Bombyx mori employing luciferase reporter assays in an ecdysteroid-responsive cultured cell line, NIAS-Bm-aff3 (aff3). The EcRE of BmEcR-B1 at −2800 comprises of two adjacent elements separated by 5 bp, E1 (15 bp) and E2 (21 bp), both of which are required for the 20E response. Further analysis using electrophoretic mobility shift assays showed that E1 binds to the EcR/USP heterodimer and that E2 may bind to the E-box (CACGTG) binding factor such as bHLH protein. The unique E1+E2-type EcRE is also detected in the promoter upstream regions of EcR-B1 from seven lepidopteran species studied. In contrast, both a 20 bp EcRE identified in the promoter of BmE75-A and a 18 bp EcRE identified in the BHR3-B promoter, contained only E1-type EcR/USP binding element but the E2 type element was not in the promoter regions of these genes. The combination of presence of the E2 element or other cis-regulatory elements in promoter regions explains the different 20E response of each class of nuclear receptor genes. Furthermore, the E1+E2 structure for EcR-B1 can be involved in a possible cross-talk between ecdysteroid and other regulatory pathways.Three distinct classes of nuclear receptors, EcR, E75, and HR3, are key regulators in the ecdysone-inducible gene activation cascade in insects. The transcription of these genes is induced by ecdysone (20E) differently, although the detailed mechanisms underlying their responses to 20E are largely unknown. We identified ecdysone response elements (EcREs) present in the promoters of genes coding BmEcR-B1, BmE75-A, and BHR3-B isoforms from  The steroid hormone ecdysteroid, primarily 20-hydroxyecdysone (20E), coordinates various developmental and physiological processes in insects Many but not all of the early- and early-late genes are members of the nuclear receptor super-family EcR and E75, categorized as early genes, show a rapid transcriptional induction by a low concentration of 20E in the absence of protein synthesis EcR, whose expression is induced earlier by a lower concentration of 20E, is further classified as early class I. E75, whose expression is induced later by a relatively higher 20E concentration during wandering stages, is classified further as early class II HR3 is induced later by a higher concentration of 20E, needs protein synthesis for its full expression, and is categorized as an early-late gene Expression of the three nuclear receptors, EcR, E75, and HR3, are induced by ecdysteroid. cis-regulatory DNA sequence called the ecdysone response element (EcRE) in their promoter region. There is currently a sizable list of ecdysone-regulated genes whose EcREs have been characterized. For example, Hsp27Eip28/29Fbp1DdcIt is known that the EcR/USP heterodimer regulates the expression of target genes through binding to Bombyx mori aff3 cells, which are responsive to 20E. We succeeded in showing that the cis-regulatory sequences responsible for 20E-dependent activation are located in an upstream region of EcR, E75, and HR3.In this study, we identified EcREs present in the promoters of three ecdysone- inducible nuclear receptor genes using a transient reporter assays in BmEcR is mapped on linkage group 10 and its entire region is included in one scaffold, Bm_scaf10. Comparing the sequences with the cDNA sequences of BmEcR isoforms, we found that there are three A-specific exons (exons A1 to A3) and one B1-specific exon (exon B1) upstream of the common exons. The transcriptional start sites for A and B1 isoforms are about 160 kb apart from each other , ch other . Similarm_scaf70 . BHR3 ism_scaf27 . To screBombyx cells, aff3 and BmN   .BmEcR is important for its transcription BmEcR-B1, BmE75-A, and BHR3-B. Seventy-two hours after the transfection of each plasmid, three different concentration of 20E  was added to aff3 cells and the cells were incubated up to 96 h. The average value of the basal activity for the firefly luciferase 2 without 20E in the medium after 96 h culture was 32171 (N = 4) for pBmEcR-B1_−3652/+248, 370221 (N = 4) for pE75-A_−5023/+83, and 30974 (N = 4) for pBHR3B_−2891/+18, respectively. To standardize the luciferase activity, we calculated the relative activities for the firefly luciferase 2 and the Renilla luciferase (fluc/rluc). Then, the ratio of the relative activities of luciferase with and without 20E in the medium was calculated and the fold induction by 20E  is shown in figures. Three constructs showed a clear 20E-dependent induction of the luciferase activity: about 30-fold-induction in BmEcR-B1 (BmE75-A (BHR3-B (cis-regulatory elements that are involved in the 20E inducible transcription. We did not detect a clear 20E response for BmEcR-A promoter regions (−3932 to +309) (data not shown) and focused on the above three promoter regions for further analyses. Transcription of BmEcR-B1 and BmE75-A constructs was induced at 2.0×10−2 µg/mL of 20E and transcription of BHR3-B at 2.0 µg/mL of 20E  but drastically reduced in a construct −2527 to +248 (1.46) (BmEcR-B1.We next tried to identify the regulatory elements responsible for 20E induction in the construct of 8 (1.46) . Similar8 (1.46) , a serie8 (1.46) , and a s8 (1.46)  showed tcis-elements for BmEcR-B1, we introduced a series of 5 bp-replacements with 5′-AAAAA-3′ (A5 mutations) in the region from −2850 to −2800 of pBmEcR-B1_−3652/+248  cell that derived from embryos, and the Spodoptera Sf9 cell  using BmEcRB1-E1 , BmEcR-B1 (B1T) and BmUSP (USPT), all of which were tagged with a V5 antigen, in aff3 cells by plasmid transfection. The cell extracts from BmEcR-A-overexpressed (AT) and BmEcR-B1-overexpressed cells (B1T) intensified shifted bands for BmEcRB1-E1, BmE75A-EcRE, and BHR3B-EcRE , respectively, but not in the intact cell extracts (lane 2). This result suggests that EcRA, EcRB1, and USP are involved in the protein–DNA complex corresponding to the supershifted band and that BmEcRB1-E1, BmE75A-EcRE, and BHR3B-EcRE are the binding sites of the EcR or USP protein. This possibility is also supported by the results with anti-USP antibodies , and by the results of the addition of two antibodies simultaneously, showing that the bands were shifted further , suggesting that the EcR-A/USP or EcR-B1/USP heterodimer binds to each element.To characterize further the binding of the EcR/USP heterodimer to BmEcRB1-E1, BmE75A-EcRE, and BHR3B-EcRE, we overexpressed BmEcR-A (ACATGG-3′5′-C) . When the promoter regions for HR3-B were compared among four Lepidoptera, we found that BHR3B-EcRE identified here and its upstream regions are highly conserved among all species  (Bombyx nuclear receptor genes show the highly conserved 14 bp tract in their 5′-side. The 14 bp consensus motif (5′-KCRGGTCWWCGMWC-3′) among Bombyx four nuclear receptor genes has 64 ( = 26) patterns of the sequences (http://sgp.dna.affrc.go.jp/KAIKObase/) and found 138 motifs which matched 100% to either of 64 sequences. We compared BmEcRB1-E1 (16 bp), BmE75A-EcRE (20 bp), BHR3B-EcRE (18 bp) with the canonical EcRE (15 bp), a consensus sequence among dozens of NTGAMCY) . The USPequences . We thenBmE75-A, BHR3-B, BmEcR-B1 and BmEcR-A were induced by the 20E treatment clearly. Among 138 motifs, there are at least 17 motifs near which gene expressions showed more than 4 fold induction by 20E  and BHR3-B (early-late class) denied this possibility, because BmE75A-EcRE and BHR3B-EcRE showed the same effect on the dose response to 20E  conserved among EcREs for three nuclear receptor genes exists at 138 locations in the silkworm genome and 296 genes surround the motifs  and E2 (21 bp), both of which are required for the 20E response. This unique structure of EcR-B1 EcRE is also conserved in other Lepidoptera, Helicoverpa, Spodoptera. Bicyclus, Danaus, Manduca, and Papilio  of BmEcRB1-E2, suggesting different binding factors between A and B1 isoforms of EcR. We found that the putative BmEcRA-EcRE binds to the EcR/USP heterodimer (data not shown), while its contribution to the 20E response has not been confirmed. We also searched E2-like sequences in other genomes, but we did not find a similar sequence in EcR-B1 promoter regions of Drosophila and other non-lepidopteran insects.We also searched the E1+E2 sequence in the silkworm genome and found a similar structure in the second intron A2 of Drosophila, it is reported that 48 transcriptional factors belong to bHLH protein and 7 transcriptional factors belong to bHLH-PAS protein Bombyx mori and the red flower beetle, Tribolium casteneum, respectively Because the introduction of a mutation into the E-box of BmEcRB1-E2 decreased the intensity of the mobility shifted band , we specBmEcR-B1 but not BmEcR-A is repressed by JH BmEcR-B1 is involved in the ecdysone- dependent developmental event, we performed EMSA with the E2 probe in cellular extracts from pupal wings, and observed the shifted band only in the proximal region extract but not in the distal region extract (data not shown). We previously observed that BmEcR-B1 is expressed in the proximal region but BmEcR-A in the distal region of pupal wings EcR-B1 is involved in the lepidopteran wing formation. We have recently observed that the Bombyx Met2 gene is transcribed more dominantly in the proximal region than in the distal region of pupal wings, supporting the above idea. In Drosophila glue protein genes, Sgs-3 and Sgs-4, the binding site of a forkhead transcription factor is located near the EcR/USP binding site in their 5′-flanking region and is essential for their ecdysone-dependent induction Drosophila Sgs-3 and Sgs-4, the coupling of EcR/USP binding to E1 and a transcription factor binding to E2 may regulate the ecdysone-dependent and tissue-specific expression of BmEcR-B1 in the silkworm pupal wings.Our former observation that the expression of Manduca sexta MHR3Manduca GV cells, but the reporter construct composed of the short promoter region including the EcRE1 did not show the 20E-dependent activation of MHR3 Manduca sexta GV1 cells, MHR3 expression is suppressed by overexpression of MsE75A, which binds monomeric response element 2 (MRE2) 2.4 kb upstream of MHR3 at a lower concentration of 20E 5′-TATTAAGGCGATGATAC-3′, −2415 to −2399), suggesting that the HR3 regulation system is conserved between the two species and this type of interaction may underlie the difference in the 20E response between E75 and HR3. Similar to EcR-B1 promoter regions (We compared the BHR3-B-EcRE identified here with putative four EcREs (1 to 4) of  regions , the EcR regions , but we BmEcR-B1 comprises of two adjacent elements E1 and E2, both of which are required for the 20E response, but BmE75-A and BHR3-B EcREs contained only an E1-type EcR/USP binding element. This indicates that the combination of presence of the E2 element or other cis-regulatory elements in promoter regions explains the different 20E response of each class of nuclear receptor genes. E2 of BmEcR-B1 EcRE includes E-box (CACGTG) and may bind to E-box binding factors such as bHLH protein. Furthermore, the unique E1+E2-type EcRE is highly conserved among the promoter upstream regions of EcR-B1 from seven lepidopteran species studied. These