream of the 51-bp core. Analysis of reporter gene transcription showed promoter activity to reside in the regions from positions −86 to +123 (rDNA I) and positions −101 to +140 (rDNA II). The promoter-containing fragments from both units could bind and compete with each other for protein(s) from nuclear extracts. Protein binding was especially dependent on the A+T-rich region upstream of the 51-bp core (positions −53 to −68). The requirement of >80 bp downstream of the tsp was striking. Although this sequence was not conserved in the two units, it could potentially fold into very long stem-loops. Both rDNAs transcribed with comparable efficiency, as measured by nuclear runon. Thus, both rDNAs share very similar organization of promoter sequences, and in exponential culture both rDNAs are transcribed. It remains to be seen whether the different IGS affect the regulation of the two units under adverse conditions.
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Mahajan, Milind C., Ghia Euskirchen, Jin Lian, Adam S. Raefski, Michael P. Snyder, and Sherman M. Weissman. "Chromatin Structure and Transcription of the Human alpha Globin Locus in Erythroid and Non-Erythroid Environments." Blood 110, no. 11 (November 16, 2007): 1774. http://dx.doi.org/10.1182/blood.v110.11.1774.1774.
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Abstract The ζ, α-1 and α-2 are the major alpha like globin genes. The ζ gene is expressed in the embryonic stage, while α-1 and α-2 genes are expressed throughout the adult life. Although the alpha like globin genes are flanked by genes that are transcribed in many cell types, their expression is restricted to the erythroid cells. Since the alpha globin genes are situated amidst the actively transcribing genes, they are supposed to be in the open chromatin configuration, even when they are transcriptionally silent in non-erythroid cells. Hence, understanding the structure of the chromatin of the alpha globin locus in erythroid and non-erythroid cells is needed to delineate the cell type and developmental stage specific regulation of expression of these genes. In the present study, we have undertaken a comparative analysis of the chromatin structure of the alpha globin locus, recruitment of transcription factors, and the transcriptional activity of the locus in enrythroid and non-erythroid cells. We have taken advantage of the availability of genomic tiling microarrays that include 50 base oligonucleotides spaced at 38 base pair intervals throughout extended regions embedding and flanking the alpha globin cluster and performed ChIP-chip analysis. The data obtained from these studies suggest that in erythroid K562 cells, Histone 3 of the alpha globin locus is acetylated at Lys 9 and dimethylated at Lys4 throughout the locus. The trimethyl Lys 4 marker was present on the promoters of transcribed genes, but not on the active HS40 enhancer. However, Pol II and its phosphory-lated forms were present on both the actively transcribing genes and the HS40 enhancer. Among the transcription factors, NF-E2 was predominantly associated with the HS40 sequences while GATA-1 was present on the alpha like promoters as well as the HS40 enhancer. The insulator binding CTCF was detected at several flanking regions of the HS40 enhancer in K562 and HeLa cells. We speculate that differential interaction among CTCF sites may play a role in regulating the effects of the HS-40 enhancer. In erythroid K562 cells, a strong HS40 enhancer formed by the virtue of the recruitment of the enhancer factors can overcome blocking by the downstream flanking CTCF site and, in analogy to suggestions in studies of Drosophia insulating elements, this might be mediated by specific interactions between upstream and downstream insulators. In the non-erythroid cells, the alpha globin locus was hypoacetylated. Along with the absence of trimethylation of the Lys 4 marker for active transcription, the methylations at Lys 9, and Lys 27 that are associated with the inactive genes were also absent. We also observed a lack of Lys 36 marker associated with the body of the transcribing genes in HeLa cells. In contrast to these observations, we have detected a robust presence of Pol II and Brg1 on the entire locus. Surprisingly, we have detected significant amount of transcriptional activity associated with parts of the theta and zeta genes and intergenic regions in HeLa, NB4 and 06990 lymphoblastoid cells. Initial studies indicate the generation of spliced polyadenylated RNA of the alpha globin locus in HeLa cells. The transcription of the locus was not uniform, but it was localized to certain regions, suggesting that the alpha globin transcription is not just a uniform leaky transcription, but that there may be hitherto unappreciated transcriptional regulatory elements within the locus.
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Twa, David D. W., Fong Chun Chan, Susana Ben-Neriah, Bruce W. Woolcock, King L. Tan, Graham W. Slack, Jay Gunawardana, et al. "Genomic Rearrangements Involving Programmed Death Ligands Are Recurrent In Primary Mediastinal Large B-Cell Lymphoma." Blood 122, no. 21 (November 15, 2013): 635. http://dx.doi.org/10.1182/blood.v122.21.635.635.
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Abstract Introduction Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive malignancy commonly diagnosed in young adult females. In recent years, mutational and gene expression profiling has established genotypic and phenotypic similarity of PMBCL with both classical Hodgkin and diffuse large B-cell lymphoma (DLBCL). In-depth analyses of genomes and transcriptomes have highlighted several inactivating mutations (SOCS1, TP53), chromosomal amplifications (2p, 9p, Xp, Xq) and translocations (CIITA) thought to be integral in establishing and/or maintaining the PMBCL phenotype. Programmed death ligands (PDL) 1 (CD274) and 2 (PDCD1LG2), which are located on chromosome 9p24.1, are two emerging genes of interest that have been shown to be altered in PMBCL and can induce T-cell anergy by binding to the receptor, programmed death 1. Here, we describe the recurrence of chromosomal rearrangements of the PDL locus in various B-cell lymphomas and explore the association of these rearrangements with transcript levels. Methods To establish the frequency of CD274 and PDCD1LG2 aberration, we conducted fluorescence in situ hybridization (FISH) on 551 clinical samples and 20 established cell lines using in-house break-apart probes. Epstein-Barr virus encoded RNA in situ hybridization was also carried out on the clinical cohort. The clinical cases, sourced from the British Columbia Cancer Agency’s Centre for Lymphoid Cancer tissue repository, consisted of 125 PMBCLs, 216 DLBCLs, 130 primary DLBCL of the central nervous system (PCNSL), 12 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL) and 68 follicular lymphomas (FL) with diagnoses based on the WHO classification. The DLBCL cohort could be further subdivided into 134 nodal DLBCLs and 82 testicular DLBCLs (T-DLBCL). Quantitative real-time PCR (qRT-PCR) was subsequently conducted on 17 cell lines and a clinical sub-cohort of 76 samples, for which fresh-frozen material was available, to determine the effect of mutations on transcript expression. We then characterized the PDL aberrations of two clinical PMBCL cases and three cell lines (DEV, L-428, L-1236), at base pair resolution, by applying the bioinformatic tools, nFuse, deFuse and destruct to both newly produced and previously published whole genome (WGS) and whole transcriptome (RNA-seq) libraries. Results FISH revealed a PDL locus (9p24.1) break-apart frequency of 20% (25/125) in PMBCL. There were no differences in any known clinical parameters or frequency of Epstein-Barr virus positivity between positive and negative PDL break-apart cases. Break-apart frequencies in other malignancies were calculated to be 3% in DLBCL, 7% in T-DLBCL and 1% in PCNSL; no positive cases were identified in either NLPHL or FL. The proportion of break-apart positive cases was significantly higher in PMBCL as compared to the other lymphomas surveyed (P < 0.05). Further, in agreement with the published literature, we observed an amplification frequency of the PDL locus in 36% (45/125) of PMBCLs. qRT-PCR established that PDCD1LG2 transcript levels were significantly higher in cases with 9p24.1 locus rearrangements compared to copy number neutral (P = 0.0003), gain (P = 0.001) and amplified cases (P = 0.005). Likewise, CD274 transcript levels were significantly higher in rearranged cases compared to copy number neutral cases (P = 0.03). Following the analysis of WGS and RNA-seq libraries, we were able to characterize four novel fusion transcripts involving the 9p24.1 locus: PDCD1LG2-NRG1 (PMBCL clinical case), PDCD1LG2-IGHV7-81 (L-1236), CIITA-PDCD1LG2 (DEV) and KIAA1432-CLDN14 (L-428). Aberrations involving both NRG1 and CIITA have previously been implicated in breast cancer and B-cell lymphomas, respectively. We also identified a translocation in another PMBCL clinical case with breakpoints in the intergenic spaces near LRMP and CD274, though this rearrangement did not produce a fusion transcript. Conclusion Taken together, our findings show that rearrangement of the PDL locus is recurrent in PMBCL, characteristic of PMBCL and leads to overexpression of PDL transcripts. Given the well-referenced function of PDLs in repressing the anti-tumor response, these data suggest that targeting the PDL axis in a subgroup of B-cell lymphomas holds clinical promise. Disclosures: No relevant conflicts of interest to declare.
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Singh, Dharam, Oleg N. Murashko, and Sue Lin-Chao. "Posttranscriptional Regulation of tnaA by Protein-RNA Interaction Mediated by Ribosomal Protein L4 in Escherichia coli." Journal of Bacteriology 202, no. 10 (March 2, 2020). http://dx.doi.org/10.1128/jb.00799-19.