observations suggest that the E1+E2 structure for EcR-B1 can be involved in a possible cross-talk between ecdysteroid and other regulatory pathways.The EcRE of Spodoptera frugiperda were maintained at 27°C in an IPL-41 insect medium (Gibco) with 10% fetal bovine serum . BmN that derived from the silkworm ovary was maintained at 27°C in a Grace's Medium Supplemented (Gibco) with 10% FBS.The NIAS-Bm-aff3 (aff3) cell line 4 cells. The activities of firefly and Renilla luciferase in cell lysates were measured by a dual-luciferase reporter assay system (Promega) using a MicroLumat Plus (Berthold). The firefly luciferase activity was normalized to the Renilla luciferase activity.The plasmid constructs were transfected into aff3 cells by a liposome-mediated method using the TransFast™ or FuGENE®-HD reagent (Promega). Forty microliters of Sf-900II SFM (Gibco) containing 100 ng of each pGL4.10 construct, the HSP70-pRL plasmid and 1.0 µL of TransFast™ were added to 5×102, 6.6 mM EDTA, 1.2 mM DTT, and 12% glycerol), then 500 fmol of the labeled probe (or with competitor DNA), and the mixture was incubated for 20 min on ice. The anti-V5 antibody or/with anti-USP antibody was added, and the mixture was incubated on ice for another 1.5 h and then separated on a 4% polyacrylamide gel containing 2.5% glycerol. The dried gel was analyzed with a high-performance autoradiography film . To produce the anti-USP antibody, a BmUSP (2N-MLDGFRDDSTPPPPFKNYC -COOH] (from 85 to 105 amino acids in A/B region) was synthesized, HPLC purified, and used to immunize rabbits (Sawady Technology (QIAGEN)).The oligonucleotides used as probes are shown in  a BmUSP  peptide Drosophila HSP70 promoter, which constitutively expresses the Renilla luciferase in aff3 cells (HSP70–pRL), was co-transfected with each experimental DNA into the firefly luciferase vector.We used the pGL4.10 vector containing a firefly luciferase2 gene as the reporter, for the promoter assay. To normalize the expression of the firefly luciferase2 in aff3 cells, the pRL vector containing the 4 polynucleotide kinase (TOYOBO) and ATP (Roche). The pBmEcR-B1_−3652/+248 plasmid was used as a basal template for making various mutants of BmEcR-B1. Similarly, the pBmE75-A_−518/+85 and pBHR3-B_−485/+18 were used as basal templates to make various mutants for BmE75-A and BHR3-B mutants, respectively. To introduce various mutations, one primer with excisions or A5 or 2 bp substitutions were used in the inverse PCR. Each inverse PCR product was amplified by iProof (Bio-Rad) with a pair of primers (EcoRV (BmEcR-B1 mutant) of a pBmEcR-B1_−3652/+248 vector. The mutated inserts of BmE75-A and BHR3-B were subcloned between SmaI and NcoI of each pBmE75-A_−518/+85 or pBHR3-B_−485/+18.The deletion and substitution mutants were constructed by inverse PCR and self-ligation with 5′-phosphorylated primers. Phosphorylated primers were made with T primers , S4, S5.EcoRI or NotI and at each end of the PCR product (EcoRI and NotI were cloned into the piZT/V5-His vector (Invitrogen). We named these constructs B1T and USPT. BmEcR-A_ORF was cloned into the piZT/V5-His vector, and then partially exchanged of B1T C-terminal by EcoRV and NotI. This construct was named AT.The BmEcR-B1_ORF and BmUSP_ORF were amplified by PCR with iProof (Bio-Rad) using primer sets designed to make sequences for  product , S4, S5.At 120 h after transfection of overexpression vectors of BmEcR-A, BmEcR-B1 and BmUSP, cell pellets were lysed in 200 µl of a sonication buffer , sonicated at 4°C for 35 s, and centrifuged at 12,000×g at 4°C for 10 min. The supernatant was collected as 20 µl aliquots and stored at −70°C.Approximately 20 µg total RNA extracted from aff3 cells under various 20E conditions was separated on a 1.0% denaturing gel , 5 mM sodium acetate, 1 mM EDTA, and 1.0% agarose), and blotted onto Biodyne A nylon membrane  in 10×SSC . The membranes were hybridized with each probe at 42°C for 16 h in 50% formamide, 10×Denhardt's solution , 50 mM sodium phosphate (pH 7.0), and 2.5 µg/ml denatured salmon sperm DNA in 5×SSC. Post-hybridization washes were carried out once each in 2×SSC with 0.1% SDS for 15 min at 42°C. PCR primers used are referred to A silkworm 4×44K oligonucleotide microarray (order made by Agilent) is tilled by 44,000 olidonucleotide probes (60-mer) for the silkworm genes. Each slide has four arrays. After the aff3 cells were incubated with 2.0 µg/mL of 20E for 3, 6, 12 and 24 h, total RNAs were extracted at each time. The concentration of total RNA was checked by a NanoDrop ND-1000 spectrometer (Thermo Fisher Scientific). The quality of total RNAs was checked by an Agilent 2100 BioAnalyzer (Agilent). Each cDNA with T7 promoter-tag was synthesized from 500 ng of total RNA according to the Agilent's protocol. cRNA labelled with a fluorescent dye (Cy3-CTP) was produced according to the Agilent's protocol and then purified with an RNeasy column (QIAGEN). Before loading onto a microarray, each labelled cRNA was fragmented in hybridization buffer at 60°C within 30 min. The array was hybridized with labelled cRNA at 65°C for 17 h and washed by Agilent Gene Expression Wash Buffer 1 and 2, respectively. All microarrays were scanned using an Agilent Microarray Scanner (Agilent). The signal intensity of each spot was measured using Agilent Feature Extraction version 9.5 (Agilent). These data were converted Excel (Microsoft) file, and then analyzed.Figure S1Cellular response to 20E of two Bombyx cultured cells and the 20E-dependent expression of three nuclear receptor genes. (A) A Bombyx cell line aff3 showed an aggregated cellular response (white arrows) to 2.0×10−2 and 2.0 µg/mL of 20E, whereas BmN did not show a remarkable cellular response to 20E. (B) Time course of 20E-dependent expression patterns of BmEcR-B1, BmEcR-A, BmE75-A and BHR3-B in aff3 cells analyzed by Northern hybridization. Total RNA was extracted from the cells incubated with 2.0 µg/mL of 20E in the medium at 3, 6, 12 and 24 h. rRNA stained by ethidium bromide is shown as equal loading. (C) Dose dependency of BmEcR-B1, BmE75-A, and BHR3-B expressions in the response to 20E were analyzed in aff3 cells. The conditions for Northern hybridization were same as (B). The probes for BmEcR-B1 and BmE75-A were hybridized to the same membrane, transferred the total RNAs extracting from aff3 cells incubated 3 hrs with each diluted 20E. BHR3-B was detected from another membrane, incubating 12 h with 20Es.(TIF)Click here for additional data file.Figure S2Identification of the ecdysone responsive region in BmEcR-B1 promoter. (A–C) Each promoter activity of 5′-deletion series (A) and excision series  of constructs for the BmEcR-B1 promoter region is shown. Each plasmid construct was transfected into aff3 cells and incubated 48 h with 2.0 µg/mL (4.2 µM) of 20E and measured the luciferase activity. The ratio of relative luciferase activities with and without 20E (fold induction by 20E) is shown on the right, as referred 1.0 at 48 h without 20E. Error bar represents SE (N = 4). “null” indicates the pGL4.10 vector.  Excisions are shown by the white squares. The sizes of the excisions are 40 bp (B) and 10 bp (C), respectively.(TIF)Click here for additional data file.Figure S3Identification of EcREs for BmE75-A.  Each promoter activity of 5′-deletion series of constructs for BmE75-A promoter region is shown. The fold induction by 20E of each construct is shown in right. Error bars (N = 4). “Null”; the pGL4.10 vector.(TIF)Click here for additional data file.Figure S4Identification of EcREs for BHR3-B. Each promoter activity of 5′-deletion series of constructs for BHR3-B promoter region is shown. The fold induction by 20E of each construct is shown in right. Error bars (N = 4). “Null”; the pGL4.10 vector.(TIF)Click here for additional data file.Figure S5Effects of swapping EcRE between BmE75-A and BHR3-B promoter regions on their responses to 20E. (A) Schematic structures of each reporter construct for EcRE swapping between BmE75-A and BHR3-B promoter regions. EcREs of BmE75-A and BHR3-B full-length reporter plasmids were replaced with each other. BHR3B-EcRE, TCGGGTCAACGAACCGGTGT; BmE75A-EcRE, TCGGGTCTTCGAACTCTCGG (B) Dose response to 20E of each construct. Each reporter plasmid was transfected into the aff3 cell and incubated with various concentrations of 20E for 2 days. The reporter activities were measured by a dual-luciferase assay. Bars represent SE (N = 6).(TIF)Click here for additional data file.Figure S6Electrophoretic mobility shift analysis for BmE75A-EcRE and BHR3B-EcRE.  Competition assay with cold probe for BmE75A-EcRE (A) and for BHR3B-EcRE (B). Mutation sites in E1 and E2 probe sequence “Normal” are shown in the gray region of “Mutant”. Two hundred femtomoles of 32P-probe were incubated with 5 µg of cell extracts and loaded onto the gel. 20E (6 h) represents extracts from cells cultured under 20E during 6 h. ×1, ×10, ×50 and ×100 represent the ratio of the cold probe amount to the 32P-probe amount. Filled arrows show the shifted bands and blank arrows show the free probes.  Super shift assay with anti-V5 or/and anti-USP antibodies for BmE75A-EcRE (C) and for BHR3B-EcRE (D). Intact: intact cell extracts. AT, B1T, and USPT represent extracts from cells that overexpressed EcRA, EcRB1, and USP, respectively. (E) Western blot analysis of the overexpressed nuclear receptors. Each protein with a V5-tag was detected by the anti-V5 antibody.(TIF)Click here for additional data file.Figure S7HR3-B (A) and E75-A (B) in Lepidoptera. (A) Highly homologous sequences, BHR3B-EcRE and EcRE1 of Manduca HR3 (MHR3-EcRE1) and its similar sequences, are shown by red and yellow boxes in the upper section, respectively. Three highly conserved regions, A, B, and C between BHR3 and MHR3 promoter regions shown by blue thick lines. EcRE2, 3, and 4, which were identified in Manduca MHR3, are shown by open box. The homologous sequences to Manduca EcRE2, 3, and 4 are not found in the promoter region of BHR3. +1, the transcriptional start site. Shaded box, the HR3 gene. Sequence comparison of each element is shown in the lower section. B. m, Bombyx mori; M. s, Manduca sexta; D. p, Danaus plexippus; P. x, Papilio xuthus. Nucleotide sequences in the region A  and the region B  are aligned. *, a consensus nucleotide among the sequences. (B) Schematic localization of E75-A of four lepidopteran insects (upper section) and sequence comparison of each element (lower section). Red, the same sequence to BmE75-A EcRE. *, a consensus nucleotide among the sequences. ATG: the translational start site.Conservation of EcREs for (TIF)Click here for additional data file.Table S1List of Accession Number.(PPT)Click here for additional data file.Table S2List of Primer.(PPT)Click here for additional data file.Table S3List of Primer.(PPT)Click here for additional data file.Table S4List of Primer.(PPT)Click here for additional data file.Table S5List of Primer.(PPT)Click here for additional data file.Table S6List of 20E-inducible genes identified by microarray analysis and the 14 bp consensus motifs.(PPT)Click here for additional data file.


Enhancer elements regulate the tissue- and developmental-stage-specific expression of genes. Recent estimates suggest that there are more than 50,000 enhancers in mammalian cells. At least a subset of enhancers has been shown to recruit RNA polymerase II transcription complexes and to generate enhancer transcripts. Here, we provide an overview of enhancer function and discuss how transcription of enhancers or enhancer-generated transcripts could contribute to the regulation of gene expression during development and differentiation. The. The3]. Transcription in eukaryotes takes place in the nucleus. Certain activities in the nucleus, e.g. rRNA synthesis, are compartmentalized. These compartments or domains are not engulfed by membranes, but rather represent dynamic centers of specialized function that are characterized by the constant influx and efflux of activities that constitute these domains. For example, there is evidence that many genes are transcribed in domains enriched for Pol II, referred to as transcription factories ,40,41. IThe nucleus is also structured with respect to the separation of inaccessible and accessible chromatin. Silent genes are located within inaccessible chromatin territories (CTs), while active genes are located at the surface of these territories ,44. ChroEnhancers activate gene expression at the level of transcription . Recent et al. [It appears that a distinct set of enhancers act by opening chromatin at promoters to increase accessibility for the transcription complex. This has recently been demonstrated for an enhancer element associated with embryonic epsilon globin gene expression in the murine β–globin gene locus . The mecet al.  demonstret al. . During Enhancers have been shown to directly recruit transcription complexes, which could be delivered to genes by looping mechanisms ,49,50,51There is also increasing evidence demonstrating that enhancer elements affect elongation of RNA Pol II by mediating the recruitment of transcription elongation factors like pTEFb or components of the FACT (facilitator of chromatin transcription) complex ,55,56. TThe above described examples illustrate that enhancers are quite diverse with respect to their roles in gene activation. For genes regulated by multiple enhancers, each enhancer could provide a unique activity, and the collective set of enhancers could therefore mediate optimal gene expression. Drosophila switched the element from being silent to being activated [The hitherto clear distinction between promoters and enhancers has become blurry due to recent evidence showing that enhancers are capable of recruiting transcription complexes and a recent demonstration that promoters can activate other promoters ,59. Tuanctivated . The conctivated ,66.Intronic enhancers have also been shown to recruit transcription complexes, which led investigators to propose the term "prohancer" for those enhancer elements that also exhibit promoter activity . A recenDespite earlier findings of enhancer mediated Pol II recruitment, it was a surprise when in 2010 the Greenberg laboratory showed that thousands of neuronal specificity enhancers recruited Pol II and initiated formation of enhancer RNAs (eRNAs) . The eRNin vivo they appeared to regulate specific target genes [Non-coding RNAs have been shown to associate with protein complexes and to directly regulate expression of genes . For exaet genes . HoweverThere are many studies showing that RNAs are involved in the formation of specific protein/ RNA complexes or nuclear domains . The ribet al. [Different classes of enhancers can be distinguished by their specific epigenetic signatures. Zentner et al.  reportedet al. ,59. Inteet al. .Enhancers are often in a poised configuration in progenitor or stem cells . In factet al. [Observations that enhancers are often primed in progenitor or stem cells suggest that these elements are the primary site of recruitment for activities involved in transcription activation, be it activities that open chromatin, activities that recruit transcription complexes to target genes or activities that stimulate transcription elongation. Moreover, a recent report demonstrated that an enhancer associated with the T-cell-specific CD4 gene is required for initiating activation of the gene locus in undifferentiated cells that do not express the target gene, but is no longer required in differentiated cells that express the CD4 gene . Likewiset al.  demonstret al. . Based oAs mentioned before, recent interaction maps reveal extensive enhancer/ promoter and promoter/ promoter interactions . RNAs geIt should be pointed out that the overall level of sequence conservation of non-coding RNA is 10-fold lower compared to mRNA . FurtherThe recent genome-wide analysis of histone PTMs and transcription factor binding revealed novel aspects of long-distance regulatory elements. Enhancers and gene proximal promoters share the ability to recruit transcription complexes and to regulate expression of genes over long distances. In hindsight, this may not be too surprising, as enhancers and promoters often harbor similar arrangements of transcription factor binding sites. However, in contrast to transcription at enhancers, it appears that promoter-mediated transcription is more efficient and leads to the production of stable RNAs. The reasons for this are currently not known, but may be due to the fact that promoters contain basal promoter elements that recruit Pol II transcription complexes with a high affinity. Furthermore, promoters generate transcripts that are conserved and contain sequences that mediate stability. It will be important to determine if transcription complex assembly at enhancers follows similar pathways as those operating at promoters. Other outstanding questions pertain to the role eRNAs may play during activation of transcription and if the process of transcription also contributes to the function of enhancers. The available data so far suggest that enhancers exhibit different activities. Therefore, it is possible that for those enhancers that are involved in modulating chromatin domains, the process of transcription is important, while for those enhancers that modulate the recruitment or activity of transcription complexes, the eRNA transcripts directly participate in the activation process.


Bacillus subtilis is an attractive host for production of recombinant proteins. Promoters and expression plasmid backbones have direct impacts on the efficiency of gene expression. To screen and isolate strong promoters, a promoter trap vector pShuttleF was developed in this study. Using the vector, approximately 1000 colonies containing likely promoters from Bacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09 exhibited the highest β-Gal activities in both Escherichia coli and B. subtilis. The activity of pShuttle-09 in B. subtilis was eight times of that of the P43 promoter, a commonly used strong promoter for B. subtilis. A sequence analysis showed that pShuttle-09 contained PluxS and truncated luxS in-frame fused with the reporter gene as well as another fragment upstream of PluxS containing a putative promoter. This putative promoter was a hybrid promoter and its β-Gal activity was higher than PluxS. Reconstructing the hybrid promoter from pShuttle-09 to PlapS further improved the β-Gal production by 60%. The usefulness of our promoter trap system is likely due to random shuffling and recombination of DNA fragments and adoption of a rapid and high-throughput screening. Thus, our data provide additional evidence to support the concept of using a promoter trap system to create new promoters. Bacillus subtilis is a non-pathogenic gram-positive bacterium and has been an attractive host for production of recombinant proteins from both prokaryotic and eukaryotic origins B. subtilis as one of main hosts for recombinant protein production is due to several reasons: generally recognized as safe as a result of its lack of pathogenicity and the absence of endotoxins; capable of secreting functional proteins directly into culture media, easy for genetic manipulation and handling, and capable of large-scale fermentation B. subtilis own proteins can be over expressed in B. subtilis in the order of several grams per liter B. subtilis is usually low B. subtilis remains a major challenge. To circumvent the problem, strong promoters, a variety of translation/secretion signals, and transcription terminators are continuously being explored. In particular, several strong promoters have been reported B. subtilis either from B. subtilis itself or from other bacillus species Promoters are important regulatory elements in a genome and control spatial and temporal expression of genes and their expression strength. In bacteria, a promoter is recognized by RNA polymerases and associated sigma factors, which may be recruited to the promoter by regulatory proteins binding to specific sites in the promoter. There are several ways to find new promoters. The advent of DNA sequencing has significantly accelerated sequencing of many bacterial genomes and prediction of promoters. However, it is not yet possible to scan a bacterial genome sequence and readily predict the expression behavior of genes belonging to a regulon, largely because an accurate prediction of promoters is difficult B. licheniformis genomic DNA for potential promoters, a strong double promoter was identified, containing a normal promoter and a hybrid promoter due to shuffling and recombination. The putative double promoter was further improved and used for protein expression both in E. coli and B. subtilis. Our results show that promoter trap systems could be suitable for finding novel strong promoters.A promoter trap system could perform shuffling and recombination of DNA fragments and screen of strong promoters simultaneously. However, this has never being reported. Fortuitously, when a promoter trap system was used in this study to screen Sau3A I cut fragments from B. subtilis 1A747 (B. subtilis PY79) was a gift from Bacillus Genetic Stock Centre of the Ohio State University. B. pumilus BYG, B. licheniformis A061, B. amyloliquefaciens and B. megaterium W-5 were isolated by our laboratory. The bacterial strains were isolated from effluent of a paper mill in Hubei province of China and partial 16S rDNA sequencing was used for identification of species in the genus Bacillus. The plasmids used in this study are listed in E. coli DH5α was purchased from Novagen . The bacterial strains were cultured in the Luria-Bertani (LB) medium at 37°C. The following concentrations of antibiotics were used for selection: 100 µg/mL ampicillin (Amp), 5 µg/mL chloramphenicol (Cm) and 50 µg/mL spectinomycin (Spec).E. coli and B. subtilis was performed by electroporation Polymerase chain reaction (PCR) primers and oligonucleotides used in this study are listed in bgaB-1 and bgaB-2, the bgaB coding region was PCR amplified from the plasmid pDL bgaB digested by BamH I and Sac I was cloned into corresponding sites of pGJ103, yielding pGJ-Bga. Then, the Apa I-BamH I-treated spectinomycin resistance gene, which was PCR amplified from the pDG1728 Bga, resulting in a promoter trap vector pShuttleF.Using the primer pair B. licheniformis was partially digested with Sau3A I, and then ligated with the promoter trap vector pShuttleF treated with the BamH I and alkaline phosphatase. The ligation mixture was transformed into E. coli DH5α and recombinants harbouring promoter fragments were screened on the LB solid medium supplemented with X-Gal (20 µg/mL).The genomic DNA of www.ebi.ac.uk); promoter region was predicted by the BPROM program .Promoters were sequenced by AuGCT Biotechnology . The sequence analysis was performed online with NCBI blast 2.0  was PCR amplified from pDL by using bgaB-fus and bgaB-2 and cloned into pshuttleF, resulting in pDET-1. Then, the hybrid promoter amplified from pShuttle-09 (using the primer pair insert-3/insert-2) was cloned into pDET-1 treated with Apa I and BamH I, resulting in pShuttle-Hyb. By using the primer pair lux-2/insert-2, an isolated promoter fragment (deletion of 5′ coding region of luxS) was amplified from pShuttle-09 and cloned into pShuttleF, yielding pShuttle-Fus1.Using the primer pair P43-1/P43-2, the P43 promoter was PCR amplified from plasmid pB43. The resultant promoter fragment was digested with Apa I and BamH I, and cloned into corresponding sites of pShuttleF, resulting in pShuttle-P43. By means of primer pairs luxS-up/luxS-down, luxP-up/luxP-down and luxM-up/luxM-down, three apr  from genomic DNA of B. subtilis, B. pumilus, B. licheniformis and B. amyloliquefaciens, respectively, yielding PaprS-1, PaprP-1, PaprL-1 and PaprA-1. Four pairs of primers  were used to amplify the PluxS and −10 region of the hybrid promoter from pShuttle-09. The upstream primers were designed about 15 bp sequence homogenous with 3′ end of PaprS-1, PaprP-1, PaprL-1 and PaprA-1, respectively, resulting in fragments luxF-1 to luxF-4. Other four pairs of primers  were employed to amplify promoters from PaprS-1, PaprP-1, PaprL-1 and PaprA-1respectively. The downstream primers were designed about 15 bp sequence homogenous with 5′end of −10 region of the hybrid promoter, resulting fragment PaprS-2, PaprP-2, PaprL-2 and PaprA-2. With the mixture pairs of PaprS-2/luxF-2, PaprP-2/luxF-3, PaprL-2/luxF-4 and PaprA-2/luxF-5 as templates, in which there were about 30 bp overlap in each mixture pair, four overlapping PCR amplifications were facilitated by means of apr-1/Lux-2, apr-3/Lux-2, apr-5/Lux-2 and apr-7/Lux-2, respectively. Recombined promoters PLapS, PLapP, PLapL and PLapA were digested with Apa I and BamH I and cloned into pShuttleF, respectively, resulting in pShuttle-PLapS, pShuttle-PLapP, pShuttle-PLapL and pShuttle-PLapA.The reconstruction of promoter was carried out by the splicing by overlapping extension PCR. Four pairs of primers, apr-1/apr-2, apr-3/apr-4, apr-5/apr-6 and apr-7/apr-8, were employed to PCR amplified −35 region of promoter PLapS flanked by Apa I and EcoR I was PCR amplified from pShuttle-PLapS. The resultant promoter was digested with Apa I and EcoR I and cloned into corresponding sites of pGJ103, yielding the expression vector pLus-Hyb. To introduce 6-His tag, synthetic oligonucleotides Tag-1 and Tag-2 were annealed. The annealed fragment with adhesive ends of Xba I and Sac I at 5′ and 3′ ends respectively was inserted into the Xba I-Sac I-treated pLus-Hyb, resulting in pLus-His.Using primers apr-1 and laps-down, the promoter PbgaB fragments were PCR amplified from pShuttleF, resulting in bgaF-1 flanked with EcoR I and Sac I and bgaF-2 with EcoR I and BamH I. bgaF-2 did not contain the termination codon. The bgaF-1 and bgaF-2 were cloned into pLus-Hyb and pLus-His, respectively, yielding pLu-bga and pLuHis-bga. Similarly, the bioI gene were PCR amplified from B. subtilis 1A747 genomic DNA by using primer pairs bioI-1/bioI-2 and bioI-1/bioI-3, resulting in bioIF-1 with EcoR I and BamH I and bioIF-2 with EcoR I and Xba I. The resultant fragments were inserted into pLus-Hyb and pLus-His, yielding pLu-bioI and pLuHis-bioI.Using primer pairs bgaB-3/bgaB-2 and bgaB-3/bgaB-T, two The β-Gal activity assay was carried out as previously described E. coli-B. subtilis shuttle vector pGJ103. The pShuttleF contained bgaB coding for a heat stable β-galactosidase  as a reporter gene. In addition, an alternative resistance selection marker, spectinomycin, was introduced into the pShuttleF beside the chloramphenicol resistance marker. Both spectinomycin and chloramphenicol resistance genes could confer the resistance selection either in E. coli or B. subtilis. Finally, an engineered BamH I restriction site was designed next to the start codon of bgaB in order to clone DNA fragments digested by the Sau3A I. Validation of the pShuttleF was achieved by inserting a commonly used strong promoter P43  in B. subtilis upstream of bgaB. The resultant pShuttle-P43 was transformed into E. coli DH5α and B. subtilis 1A747 and blue colonies on solid LB plate supplemented with X-gal were isolated. The bgaB directed by the P43 in pShuttleF was duly expressed in E. coli and B. subtilis . Taken together, these results suggested that pShuttle-09 contained a putative strong promoter from B. licheniformis chromosomal DNA and drove expression of the reporter gene both in E. coli and B. subtilis.In order to identify new promoters, Sau3A I-digested B. licheniformis genome sequence (NC_CP000002.3) with 96% of homology. According to the annotation of B. licheniformis genomic DNA sequences, the inserted sequence contained two fragments (ylyB gene (about 109 bp) and a 5′end partial sequences of luxS (about 200 bp).To characterize the putative promoter DNA element in pShuttle-09, the inserted fragment was enzyme cut and sequenced. The sequencing result showed that the inserted fragment was 305 bp long. Sequence alignment showed that the inserted sequence in pShuttle-09 belonged to ragments  correspoluxS gene was fused with bgaB and a typical SD was present 7 bp upstream of the start codon of the luxS gene. It contained typical −35 and −10 elements recognized by a Sigma Factor σA. Therefore, the promoter PluxS might be the control element of bgaB in pShuttle-09. However, further in silico prediction of promoters (luxS) was located in the 5′ non-translational region of luxS gene. Interestingly, the second putative promoter was located on two sides of the second Sau3A I-cutting site in pShuttle-09. The −35 element was located within the coding region of ylyB, while the −10 element was located upstream of the core promoter region of luxS.Sequence analyses  also shoromoters  indicateluxS) on the reporter gene expression, a PluxS-i vector was constructed by sub-cloning a 200 bp PluxS fragment into pShuttleF. For facilitating a rigorous comparison, promoters of luxS were also cloned from genomic DNA of B. subtilis, B. pumilus and B. megaterium. The β-Gal production driven by PluxS from B. subtilis, B. pumilus and B. megaterium were about 78%, 63% and 70% of that from B. licheniformis  in B. subtilis (To determine efficiencies of pLus-Hyb and pLus-His, bgaF-1 and bgaF-2 were cloned into pLus-Hyb and pLus-His, respectively, to facilitate β-Gal expression. The production of β-Gal from the pLu-bga reached 26037±1037 Miller U/mL , indicat pLu-bga  or purifuHis-bga  further subtilis . TherefoB. licheniformis through a promoter trap system. The double promoter contained PluxS and a hybrid promoter and can be used for protein expression in B. subtilis. These results support the notion that it is possible to shuffle, generate and select a strong prokaryotic promoter simultaneously, by applying a promoter trap system to randomly cut DNA fragments from a genome.The major finding from this study is that an artificial double promoter was obtained from bgaB rather than the bgaB gene with a SD as a reporter. So far, the promoter-less bgaB gene has been used as a reporter in several promoter trap systems bgaB gene with a SD. In our system, promoter fragments inserted into the upstream of bgaB could not drive expression of β-Gal unless the cloned promoter fragments contain SD. This design might limit the number of recombinant clones which can be screened. Nevertheless, approximately 1000 blue recombinant colonies were observed in this study. The strength of cloned promoters was conveniently determined via coloration caused by production of β-Gal when a recombinant harbouring a promoter fragment was cultured on solid LB supplemented with X-gal. Further quantitative determination of putative promoter strength was easily achieved by measuring the activity of β-Gal.The interest in promoters stems from myriad opportunities for controlling gene expression. Our promoter trap system was designed to increase the stringency of screening by employing the coding region of B. licheniformis genomic DNA was used as a potential promoter source in this study. Recombinant protein expression in B. subtilis can be driven by promoters from B. subtilis or from other bacillus species. To date, several strong exogenous promoters for the B. subtilis system have been isolated and used to construct expression vectors xyl and Pspac have been widely used as expression control elements in B. subtilisB. subtilis.ylyB gene and a −10 element from non-coding sequence of luxS gene. Our hybrid promoter is artificially created and quite different from reported hybrid promoters, which contain elements from two different promoters. It is well known that a hybrid promoter could be far more efficient than either one of the parental promoters tac promoter in order to express B. licheniformis aminopeptidase in B. subtilis and found that the activity of the hybrid promoter was increased approximately threefold in comparison with the tac promoter. Nevertheless, the study on hybrid promoters is limited, presumably due to lack of suitable ways to create hybrid promoters.Our results exemplified the potential of a promoter trap system to create a strong hybrid promoter. A hybrid promoter is defined in this study as a promoter consisting of different core elements from two different genes. One promoter identified in this study is a hybrid promoter assembled artificially from a −35 region from the coding sequence of luxS promoter in our study contributed to transcription of the reporter gene in an additive way. A double promoter in this study is defined as the combination of two promoters which control transcription of same gene. This definition is different from most overlapping promoters often seen in bacteria. Analyzing the database of the E. coli genome, Bendtsen B. subtilis. Kang B. subtilis NA64 or maltogenic amylase from B. licheniformis downstream of the Hpa II promoter elevated the TSaGT productivity by 11- and 12-fold, respectively, in comparison with the single Hpa II promoter system. Li phaCAB genes with the 5 copies of core-tac-promoters resulted in an engineered E. coli that can accumulate 23.7% polyhydroxybutyrate of the cell dry weight in batch cultivation. On the other hand, Wu The results from this study also demonstrated the capability of a promoter trap system to create a double promoter. Interestingly, both the hybrid promoter and PIn conclusion, our data support the concept of using a promoter trap system to discover new promoters. The usefulness of a promoter trap system is likely due to random shuffling and recombination of DNA fragments and adoption of a rapid and high-throughput screening. It is tempting to speculate that more novel promoters could be found if reaction conditions for cutting and ligation could be adjusted to increase chances for shuffling DNA fragments.


HLA-DRB1 is the most polymorphic MHC (major histocompatibility complex) class II gene in human, and plays a crucial role in the development and function of the immune system. Extensive polymorphisms exist in the promoter and 3′-UTR of HLA-DRB1, especially a LTR  element in the promoter, which may be involved in the expression regulation. However, it remains unknown how the polymorphisms in the whole promoter region and 3′-UTR to regulate the gene expression. In this study, we investigated the extensive polymorphisms in the HLA-DRB1 promoter and 3′-UTR, and how these polymorphisms affect the gene expression in both independent and jointly manners. It was observed that most of the haplotypes in the DRB1 promoter and 3′-UTR were clustered into 4 conserved lineages , and showed high linkage disequilibrium. Compared with H1 and H2 lineage, a LTR element in the promoter of H3 and H4 lineage significantly suppressed the promoter activity, whereas the activity of the linked 3′-UTR increased, leading to no apparent difference in the final expression product between H1/H2 and H3/H4 lineage. Nevertheless, compared with the plasmid with a promoter and 3′-UTR from the same lineage, the recombinant plasmid with a promoter from H2 and a 3′-UTR from H3 showed about double fold increased luciferase activity, Conversely, the recombinant plasmid with a promoter from H3 and a 3′-UTR from H2 resulted in about 2-fold decreased luciferase activity. These results indicate that the promoter and 3′-UTR of HLA-DRB1 may antagonistically regulate the gene expression, which may be subjected to stabilizing selection. These findings may provide a novel insight into the mechanisms of the diseases associated with HLA-DRB1 genes. Major histocompatibility complex (MHC) plays a crucial role in the regulation of immune response. Since the landmark discovery of MHC restricted T-cell recognition by Zinkernagel and Doherty DRB1, DRB3, DRB4 and DRB5), HLA-DRA is monomorphic, whereas HLA-DRB, especially DRB1, is highly polymorphic DRB3, DRB4 and DRB5) HLA-DR molecules are constitutively expressed on the cell surface of B lymphocytes, thymic cortical epithelial cells, dendritic cells (DCs), monocytes and other antigen presenting cells (APC), with the function of presenting antigens T-cells http://www.nlm.nih.ncbi.org) and assigned a GenBank accession number of FJ464430 to FJ464461. Phylogenetic analysis showed that the sequences from both promoter and 3′-UTR were clustered apparently into 4 deep clades, which were designated as H1, H2, H3 and H4 for promoter clades . Fu and Li's D* test statistic is 2.09095 ( P<0.02) and Fu and Li's F* test statistic is 2.73971 (P<0.02). In 3′-UTR, Tajima's D is 2.3799 with a statistical significance P<0.05, Fu and Li's D* and F* is 1.8129 and 2.4305, respectively, and both with a statistical significance . Fu and Li's D* test statistic is −3.26961 ( P<0.02) and Fu and Li's F* test statistic is −3.47765 (P<0.02). Tajima's D, Fu and Li's D* and F* test for UTH1 are −1.84699, −2.50331 and −2.65713, respectively (P<0.05)  . These r(P<0.05) . The aboAt least one SNP was observed in each of the cis-elements such as TATA box, CAAT box, Y box, X box and W box . We compP<0.05), suggesting the polymorphisms in the 5′-UTR could affect the promoter activity  (P<0.001) (P<0.05)  from each DRB1 lineage into pGL3.0 basic vector, and simultaneously constructed several recombinants by inserting the LTR into the promoter sequence from H2 lineage. Compared with the constructs of H3AIC (from H3 lineage) and H4AIC (from H4 lineage) which comprise a LTR element, the long promoter constructs H1AC (from H1 lineage) and H2AC (from H2 lineage) which contain no LTR element showed significantly increased expression activity (P<0.01) . FurtherP<0.001) . The abo(P<0.05) . Another(P<0.05) . These fIn order to find out whether the polymorphisms in 3′-UTR of HLA-DRB1 are involved in the regulation of gene expression, the luciferase activities with various 3′-UTR constructs were determined in 293T and Raji cells. A significant higher luciferase activity was observed for the 3′-UTR from DRB1 lineages of UTH3 and UTH4 than that from DRB1 lineages of UTH1, UTH2 and UTHx . SimilarIn previous studies, it was reported that polymorphisms in cis-elements located in the core promoter of HLA-DRB1 genes led to apparent expression difference It is very interesting that the impact of the polymorphisms in the 3′-UTR on the gene expression is just reverse to that in the promoter, suggesting antagonistic regulation between the promoter and 3′-UTR of HLA-DRB1 gene. This regulatory mechanism ensures the 4 distinct HLA-DRB1 lineages, although with different promoter activity, may have a similar final expression product. So we proposed that it may be very important to maintain an identical expression level for two distinct HLA-DRB1 alleles in an individual, and HLA-DRB1 gene was subjected to stabilizing selection in the evolutionary history.g7 gene product to I-Ak product in mixed MHC mice determined the difference in autoproliferative response, and the higher ratio would cause more autoreactive T-cells to evade negative selection into the periphery and readily elicit autoimmune response Stabilizing selection between the alleles from distinct DRB1 lineages suggested that the maintenance of equal expression of two distinct DRB1 alleles in an individual must have a very important biological significance. Although the expression of most genes in primate and model organisms was demonstrated to be subjected to stabilizing selection The promoter regions of MHC II genes were subjected to Balancing selection DRB1*10:01:01 which was recombined with the allele on the DR52 haplotype in intron 1 and has the most recent common ancestor with the alleles on DR51 and DR52 haplotype The nucleotide diversities between lineage 12 (lineage 1 and lineage 2) and lineage 34 (lineage 3 and lineage 4) in the 3′-UTR region is much higher than that in the promoter region, suggesting that the 3′-UTR and the promoter were subjected to different natural selection. The 3′-UTR from lineage 3 and 4 might be subjected to positive selection so as to evade the suppression to counteract the inhibition of its promoter activity. UTHx belongs to In conclusion, the expression of HLA-DRB1 may be antagonistically regulated by its coordinately evolved promoter and 3′-UTR under stabilizing selection. The polymorphic pattern in the DRB1 promoter and 3′-UTR was shaped by multiple natural selection, such as purifying selection, positive selection, stabilizing selection, and hitch-hiking by the polymorphic sites in the exons. The findings in this study implicated that balancing the expression of two different MHC II alleles in an individual may be very important, which provide a novel insight into the pathogenic mechanism of MHC involving in many diseases.Whole blood samples were collected from 26 Han Chinese volunteers from the different provinces. A written informed consent was obtained from each participant, and this study was conducted with the approval of the Institute Research Medical Ethics Committee of Sun Yat-Sen University. Genome DNA was extracted with Blood DNA kit (D3392-02) purchased from OMEGA Bio-tek, Inc., Guangzhou, China.DRB1*15:01:01, GenBank accession no. NG_002432), DR52 , and DR53 . Sequence alignment was performed with ClustalX 1.81, and all primers were designed with primer premier 5.0. The primer pairs and their relative positions were shown in We downloaded 3 HLA-DRB1 reference sequences which located at different DR haplotypes, namely DR51  for 30 seconds and extension at 68°C for y (the “y” means the extension time for amplifying different length of fragments) minutes, then elongating at 68°C for 10 minutes. The PCR products were cleaned with Gel extraction Kit , then sequenced directly and finally cloned into T-easy vector .To investigate linkage relationship among promoter, exon 2, and 3′-UTR, we designed 2 universal primer pairs (and) to amplify the fragment containing promoter region and exon 2, as well as the fragment containing 3′-UTR and exon 2 . Then thThe allele specific PCR product was purified by gel extraction with DNA Gel Extraction Kit  followed by sequencing with haplotype specific primers  on an ABHind III and Nco I site, so they do not contain the 5′-UTR of luciferase reporter gene from the vector itself  element is about 1.1 kb, which was inserted into the promoter region of H3 and H4 lineages upstream at the position −938 to −2065. Here, we defined the position of LTR elements as indel site, and use an “A” represent the distal promoter that located upstream of the indel site, an “I” indicates the LTR elements, a “C” denotes the proximal promoter that located between translation start site and indel site, and H1-4 represent the 4 distinct promoter lineage. To investigate whether the LTR element influences on the transcription activity of the promoter, we constructed 4 constitutive promoter vectors designated as H1AC, H2AC, H3AIC and H4AIC, respectively. Simultaneously, we constructed four recombinant plasmids namely H2AIC, IH2C, H3AC and IH3C, respectively (shown on the left of Xba I and BamH I site. We successfully constructed four different kinds of vectors and confirmed its fidelity by sequencing. 1) The vector in which the 3′-UTR and 5′-UTR of the luciferase gene were simultaneously substituted for that from different DRB1 lineages (We amplified the fragment containing the complete 3′-UTR and about 300 bp long downstream sequence from the poly(A) signal site from each 3′-NCR lineage of HLA-DRB1, which is about 586–594 bp long. We denoted the fragments of 3′-UTR from UTH1, UTH2, UTH3, UTH4 and UTHx as 3′U1, 3′U2, 3′U3, 3′U4 and 3′Ux, respectively, and constructed them into pGL3.0 Basic vector or pGL3.0 control vector between lineages . 2) The lineages . 3) The lineages .All plasmids for transfection were extracted with Qiagen Midi Kit-25  according to manufacturer's instruction. The concentration of each construct was measured with Spectrophotometer ND-1000. Burkitt's lymphoma B cell line Raji was propagated with RPMI 1640 culture medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. All promoter constructs and an internal control (pRL-TK vector) were co-transfected into Raji cells by electroporation, and into 293T cells with lipofactamine2000 (Invitrogen). The electroporation was performed as following: the mix of 200–400 µl cells and plasmids were transferred into a 4 mm cuvette pre-cooled on ice and discharged for 25 ms at 160 V on a Genepulser Xcell (Bio-Rad). Then, the cells were transferred to wells containing 2 ml RPMI 1640 complete culture medium and incubated at 37°C in a CO2 incubator with moist ambient air for 36–48 hours. The procedure of transfection with lipofactamine2000 was according to the manufacturer's instruction.Transfected cells were harvested and schizolysed with passive lysis buffer attached with the Dual-Luciferase reporter assay system (Promega). Then, the enzyme activity of the reporter gene including the internal control was assayed with the Dual-Luciferase reporter assay system on a chemoluminescence meter . The fluorescence intensity representing promoter activity of each lineage was normalized by internal control and comparison was performed among all lineages.Sequence assembly was performed manually with BioEdit version 7.0 and the sequence alignment was carried out with clustalX 1.81. The polymorphism level, π (the average number of pairwise nucleotide differences per site) and θw (the number of segregating sites per nucleotide), was calculated with DnaSP version 4.50.3 P<0.05.Data for analysis of promoter activity were expressed as the mean ± standard error of mean (s.e.m.) from at least 3 separate experiments. Unless otherwise noted, the differences between groups were analyzed using Student t test when only 2 groups, or assessed by one-way ANOVA when more than 2 groups were compared. All tests performed were two-sided. Differences were considered statistically significant at Figure S1Phylogenetic Analysis for the 3′-UTR and 3′-UTR Downstream Sequences (3′-UTR-DS). Taxon names that designated with UTH plus numbers represent haplotypes containing 3′-UTR or 3′-UTR downstream sequences. UTHx signifies that the haplotype is distinct from the others. The arrow indicated the different position of the UTHx haplotype on the 3′-UTR tree and 3′-UTR-DS tree. The chimpanzee and rhesus sequences were used as reference sequences. The haplotypes belonging to the same lineage were denoted with opposite bracket.(JPG)Click here for additional data file.Figure S2Slide Window Analysis for Nucleotide Polymorphisms and Frequency Spectrum Analysis for the Promoter Sequences of HLA-DRB1. A mutation of size i means that is occurs i times in our sample of 52 sequences. The thin line represents the expected frequency spectrum in neutral equilibrium, and the black bars denote the observed values.(JPG)Click here for additional data file.Figure S3Polymorphism in the Core Promoter Region of Partial DRB Alleles from human, chimpanzee, rhesus and marmoset. The numbering is relative to the transcription start site. The cis-element box was encircled with a pane and the core sequence in a cis-element box was marked with underline.(JPG)Click here for additional data file.Figure S4The Sketch of the Relative Position of Primers at HLA-DRB1 Gene. The number – represents the primers at certain position, for example, represents the 5 primers designated as H1ASP1R, H21ASP1R, H22ASP1R, H3ASP1R and H4ASP1R which are specific for different sequence lineage. The distance marked in the figure denotes the approximate size of PCR product which varying for different lineage.(JPG)Click here for additional data file.Figure S5The Sketch Map of Construction of HLA-DRB1 Promoter Vectors. Only the reporter gene and its 5′ flanking region in which the multiple clonal sites are located, were shown on the sketch. The numbers in the parenthesis following the names of restriction endonuclease (REN) denote the relative positions of the RENs at the vector sequence. The rectangles below multiclonal sites represent the fragments of promoters or 5′UTRs from HLA-DRB1 genes, which have been inserted into the REN sites denoted by triangles. Three different kinds of promoter vectors were constructed and shown on A, B and C, respectively.(JPG)Click here for additional data file.Figure S6The Sketch Map of Construction of HLA-DRB1 3′UTR and recombinant Vectors. Only the reporter gene and its 5′ flanking region in which the multiple clonal sites are located were shown on the sketch. The numbers in the parenthesis following the names of restriction endonuclease (REN) denote the relative positions of the RENs at the vector sequence. The rectangles below multiclonal sites represent the fragments of promoters or 3′UTRs from HLA-DRB1 genes, which have been inserted into the REN sites denoted by triangles. Three different kinds of vectors were constructed and shown on A, B and C, respectively.(JPG)Click here for additional data file.Table S1Neutral test in DRB1 promoter region and 3′ non-coding region with our data.(XLS)Click here for additional data file.Table S2All primer sequences involved in this paper.(XLS)Click here for additional data file.


Core promoters with adjacent regions of the human genes CDC6, POLD1, CKS1B,MCM2, and PLK1 were cloned into a pGL3 vector in front of the Photinus pyrailsgene Luc in order to study the tumor specificity of the promoters. The clonedpromoters were compared in their ability to direct luciferase expression indifferent human cancer cells and in normal fibroblasts. The cancer-specificpromoter BIRC5 and non-specific CMV immediately early gene promoter were usedfor comparison. All cloned promoters were shown to be substantially more activein cancer cells than in fibroblasts, while the PLK1 promoter was the mostcancer-specific and promising one. The specificity of the promoters to cancercells descended in the series PLK1, CKS1B, POLD1, MCM2, and CDC6. Thebidirectional activity of the cloned CKS1B promoter was demonstrated. Itapparently directs the expression of the SHC1 gene, which is located in a“head-to-head” position to the CKS1B gene in the human genome. This featureshould be taken into account in future use of the CKS1B promoter. The clonedpromoters may be used in artificial genetic constructions for cancer genetherapy. The design of genetically engineered vectors that express products that aretoxic for tumor cells holds an important position among the topical directionsin the development of antitumor agents. These vectors need to containcancer-specific regulatory elements that can ensure both the expression of thetherapeutical gene in the maximum possible number of tumors and the absence ofexpression in normal tissues. Today, the number of promoters known to havethese properties is limited.CDC6, POLD1, CKS1B, MCM2,and PLK1 genes were used for cloning.While searching for new cancer-specific promoters, we have put forward ahypothesis that many promoters participating in DNA replication may exhibittumor specificity, since the disturbed regulation of cell division isconsidered to be the common property of all tumors. In order to verify thishypothesis, we cloned the promoters of several genes participating in DNAsynthesis and cell division and assessed the ability of these promoters todirect the expression of the reporter gene in normal and tumor cells ofdifferent origins. Promoters of the CDC6 gene product is the homologue ofSaccharomyces cerevisiae CDC6, a protein essential for theinitiation of DNA replication. CDC6 regulates the early stages of DNAreplication and helps control the check-point determining the termination ofDNA replication before mitosis begins. A disturbed regulation of CDC6expression is associated with a high risk of cancer development . The POLD1 gene encodes the catalytic subunit of DNApolymerase δ, which participates in the replication and reparation of humangenomic DNA. This subunit exhibits polymerase (synthesis of DNA) andexonuclease (in the 3’–>5’ direction) activities. Moreover, POLD1participates in the completion of the Okazaki fragments initiated by the DNApolymerase α/primase complex. The frequency of the development of spontaneoustumors is higher in mice with a deficient DNA polymerase δ function [3]. TheCKS1B protein is a component of CDC28 protein kinase required for embryogenesisand correct alternation of the phases of the somatic cell cycle [4]. CKS1Bforms a complex with the CDC2 protein and regulates the transcription of theCDC20 gene. The interaction between CKS1В and the SKP2–cyclinE-p27KIP complex ensures ubiquitination and degradation of p27 which is thecell blocker in the G0/G1 phase in response to different signals andunfavorable factors and the regulator of cell mobility and apoptosis [5]. TheCKS1B gene localizes head-to-head with the SHC1gene and presumably uses the bidirectional promoter shared with thisgene [6]. SHC1 gene products are known to regulate thetransfer of mitogenic signals in the cell, to participate in p53-dependentapoptosis under oxidative stress, and to regulat the lifespan. The proteinр66Shc plays an important role in carcinogenesis and tumor dissemination [7].The MCM2 gene encodes one of the subunits of the MCM2-7protein complex, which is required for the initiation of DNA replication,formation of the replicative fork, and recruitment of the other proteins thatparticipate in DNA replication. By interacting with the other proteins of theinitiation complex, MCM2 regulates its helicase activity [8]. The promoters ofthe SHC1 and MCM2 genes are not characterizedyet in detail. PLK1 (polo-like kinase 1) – serine/threonine protein kinase 1 –has several crucial functions during the M-phase of the cell cycle, includingcentrosome maturation, mitotic spindle assembly, and regulation of mitotic exitand cytokinesis. The PLK1 protein is required for cell restoration after DNAdamage and when it enters mitosis [9]. The listed properties of the six genesand their increased expression in a number of human tumors  provide grounds for hoping that the selected promoters could exhibit bothtumor specificity and versatility with respect to tumors and would be able toact as regulator elements within genetically engineered anti-tumor constructs.The Table. All the primers were synthesized onan ABI 3900 synthesizer (Applied Biosystems). The amplified DNA fragments werecloned into the vector in pAL-TA  and re-cloned into the pGL3Basic Vector  at the proper restriction sites in front of thePhotinus pyralis luciferase gene. Plasmid clones containingpromoters in the required orientation were selected by restriction analysis andDNA sequencing. The resulting clones with promoters of the CDC6, CKS1B,and PLK1 genes contained no nucleotide substitutions,while the clones with the promoters POLD1 and MCM2contained one and two substitutions with respect to the nucleotidesequences, respectively, which are listed in NC BI GenBank. We used the plasmidclones containing the cloned promoters to transfect the following cell lines:A375 , A431 , A549 , Calu1 ,HepG2 , HT1080 , Panc-1, and normal fibroblasts IVL-7C.Fibroblasts IVL-7C were obtained from the morphologically normal tissue of thelung of a patient who had undergone surgical resection of his lung cancer atthe Blokhin Cancer Center, Russian Academy of Medical Sciences, using thepreviously described procedure . Hence, when cloning the CKS1B promoter, weadditionally selected the pGL3 plasmid clone containing this promoter inreverse orientation. The coordinates of the cloned DNA fragment with respect tothe TSS of the SHC1 gene were . In order to verifythe promoter activity of the cloned fragment, we used this clone to transfectA549 cells and normal fibroblasts and measured the chemiluminescence of thecell lysates. The results are shown in Fig. 2. The results ofdetermining the activity of the incorporated CDC6 promoterwith the reverse orientation (CDC6-rev), which was identicalto that of the control vector pGL3-BV, and the BIRC5 promoterin direct orientation are given here for the sake of comparison. It turned outthat the activity of the cloned SHC1 promoter was higher thanthat of the CKS1B promoter both in the tumor cells and innormal fibroblasts. The activity of the SHC1 promoter in A549adenocarcinoma cells was approximately eightfold higher than that in normalfibroblasts, which is lower than the activity of the CKS1Bpromoter but is comparable to the cancer specificity of theBIRC5 promoter (not shown). Since the range of tumors with anincreased expression level of the SHC1 gene is smaller thanthat of the tumors with an increased expression level of the other genes thatwere used in our work , we did not study the SHC1promoter using other cells.As mentioned previously, the PLK1 promoter, was comparable to the indices for theBIRC5 promoter that had been studied previously. Thespecificity of the promoters slightly decreased for the series CKS1B,POLD1, MCM2, CDC6. The PLK1 promoter exhibitedconsiderably higher cancer specificity; the expressionlevels ofthe reporter gene controlled by the PLK1 andBIRC5promoters in the tumor cells were approximately identical. This makesthe PLK1 promoter superior toother promoters andprovides grounds to regard it asthe most promising promoter fordesigning geneticallyengineered anti-tumor constructs.Thus, the promoters of five genes that regulate DNA replication and celldivision, exhibiting tumor-specific expression, and significantly contribute tocarcinogenesis had been cloned. When cells were transfected with plasmidvectors expressing the luciferase gene under the control of these promoters,the promoters exhibited a considerably higher activity in tumor cells ofdifferent origins as compared to their activity in normal fibroblasts. Theactivity and tumor specificity of the cloned promoters, except for theCKS1B/SHC1 promoter. One should takethese features into account when designing genetically engineered vectors withthis promoter, since its bidirectional activity may result in undesirabletranscription of the vector sequences in a direction reverse to that of thetherapeutical gene. On the other hand, this promoter can be used tosimultaneously express two therapeutical genes in tumors or to designanti-tumor constructs that have binary effect with a more complex regulation.Further investigation into the CKS1B/SHC1 promoter and thegenes whose expression it directs in various tissues and tumors is required.We have also demonstrated the bidirectional activity and high cancerspecificity of the cloned in vivo, one needs to study the cloned promoters in invivo models in order to draw unambiguous conclusions about the cancerspecificity of the promoters.It should be mentioned that only non-tumor control was used in this study. Taking into account the source of the cells  and the fact that theproperties of cells dividing in culture may differ from their propertiesThe significant length of the promoters (1016–2373 bp) allows one to putforward a hypothesis that they contain key elements in transcriptionregulation, such as core promoters and proximal regulatory elements. However,it is entirely possible that there are additional remote regulatory elements,such as enhancers, silencers or repressors, which also participate in theregulation of the activity of these promoters. A comparison of the endogenousactivity of the corresponding genes in various cell lines and tissues with theresults obtained in our study will allow one to assess the relativecontribution of the promoter and additional regulatory elements.


Eukaryotic translation initiation factor 4G (eIF4G) is thought to influence the translational efficiencies of cellular mRNAs by its roles in forming an eIF4F-mRNA-PABP mRNP that is competent for attachment of the 43S preinitiation complex, and in scanning through structured 5' UTR sequences. We have tested this hypothesis by determining the effects of genetically depleting eIF4G from yeast cells on global translational efficiencies (TEs), using gene expression microarrays to measure the abundance of mRNA in polysomes relative to total mRNA for ~5900 genes.Although depletion of eIF4G is lethal and reduces protein synthesis by ~75%, it had small effects (less than a factor of 1.5) on the relative TE of most genes. Within these limits, however, depleting eIF4G narrowed the range of translational efficiencies genome-wide, with mRNAs of better than average TE being translated relatively worse, and mRNAs with lower than average TE being translated relatively better. Surprisingly, the fraction of mRNAs most dependent on eIF4G display an average 5' UTR length at or below the mean for all yeast genes.7 G cap- and poly(A)-binding factors (eIF4E and PABP), is not essential for translation of most  mRNAs but enhances the differentiation of translational efficiencies genome-wide.This finding suggests that eIF4G is more critical for ribosome attachment to mRNAs than for scanning long, structured 5' UTRs. Our results also indicate that eIF4G, and the closed-loop mRNP it assembles with the m Attachment of the 43S complex at the mRNA 5' end is stimulated by the eIF4F complex, comprised of cap-binding protein eIF4E, the scaffold subunit eIF4G, and the DExD/H-box helicase eIF4A, which is thought to provide a single-stranded region in the mRNA for recruiting the ribosome. Binding sites in eIF4G for either eIF3  or eIF5 and eIF1 (in yeast) are thought to facilitate recruitment of the 43S PIC to eIF4F bound at the cap structure. eIF4G also harbors a binding site for the poly(A)-binding protein (PABP) that, together with an RNA binding domain in the middle region of mammalian eIF4G, increases the stability of eIF4F binding to the mRNA 5' end and also mediates circularization of mRNA in the activated eIF4F·mRNA·PABP mRNP -Met incorporation under these conditions  by a factor > 5 compared to the P/M ratio for the WT strain under the same conditions Figure . This iss Figure . Thus, iAlthough a significant level of translation continues following the extensive depletion of eIF4G in the degron mutant Figure , it was RPL41A and RPL41B mRNAs were examined because their coding sequences, of only 78 nt , and broken by vortexing with glass beads. Polysomes were separated by loading whole cell extracts (WCEs) onto 4.5-45% sucrose gradients and centrifuged in a SW41Ti rotor (Beckman) at 39,000 rpm for 2.5 h at 4°C as described previously  4G/2-[ΔΔCt(HP-T)] WT)(0.114/0.308), where 0.114 and 0.308 are the fractions of 18S rRNA present in heavy polysomes in the mutant and WT cells, respectively, ΔΔCt(HP-T) = ΔCt(HP)norm -ΔCt(T)norm, ΔCt(HP)norm = Ct(HPGOI)-Ct(HP18S), and ΔCt(T)norm = Ct(TGOI)-Ct(T18S). The primers employed for qRT-PCR analysis of the mRNAs for these genes are listed in Table S3  and total RNA isolated from WT and eIF4G-depleted cells. Spreadsheet 1 "microarray of 5869 ORFs" contains the data from microarray analysis of 5869 ORFs using HP and total RNA samples from WT and eIF4G mutant strains, from projects  I-III. Columns A-C contain the Nimblegen sequence identification number, SGD ORF name, and functional annotation, respectively. Columns D-F contain the log2 of signal intensity for the HP RNA sample from the eIF4G mutant in project I (log4G-HP_I), the cognate value for total RNA (log4G-T_I), and the corresponding TE value (TE 4G_I) calculated as the ratio of these two intensities (HP/T), respectively. Columns G-I contain the corresponding data found in D-F for the HP and T RNA samples but from the WT strain in project I. Columns J-L and M-O contain the corresponding data found in D-F and G-I but from project II, and columns P-R and S-U contain the cognate data from project III. Columns V and W contain the mean TE4G and mean TEWT values, respectively, obtained by averaging the TE values from projects I-III. The log2 of the values in columns V and W were plotted in Figure 4G to TEWT values from projects I, II, and III, respectively, contained in columns F and I, L and O, and R and U, respectively. Column AA contains the average of columns X-Z, AB contains the S.E.M. calculated from the data in X-Z, and column AC contains the results of a two-tailed Students t-test conducted to determine if the set of TE4G values in columns F, L, and R are significantly different from the set of TEWT values in columns I, O, and U. Spreadsheet 2 "t-test calcs" contains these last calculations. Spreadsheet 3 "" contains the 94 genes with mean TE4G/TEWT values (in column AA) ≤ 0.71, and also includes in columns AD-AI the lengths and MFEs of 5' UTRs, and numbers of uORFs, all from [4G/TEWT values (in column AA) ≥ 1.4, with the identical column definitions described above for sheet 3. Spreadsheet 5 "Lawless UTRs" contains the data from Additional file 9 of Lawless et al [all from , and 3' all from . Spreadsss et al . Spreadsss et al .Click here for fileComparison of translational efficiencies between WT and eIF4G degron mutant. -Figure S1: Comparison of the ratios of TE values between the WT and eIF4G degron mutant determined by qRT-PCR and microarray analysis of selected mRNAs. -Figure S2: Comparison of translational efficiencies for all genes with ORF lengths < 625 nt between WT and eIF4G degron mutant cells. -Figure S3: Inverse correlation between translational efficiency and coding sequence length is dampened by eIF4G depletion, especially for genes with short ORFs. -Table S1: Variances from MA plots of microarray data. -Table S2: PCR Primers for measuring polysome distributions of selected mRNAs. -Table S3: Primers for qRT-PCR determination of TE4G/TEWT ratios and comparison of mean TE4G/TEWT ratios determined by microarray versus qRT-PCR analysis.Click here for fileExcel file containing results of microarray analysis of translational efficiencies using RNA from "light polysomes" (LP) and total RNA isolated from WT and eIF4G-depleted cells. Spreadsheet 1 "Project averages LP over T_4G,WT" contains our data from microarray analysis of 5868 ORFs using LP and total RNA samples from WT and eIF4G mutant strains, from all three projects (I-III). Columns A-B contain the Nimblegen sequence identification number and SGD ORF name, respectively. Columns C-F contain the mean signal intensities, averaged over all three technical replicates and all three projects  for the LP and total RNA samples from the eIF4G mutant (C-D) and from WT (E-F), respectively. Columns G and H contain the mean TE4G and mean TEWT values, respectively, calculated as the ratio of column C to column D (column G), or the the ratio of column E to column F (column H). Column I is the ratio of column G to column H. Spreadsheet 2 "Arava et al, 1-3 ribos per mRNA" contains the list of 564 genes whose polysomal mRNAs contain predominately 1-3 ribosomes, from Arava et al [va et al . SpreadsClick here for file


MicroRNAs are small noncoding RNAs that regulate genes post-transciptionally by binding and degrading target eukaryotic mRNAs. We use a quantitative model to study gene regulation by inhibitory microRNAs and compare it to gene regulation by prokaryotic small non-coding RNAs (sRNAs). Our model uses a combination of analytic techniques as well as computational simulations to calculate the mean-expression and noise profiles of genes regulated by both microRNAs and sRNAs. We find that despite very different molecular machinery and modes of action , the mean expression levels and noise profiles of microRNA-regulated genes are almost identical to genes regulated by prokaryotic sRNAs. This behavior is extremely robust and persists across a wide range of biologically relevant parameters. We extend our model to study crosstalk between multiple mRNAs that are regulated by a single microRNA and show that noise is a sensitive measure of microRNA-mediated interaction between mRNAs. We conclude by discussing possible experimental strategies for uncovering the microRNA-mRNA interactions and testing the competing endogenous RNA (ceRNA) hypothesis. MicroRNAs are short sequences of RNA . Furthermore, we confine ourselves to the biologically-relevant parameter sets where the mRNA-microRNA association rate, Note that to derive ntration . Such beThe factor et al.Comparing decay patterns of mRNAs repressed by microRNAs to free mRNAs provides a method to determine the existence and efficiency of microRNA binding. For example, Braun Gene regulation is inherently stochastic due to the small number of molecules present in the cell. Noise has important phenotypic consequences for a variety of biological phenomena To better understand the effect of catalytic interaction on noise, we calculated protein noise versus As a final test of the robustness of system on parameters, we studied protein mean and noise for a range of parameters using LNA. Experimental evidence suggests that mRNAs are often produced in bursts where the probability that a gene is in the on state is denoted by In the analysis that follows, we assume that gime see . Here noet al.However note that it is possible to design a very slow gene with the same mean value as here, by choosing small on and off rates, To gain further insight, we have derived asymptotic formulas for the noise in the repressed and expressing regime. In the expressing regime with large mRNA transcription, we define the small parameter with Materials and Methods) and it is assumed that mRNAs degrade much faster than proteins . When Materials and Methods). Thus, we conclude that in the repressed regime, non-coding RNAs reduce noise. In particular, proteins are now produced from mRNAs in a smaller burst with typical size given by where Materials and Methods). Our analysis show that the width of noise peak at the crossover regime is independent of recycling ratio aks down . The colThis scaling of the mean protein number can be better understood if we define where ch other . It is aRecently, the competing endogenous RNA (ceRNA) hypothesis proposed that microRNAs may play a crucial role in the cell in global gene regulation by inducing interactions between mRNA species A schematic of our simplified model is shown in withand variances reflecting the Poisson nature of interactions:Materials and Methods). We also examined the case where the mRNAs have different binding affinities for the microRNA, The binding of microRNAs to mRNAs is controlled by the interaction rates ults see Materials and Methods ). This region splits the transcription rate space As in the single-mRNA species case, the behavior of the system can be divided into regimes depending on whether the combined normalized transcription rate of both mRNA species is bigger or smaller than the sRNA transcription rates. We find the crossover regime to happen at r regime . Since tAs an alternative experimental strategy for testing the ceRNA hypothesis, we studied mRNA decay in the context of ceRNA hypothesis, by tracking the mean mRNA number as a function of time before it reaches steady state. In this work, we studied gene regulation by inhibitory non-coding RNAs. Whereas gene regulation by prokaryotic sRNAs has been extensively studied et al. et al. did not normalize the sRNA transcription rate Our results for the mean expression profile is consistent with recent experimental studies One of the striking results of our calculation is the similarity between sRNA-regulated and microRNA-regulated genes. This similarity is somewhat surprising given that microRNAs and sRNAs are found in different kingdoms (prokaryotes versus eukaryotes) and utilize distinct biochemistry and molecular machinery. Eukaryotic microRNAs use complicated nuclear machinery to export microRNA into cytoplasm and bring it to mature state by incorporating the RNA strand into the RISC complex. In contrast, prokaryotic sRNAs undergo relatively little post-processing and bind mRNAs spontaneously or via the chaperone protein Hfq. Given these extensive differences, the similarity between the expression characteristics of microRNA-regulated and sRNA regulated genes are suggestive of convergent evolution.Our calculations show that mRNAs regulated by catalytic non-coding RNAs have large peaks in the intrinsic noise. This differs significantly from results that would be derived from more traditional treatments of catalytic interactions based on the Michaelis-Menten or Hill equations. The underlying reason for this difference is that traditionally, the Michaelis-Menten equations are derived under the assumption that substrates of enzymes are in excess compared to the enzymes themselves. In contrast, here we are interested in the case where the number of enzymes (microRNAs) and number of substrates (mRNAs) are comparable. This accounts for the sharp peak in noise observed in the crossover regime in our model that is absent in traditional treatments of enzyme kinetics.Our calculations also suggest a strategy for testing the ceRNA hypothesis Finally we would like to note that the results discussed in this paper are based on a simplified version of the actual biochemical interactions with the goal of incorporating different mechanisms of gene regulation by noncoding RNAs into a simple mathematical framework. Adding further levels of complexity to the model can result in more realistic results, however the use of simple models can still provide insights into qualitative behavior of the system. To determine the extent to which our results hold under more realistic biological assumptions further theoretical and experimental work is necessary. The microRNAs discussed in this paper correspond to the RNA interference (RNAi) process and repress genes by binding to mRNAs. However, more recently, a small subset of microRNA processes, known as RNA activation (RNAa), has been discovered that can activate genes by binding to mRNAs Finally, we note that prokaryotic and eukaryotic cells are likely to be described by very different parameter sets within our model. In this paper we largely focused on changing the recycling ratio We can convert between concentration and particle number by multiplying by a volume, Linearization of where To find the solution of this equation we apply a Fourier transform and solve the resulting equation:where tilde denotes Fourier transform. So we have:where orSince We can rewrite Q as:withso we can write:where As noted in the main text and In the expressing regime with large mRNA transcription rate we demand To find the eigenvalues of the transfer matrix the eigenvalues of this matrix satisfy the following equation:wherethis equation can be analytically solved by expanding with one fast mode and two slow modes. Next we will calculate the noise by expanding plugging this result into Now make explicit substitutions for The terms on the first line above are order where in the last line it is assumed that in the expressing regime In this regime with mRNA transcription very small, we demand Next we expand the transfer matrix after some calculation we find the following closed form for eigenvalues of wherethis equation can be analytically solved by expanding which has one fast mode and two slow modes. Next we calculate the noise by expanding plugging this result into in the main text we have used the shorthand for the second term on the right hand side such that For two mRNAs, we linearized withand:where andWe find corresponding two point correlation functions by use of the following equation where we saw a good agreement between these results and Gillespie simulations. The two methods showed at most a deviation of 30% from each other (see For the steady state values of concentrations we have:which results inAfter some calculations we derive the following polynomial for sRNA concentration, withFurthermore, for ease of notation, in what follows we will use the following definitions:In this regime with with the only positive solution being In this regime which has the following asymptotic solutions:with This results in Crossover regime is roughly where the asymptotic solution of mean noncoding RNA in the repressed regime,