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ABSTRACT Escherichia coli ribosomal protein (r-protein) L4 has extraribosomal biological functions. Previously, we described L4 as inhibiting RNase E activity through protein-protein interactions. Here, we report that from stabilized transcripts regulated by L4-RNase E, mRNA levels of tnaA (encoding tryptophanase from the tnaCAB operon) increased upon ectopic L4 expression, whereas TnaA protein levels decreased. However, at nonpermissive temperatures (to inactivate RNase E), tnaA mRNA and protein levels both increased in an rne temperature-sensitive [rne(Ts)] mutant strain. Thus, L4 protein fine-tunes TnaA protein levels independently of its inhibition of RNase E. We demonstrate that ectopically expressed L4 binds with transcribed spacer RNA between tnaC and tnaA and downregulates TnaA translation. We found that deletion of the 5′ or 3′ half of the spacer compared to the wild type resulted in a similar reduction in TnaA translation in the presence of L4. In vitro binding of L4 to the tnaC-tnaA transcribed spacer RNA results in changes to its secondary structure. We reveal that during early stationary-phase bacterial growth, steady-state levels of tnaA mRNA increased but TnaA protein levels decreased. We further confirm that endogenous L4 binds to tnaC-tnaA transcribed spacer RNA in cells at early stationary phase. Our results reveal the novel function of L4 in fine-tuning TnaA protein levels during cell growth and demonstrate that r-protein L4 acts as a translation regulator outside the ribosome and its own operon. IMPORTANCE Some ribosomal proteins have extraribosomal functions in addition to ribosome translation function. The extraribosomal functions of several r-proteins control operon expression by binding to own-operon transcripts. Previously, we discovered a posttranscriptional, RNase E-dependent regulatory role for r-protein L4 in the stabilization of stress-responsive transcripts. Here, we found an additional extraribosomal function for L4 in regulating the tna operon by L4-intergenic spacer mRNA interactions. L4 binds to the transcribed spacer RNA between tnaC and tnaA and alters the structural conformation of the spacer RNA, thereby reducing the translation of TnaA. Our study establishes a previously unknown L4-mediated mechanism for regulating gene expression, suggesting that bacterial cells have multiple strategies for controlling levels of tryptophanase in response to varied cell growth conditions.
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Cui, Jie, Yamin Zhang, Xiaoyue Ren, Lei Jin, and Hongyi Zhang. "TBX1 Functions as a Tumor Activator in Prostate Cancer by Promoting Ribosome RNA Gene Transcription." Frontiers in Oncology 10 (January 26, 2021). http://dx.doi.org/10.3389/fonc.2020.616173.
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TBX1 belongs to an evolutionarily conserved family of transcription factors involved in organ development. TBX1 has been reported to have a hypermethylated cytosine guanine dinucleotide island around its second exon, which was related to prostate cancer (PCa) progression. However, the role and exact mechanism of TBX1 in PCa remains unknown. Using human prostate samples, online data mining and multiple in vitro and in vivo models, we examined the biological role and underlying mechanisms of TBX1 in PCa. TBX1 was highly expressed in PCa tissues, and high TBX1 expression was positively associated with Gleason score, pathological tumor stage, pathological lymph node stage, extraprostatic extension and disease/progression-free survival. In vitro and in vivo data demonstrated that TBX1 silencing inhibits PCa cell proliferation and colony formation and increases the cell population at the G0/G1 phase. The exogenous expression of TBX1 rescued these phenotypes. Mechanistically, TBX1 silencing suppressed the expression of 45S ribosomal RNA (rRNA), which was rescued by the exogenous expression of TBX1. TBX1 silencing inhibited the monomethylation of histone 3 lysine 4 (H3K4me1) binding with the non-coding intergenic spacer (IGS) regions of ribosomal DNA (rDNA) and the recruitment of upstream binding factor to the promoter and IGS regions of rDNA. The drug-induced enhancement of H3K4me1 counteracted the effect of TBX1 silencing. These findings indicate that TBX1 exerts its tumor activator function in PCa cells via epigenetic control, thereby promoting rRNA gene transcription. Thus, TBX1 may represent a prognostic biomarker and therapeutic target for PCa patients.
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Sayou, Camille, Gonzalo Millán-Zambrano, Helena Santos-Rosa, Elisabeth Petfalski, Samuel Robson, Jonathan Houseley, Tony Kouzarides, and David Tollervey. "RNA Binding by Histone Methyltransferases Set1 and Set2." Molecular and Cellular Biology 37, no. 14 (May 8, 2017). http://dx.doi.org/10.1128/mcb.00165-17.
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ABSTRACT Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo. High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional posttranscriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity.
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Monahan, Kevin, Ira Schieren, Jonah Cheung, Alice Mumbey-Wafula, Edwin S. Monuki, and Stavros Lomvardas. "Cooperative interactions enable singular olfactory receptor expression in mouse olfactory neurons." eLife 6 (September 21, 2017). http://dx.doi.org/10.7554/elife.28620.
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The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and trans downregulation of OR transcription, whereas pre-assembly of a multi-enhancer hub increased the frequency of OR choice in cis. Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs.