CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter  contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine  CYP3A genes: the xenosensors pregnane X-receptor , the constitutive androstane receptor , the hepatocyte nuclear factor-4 (HNF-4), HNF-3, the vitamin D receptor , and the CCAAT/enhancer binding protein-α (C/EBPα) thiazole-5-carbaldehyde O-oxime (CITCO) and 5--3-phenyl-4,5-dihydroisoxazole (FL81) have been documented [The established activators of human and mouse CAR and PXR have been reviewed –31. DEX,cumented –34. OligCYP3A28 promoter is described in detail in The cloning of ~10 kbp Putative binding sites for CAR and PXR heterodimers with RXR (DR3-5 and ER6-9 motifs) were identified with NUBIscan and NHR scan algorithms ,36. MatICYP3A28 promoter and containing the putative NR and transcription factor (TF) binding sites, were amplified using TaKaRa LA Taq Hot Start DNA polymerase  and specific primers  and full-length PXR (XREM-3A4-luc) have been previously described ,39 and uIn brief, site-directed mutagenesis of the ER6 and HNF-4 (DR1) sites was performed using mutagenic primers , amplifihCAR and hPXR expression [2) and cultured overnight to have 50–70% confluence. HepG2 cells were transfected using PEI25 and a reverse transfection method [CYP3A28 reporter constructs, or with positive control reporters hPBREM-tk-luc or hCYP3A4-XREM-luc, or with negative control reporter pGL4.10-luc (500 ng/well) and expression vector pCI-neo, bCAR or bPXR (100 ng/well). C3A cells were transfected using the calcium phosphate precipitation method [CYP3A28 reporter constructs or positive/negative control reporters (50 ng/well) and expression vectors (25 ng/well) as with HepG2. DMEM complemented with HyClone 5% delipidated serum  was used for the treatment with the PXR agonist SR12813 (10 μM) or its vehicle . After 24 hours, cells were lysed and assayed for luciferase and β-galactosidase activities with the VICTOR2 multiplate reader  as described before [HepG2 (ATCC HB-8065) and C3A (ATCC CRL-10741) cells, characterized by very low constitutive pression ,41, weren method , with con method  with cond before .CYP3A28 expression. BFH12 cells (passages 16–21) were cultured in Williams’ E medium containing 5% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, 2 mM L-alanyl-L-glutamine, 100 nM DEX and 0.2 U/mL insulin, at 37°C and 5% CO2 in a humidified atmosphere as described [4 cells/well). At day 4, the medium was changed with complete medium but without DEX. At day 7, stock solutions of PXR ligands, solubilized in DMSO and diluted in culture medium, were added into the wells. Cells treated with 0.1% DMSO  were used as control.The novel bovine SV40 large T-antigen-transduced fetal hepatocyte-derived cell line BFH12  was usedPreliminary induction studies were performed using DEX, PCN, RIF, RU486 and SR12813 at a fixed concentration (10 μM), according to , and difRXRα, the gene-specific primers forward (F): 5’-GCGTACTGCAAACACAAGTACC- 3’ and reverse (R): 5’-AGGCACTTGAGGCCAATG-3’ were selected, while for CYP2B22, CYP3A28, CAR, PXR and RPLP0 , previously published bovine primers were used [CAR assay, 300 nM/300 nM for CYP2B22, CYP3A28, RXRα and RPLP0, 600 nM/600 nM for PXR. For each qPCR assay, negative controls  were run. PCR amplification was performed in duplicate. The ΔΔCt method [Total RNA was extracted with the RNeasy Mini kit (Qiagen) following manufacturer’s instructions and quantified by NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). Complementary DNA (1 μg) was synthetized using the High Capacity cDNA Reverse Transcription kit . The quantitative real-time PCR (qPCR) amplification was carried out in a final volume of 10 μL, using 12.5 ng of cDNA, the Power SYBR Green PCR Master Mix (Applied Biosystems) and Stratagene Mx3000P thermal cycler . Standard qPCR conditions were used. For the amplification of ere used ,17,25. Dt method  was used2 flasks at a density of 5 x 103 cells/cm2. At day 4, the medium was replaced with fresh medium without DEX. At day 7, cells were incubated for 6 hours with 100 μM RU486, 30 μM FL81 or 0.1% DMSO. Then, the cells, after trypsinization, were subjected to ChIP assay. The process for ChIP assay is detailed in BFH12 cells were seeded in 75 cmCYP3A28 fragment activity in HepG2 cells, were performed at least in triplicates. Data were normalized to the pGL4.10 empty vector and pCI-neo when no bPXR or bCAR were co-transfected ; pGL4.10 co-transfected with bPXR and SR12183 activities were used for experiments involving bPXR; pGL4.10 co-transfected with bCAR were used to normalize luciferase activity in bCAR transfections. An arbitrary value of 100 was set for pGL4.10 + pCI-neo, pGL4.10 + bPXR + SR12183, pGL4.10 + bCAR.All transfections, apart from the initial screening of P value < 0.05 was considered as statistically significant.For the statistical analysis of data from gene reporter assays and induction studies, the Student’s t-test or the analysis of variance (ANOVA), followed by the Tukey’s post-test was used . A CYP3A28 transcription start site , one potential binding site for CAR at position -4338/-4323 bp was detected by MatInspector. This element was flanked by an HNF-3β site (5’) and two HNF-4 sites (3’). A putative binding site for C/EBPα was also predicted in F2 at -3288/-3273 bp. NUBIscan identified a DR5 motif at -5541/-5525 bp within the fragment F3 (-6899/-4937 bp)  . Additio4937 bp) .CYP3A28 PP (-283/+71 bp) fused upstream of the luciferase reporter and appended by different upstream sequences  to the PP appeared to decrease NR-dependent activation. This result was particularly evident for the fragment F2 (-4998/-3133 bp), where the basal activity and the NR-mediated activation of CYP3A28 PP were completely abolished. This suggests the presence of silencing elements in F2. In contrast, bCAR and bPXR increased PP+F3 reporter activity of 70% and 150%, respectively, compared to empty pCI-neo. The responsiveness of F3 (-6899/-4937 bp) to both bPXR and bCAR indicated that the NR elements within this fragment should be analyzed further.The relevance of these putative NR sites was examined by reporter gene assays using the CYP3A28 PP (-284/+71 bp) and F3 (-6899/-4937bp) trans-activation by bPXR, the constructs PP and PP+F3 were extensively tested in C3A cells by co-transfecting the relevant expression vectors . The C3A cells were chosen over HepG2 because of their better functionality in reporter gene-based assays [P < 0.05; see To verify the consistency of d assays . The traP < 0.01; Next, the ER6, HNF-1 and HNF-4 elements located in the PP construct were deleted. This shorter PP  displayed slightly decreased activity (-25%) in bPXR co-transfected cells, and significantly reduced activation (-57%) by SR12813  present in F3 (-6899/-4937 bp) was performed to clarify its role in the observed response to bPXR and SR12813. The PP_del and the mutated PP_mER6 were used in combination to eliminate or reduce, respectively, the contribution by the intact PP. Both basal (empty pCI-neo) and bPXR-mediated activity of PP_del+F3mDR5 and PP_mER6+F3 were reduced by about 50%, indicating the presence of some important regulatory elements in F3. The mutation of DR5 (mDR5) reduced the reporter activity by 18% only in the context of PP_mER6 that contained a HNF-4 site but not in the context of PP_del that lacked both the HNF-4 and the proximal ER6 sites. This suggested that DR5 may have only a marginal role in bPXR-mediated activation that also depended on the intact CYP3A28 5’-flanking regions was examined by transient transfection of C3A cells. As shown in P < 0.05) as compared to the empty expression vector pCI-neo.The ability of bCAR to transactivate the The same set of deleted and mutated constructs used to assess bPXR-dependency was employed to investigate bCAR-dependent activity . HoweverCYP3A28 inducibility in the recently available BFH12 cell line, cells were exposed to prototypical CYP3A inducers and their time- and dose-responses were determined.To assess CYP3A28 mRNA expression was modestly but significantly induced by 10 μM RU486 after 6, 12 and 24 hours of exposure (P < 0.001)  were non-toxic after 6 hours of incubation (Alamar blue cytotoxicity test). A dose-dependent increase of CYP3A28 mRNA expression was observed only for RU486 (P < 0.01) and at 100 μM . SR12813 and RIF did not affect the expression of CYP3A28 mRNA  CYP2B22 mRNA levels after 12 hours of incubation; however, no transcriptional effects on CYP3A28 mRNA were ever noticed after 6 or 12 hours of incubation (Among the selected bCAR activators (CITCO and FL81), only the highest tested concentration of FL81 (30 μM) significantly increased , RXRα was bound to both ER6 and DR5 within the PP and the distal F3 fragment, respectively , probably due either to the low constitutive expression of NRs in BFH12 cells or the weak responsiveness of this fetal cell line to prototypical CYPs inducers, as described above.CYP3A28 transcription was investigated through DNA re-sequencing, in silico prediction of NR/TF binding sites, gene reporter assays of wild-type and mutated promoter fragments, induction studies in a bovine-derived hepatic cell line and ChIP assays.In the present study, NR-mediated regulation of bovine CYP3A28 expression via an ER6-type motif at -156 bp within the PP. This ER6 element is conserved among most of the CYP3A promoters in primates and other placental mammals [CYP3A4 reporters [CYP3A4 activation is also dependent upon distal enhancer modules XREM and CLEM [in silico analysis did not identify XREM and CLEM modules within the 10 kbp CYP3A28 promoter, although CAR- and PXR-responsive upstream regions were present (fragment F3). In a bioinformatic study using human CYP3A4 sequences as a query [CYP3A. In similar analyses, we found a CLEM-like sequence located ~34 kb upstream of the CYP3A24 sequence or ~77 kb upstream of the CYP3A38 gene, but not in the vicinity of the CYP3A28 promoter. Therefore, the lack of CLEM that could synergize with the proximal ER6 element might explain its modest responsiveness to bCAR or to its moderate induction in vivo [We showed that the bPXR is involved in the transcriptional regulation of  mammals . It conf mammals  and bindeporters –50. CYP3and CLEM ,38,51. O a query , the pro in vivo .CYP3A28 PP (bases -284 to +71) was activated by the ectopically expressed bPXR, the reporter gene expression increased about 2- to 3-fold. The extent of activation was about the same magnitude as with the human CYP3A4 ER6 element [CYP3A28 PP, in accordance with the recent profiling of bPXR ligand-binding domain [When the  element . RIF faig domain .CYP3A28 PP construct. A putative DR5 element (-5525 bp) and several HNF-3, HNF-4 and C/EBPα binding sites could contribute to F3 bPXR and bCAR responsiveness, because also DR5 elements can serve as functional sites for CAR and PXR [The distal fragment F3 -6899/-4937 bp) conferred a higher basal activity and modest 2-fold responsiveness to bPXR to the 37 bp conlement -55 bp and  and PXR .The additional NR-like elements elsewhere in the promoter  were also surrounded by LETF sites, but we did not detect any responsiveness to either bCAR or bPXR in co-transfection analyses. They cannot be completely ruled out, because reporter plasmids are not organized in the same fashion as in the natural chromatin context .CYP3A4 gene, and at least in case of CLEM, the proximal CYP3A4 promoter activity was decreased [CYP3A28 promoter context.The upstream region F2 (-4998/-3133 bp) was found to suppress the basal and bPXR- and bCAR-dependent reporter activity, even though it contained HNF-3, HNF-4 and CAR binding sites. Similar clusters of HNF-4 sites are present in the human ecreased . Althougecreased , they caecreased ,58. TherCYP3A28 reporter gene activity (fragments PP and F3). The deletion of the ER6, HNF-1 and HNF-4 binding sites in the CYP3A28 PP eliminated the bPXR-mediated activation by SR12813. Upon mutagenesis, the ER6 motif but not the HNF-4 site appeared to be critical. This suggested that the ligand-activated bPXR is able to recognize ER6 and that HNF-1 could participate in bPXR-mediated activation. Recently, an HNF-1 binding site located at -32 kbp upstream of the rat CYP3A1 was proved to act synergistically with DR4 and DR2 motifs to mediate the transactivation by rat CAR [Next, we focused on two NR site-containing regions that increased the  rat CAR .CYP3A28 mRNA and a strong activator of bPXR [CYP3A28 mRNA, which was consistent with their poor ability to activate bPXR [in vivo and in liver slices [To confirm the relevance of the ER6 motif, ChIP assays were performed in BFH12 cells treated with RU486, the most potent inducer of  of bPXR . Among oate bPXR  or weak r slices ,25,60.ChIP assays in DMSO- and RU486-treated cells showed that RU486 weakly increased the recruitment of RXRα, the obligate heterodimer partner for CAR and PXR, for the binding to ER6. It was not possible to reliably detect binding of PXR, probably due to its low expression in BFH12 cells and its modest cross-reactivity with the anti-human PXR antibody. The contribution of HNF-4 in the PP activation was not evaluated by ChIP, because of its slight mRNA expression in BFH12 cells . ConversCYP3A28 and CYP2B22 mRNA expression in BFH12 cells, comparatively lower than that elicited by FL81 in human hepatocytes [Concerning DR5, the binding site located at -5525 bp in F3, luciferase assays did not clearly identify it as the element responsible for the bPXR- or bCAR-dependent activation. It is thus possible that many other LETF sites in this 1.5-kbp fragment cooperate with either DR5 or the proximal ER6 to confer NR responsiveness. Therefore, a deeper characterization of the fragment F3 is required. Nevertheless, ChIP experiments suggested that DR5 motif could recruit both CAR and PXR. DR5 seemed the main DNA binding site recognized by CAR, and the immunoprecipitation of both PXR and RXRα was qualitatively and slightly increased by RU486 treatment. Unfortunately, FL81 did not affect CAR-mediated DR5 immunoprecipitation, probably for two reasons. First, its rather modest increase (1.4-fold) of atocytes ; second,atocytes , as demoatocytes .in vitro model because it does not express the whole set of LETFs and CYPs and does not fully respond to prototypical CYP inducers. As an example, the CYPs induction by PB, a hallmark of CAR-mediated regulation in vivo in calves [CYP3A28 mRNA expression. Secondly, as negative controls, we used no-antibody control (beads only) and a negative locus (CYP3A28 exon 13) as already reported in previous publications [The limitations of the present study are mainly attributable to ChIP assay results. First, a low specific antibody enrichment of ER6 and DR5 regions was generally obtained. This result could be justified by the fact that antibodies specific to bovine NRS were not available; moreover, the present bovine liver cell line (the only available at present) probably is not the optimal n calves ,15, in rn calves ) could nn calves  did not ications –65 insteCYP3A28 gene promoter, the proximal ER6 motif and a distal element F3. We hypothesized after protein/DNA interaction investigations that bCAR, despite the weak response in reporter assays, is recruited to the DR5 element in the distal F3 fragment. However, further molecular studies are needed to confirm ChIP results here obtained.In summary, we have localized two regions potentially responsive to bPXR in the CYP3A regulation and it opens fresh avenues for more detailed analysis.Overall, this work represents the first mechanistic study on bovine S1 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S1 FigCYP3A coding genes are known: CYP3A28, CYP3A38, CYP3A48 and the predicted CYP3A24. The GenBank IDs and nomenclature are displayed together with the nomenclature proposed by [Four posed by .(PDF)Click here for additional data file.S2 FigCYP3A28 promoter region was able to solve the first gap starting at –1492 bp revealing a sequencing artefact (A), as well as the second gap at base –2988 with a new genomic sequence (B). Two of the five single nucleotides polymorphisms (-2899T>G and -2981_-2979insA) are here shown circled.The re-sequencing of (PDF)Click here for additional data file.S3 FigCYP3A28 proximal promoter (PP) and the contribution of the binding motif DR5 identified in F3. The parental PP was deleted of the whole putative region containing several TF binding-sites resulting in the PP_del; through site-direct direct mutagenesis the ER6 (PP_mER6) and DR1 (PP_mDR1) motifs were inactivated. The parental PP+F3 was deleted of the whole putative region containing several TF binding-sites resulting in the PP_del+F3; through site-direct direct mutagenesis the ER6 (PP_mER6+F3), the DR5 motif (PP+F3_mDR5 and PP_del+F3_mDR5) or both (PP_mER6+F3_mDR5) were inactivated. Details are reported in Several constructs were produced to study the binding elements identified in the (PDF)Click here for additional data file.S4 FigCYP3A28 was detected by qPCR in control (0.1% DMSO) and treated cells, using RPLP0 as internal control gene. The relative expression of DMSO-treated cells was set to 1 and its value was used for the normalization of the other groups. Data are expressed as the mean ± SD of three independent experiments . Statistical analysis: ANOVA + Tukey’s post test. Significance was defined as P < 0.05: *; P < 0.01: **; P < 0.001: ***.BFH12 cells were treated with different CYP3A inducers  at the fixed concentration 10 μM for 0 (A), 1 (B), 3 (C), 6 (D), 12 (E) and 24 (F) hours. The expression of (PDF)Click here for additional data file.S5 FigCAR (A), PXR (B) and RXRα (C) was detected by qPCR in control (0.1% DMSO) and treated cells, using RPLP0 as internal control gene. The relative expression of DMSO-treated cells was set to 1 and its value was used for the normalization of the other groups. Data are expressed as the mean ± SD of three independent experiments . Statistical analysis: ANOVA + Tukey’s post test.BFH12 cells were treated with different CYP3A inducers  at the fixed concentration 10 μM for 0, 1, 3, 6, 12 and 24 hours. The expression of (PDF)Click here for additional data file.S6 FigCYP3A28 was detected by qPCR in control (0.1% DMSO) and treated cells, using RPLP0 as internal control gene. The relative expression of DMSO-treated cells was set to 1 and its value was used for the normalization of the other groups. Data are expressed as the mean ± SD of two independent experiments . Statistical analysis: ANOVA + Tukey’s post-test.BFH12 cells were treated with different concentrations of SR12813  and RIF  for 6 hours, as described in Materials and Methods. The expression of (PDF)Click here for additional data file.S7 FigCYP3A28  and CYP2B22  mRNA was detected by qPCR in control (0.1% DMSO) and treated cells, using RPLP0 as internal control gene. The relative expression of DMSO-treated cells was set to 1 and its value was used for the normalization of the other groups. Data are expressed as the mean ± SD of three independent experiments . Statistical analysis: ANOVA + Tukey’s post-test. Significance was defined as P < 0.01: **; P < 0.001: ***. A, C: 6 hours of incubation; B, D: 12 hours of incubation.BFH12 cells were exposed to different concentrations  of the bCAR activator, FL81, for 6 and 12 hours. The expression of (PDF)Click here for additional data file.S8 FigBovine liver cytosolic and nuclear extracts were isolated according to Renisalo et al. (2012) with minor modifications. Proteins (30 μg) were subjected to immunoblotting analysis following the protocol previously published by . Membran(PDF)Click here for additional data file.S9 FigCYP3A28 DNA region without NR binding sites, is reported in the graph. In all experiments, negative and positive controls behaved as expected.BFH12 cells were exposed to 0.1% DMSO and 100 μM RU486 for 6 hours. Chromatin was isolated, subjected to ChIP using anti-human RXRα antibody and quantified by qPCR as described in (PDF)Click here for additional data file.S10 FigCYP3A28 DNA region without NR binding sites, is reported in the graph. In all experiments, negative and positive controls behaved as expected.BFH12 cells were exposed to 0.1% DMSO for 6 hours. Chromatin was then isolated, subjected to ChIP using anti-human CAR antibody and quantified by qPCR as described in (PDF)Click here for additional data file.S11 FigCYP3A28 DNA region without NR binding sites, is reported in the graph. In all experiments, negative and positive controls behave as expected.BFH12 cells were exposed to 0.1% DMSO and 100 μM RU486 for 6 hours. Chromatin was isolated, subjected to ChIP using anti-human PXR antibody and quantified by qPCR as described in Material and Methods. Results for ER6 and DR5 DNA regions are reported in panel A and B, respectively. Data are normalized to input DNA and expressed as % ChIP/input. The experiment was performed four times independently, and similar results were obtained. The data shown derived from a representative experiment. Chromatin samples from control cells immunoprecipitated with or without Histone H3 antibody are shown as Histone H3 and beads, respectively. A further negative control (exon 13), representing a (PDF)Click here for additional data file.


Mycobacteria and Corynebacteria, reflecting their importance during infection by the human pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Corynebacterium diphtheriae. In the antibiotic‐producing Streptomyces, several Wbls have important roles in the regulation of morphological differentiation, including WhiB, a protein that controls the initiation of sporulation septation and the founding member of the Wbl family. In recent years, genome sequencing has revealed the prevalence of Wbl paralogues in species throughout the Actinobacteria. Wbl proteins are small  and each contains four invariant cysteine residues that bind an O2‐ and NO‐sensitive [4Fe–4S] cluster, raising the question as to how they can maintain distinct cellular functions within a given species. Despite their discovery over 25 years ago, the Wbl protein family has largely remained enigmatic. Here I summarise recent research in Mycobacteria, Corynebacteria and Streptomyces that sheds light on the biochemical function of Wbls as transcription factors and as potential sensors of O2 and NO. I suggest that Wbl evolution has created diversity in protein–protein interactions, [4Fe–4S] cluster‐sensitivity and the ability to bind DNA.The WhiB‐like (Wbl) family of proteins are exclusively found in Actinobacteria. Wbls have been shown to play key roles in virulence and antibiotic resistance in  Streptomyces coelicolor (Sc)  there are seven paralogues and in Corynebacterium glutamicum (Cg) there are four.The WhiB‐like (Wbl) family of proteins, of which WhiB is the founding member, are exclusively found in Actinobacteria. Following the initial characterisation of WhiB in  Chater, , multiplStreptomyces are prolific producers of antibiotics and their production is both temporally and genetically linked to a complex process of differentiation . Here I outline the findings of research into the five major classes of orthologous Wbls  and expression of the genes encoding both proteins increases under heat and oxidative stress, suggesting that WhcECg and SpiE collectively contribute to mediating the cellular response under these conditions  in Streptomyces has yet to be investigated. In vitro, holo‐WhiB1Mtb cannot bind to DNA but the apo‐form is able to bind and repress transcription from its own promoter  is required to make the C‐terminal helix available for interaction with DNA. In support of this, NMR of apo‐WhiB1Mtb, prepared via nitrosylation, suggests a substantial conformational change upon addition of NO , via region 4 of the sigma  coupled with transcriptional profiling of whiA and whiB mutants revealed that WhiA and WhiB have identical regulons, binding to the promoter regions of the same ~250 transcriptional units, including those involved in cell division, e.g. ftsZ, ftsW  and cannot bind to DNA, meaning its function in WhiA is unknown  of apo‐WhiB3 binds DNA very strongly. Thus, the activity of WhiB3Mtb as a transcription factor may be controlled by a thiol‐based redox switch , the [4Fe–4S] cluster of WhiDSc reacts with eight molecules of NO. This nitrosylation is extremely rapid, with the reaction occurring 104 times faster than that with O2  in the oxidative stress response pathway  or in vitro (e.g. by EMSA).The WhiB4 orthologue, WblA, is one of three Wbl‐family members that regulate differentiation in hae Fig. . In S. cM. tuberculosis WhiB4 and S. coelicolor WblA, the Corynebacterium orthologue, WhcA, appears to negatively regulate the expression of genes involved in the response to oxidative stress   promoter, sigRp1, thereby increasing the expression of the shorter (but stable) isoform of SigR  are also likely to be targets of WhiB7Mtb since expression of both also increases upon addition of translation‐inhibiting antibiotics  motif, in line with the observed SigA‐WhiB3 interaction  are located close to sigma factor genes and one wbl gene, located on the SCP1 plasmid, encodes a protein (WblP) consisting of a Wbl domain fused to an ECF sigma factor  , while WhiA binds further upstream akin to a Class I activator. It may therefore be a WhiA‐WhiB‐SigA tripartite complex that regulates gene expression to initiate sporulation septation in Streptomyces. Further research is required to establish whether other members of the Wbl family function in concert with partner proteins and what the functional consequences of such interactions are.As highlighted in this review, another key contributory factor may be the need for Wbls to interact with partner proteins. Several Wbl paralogues have been shown to interact with the primary sigma factor, SigA. Other protein partners for Wbls have also been identified; SpiE interacts with WhcE in S. venezuelae WhiB and M. tuberculosis WhiB7 to bind DNA . Extending their work reported in this review, the authors demonstrate, in vitro, that under conditions of oxidative stress, disulphide‐linked oligomerisation of WhiB4Mtb stimulates the protein’s ability to condense DNA. In line with these findings, in vivo ChIP‐seq coupled to microarray transcriptional profiling revealed non‐specific binding of WhiB4Mtb to GC‐rich genomic regions leading to both direct and indirect effects on transcription. These findings suggest that WhiB4Mtb links the oxidative stress response of Mtb to DNA organisation and gene expression.Whilst this review was under consideration, Chawla


Human telomerase reverse transcriptase (hTERT) is the core subunit of human telomerase and plays important roles in human cancers. Aberrant expression of hTERT is closely associated with tumorigenesis, cancer cell stemness maintaining, cell proliferation, apoptosis inhibition, senescence evasion and metastasis. The molecular basis of hTERT regulation is highly complicated and consists of various layers. A deep and full-scale comprehension of the regulatory mechanisms of hTERT is pivotal in understanding the pathogenesis and searching for therapeutic approaches. In this review, we summarize the recent advances regarding the diverse regulatory mechanisms of hTERT, including the transcriptional , post-transcriptional  and post-translational levels (phosphorylation and ubiquitination), which may provide novel perspectives for further translational diagnosis or therapeutic strategies targeting hTERT. Telomeres are located at the ends of chromosomes and their major functions are to maintain chromosomal integrity and genome stability . TelomerNumerous researches have been performed to investigate the regulation of hTERT transcription through direct binding of transcription factors to the wild-type (WT) hTERT promoter. Canonical positive regulators include the multifunctional transcription factors c-MYC, NF-κB, and STAT3. c-MYC dimerizes with its partner MAX and binds to the E-box sequences on the hTERT core promoter region, which activates the transcription of hTERT –14. BesiInterestingly, several transcription factors play dual roles in hTERT transcription. One representative is SP1. It has been reported that SP1 can activate hTERT expression by binding to five GC-box motifs at the hTERT promoter region in telomerase-positive cells , 32, 33.In addition to the common transcriptional regulation manners described above, genetic mutations and/or epigenetic modifications (methylation and acetylation) provide other regulatory modes to precisely alter the transcription of hTERT.Mut) GBM cell lines [Mut HCC cells could specifically enhance the levels of GABPA-bound genes (such as hTERT) other than other ETS transcription factor-regulated genes [hTERT promoter mutations are frequently recurring events in many cancer types , includill lines . Besidesll lines . In addill lines . Interesed genes , 53. Noted genes , 55. In ed genes , 57. In The hTERT promoter mutations also frequently accompany with the mutations of other oncogenic genes or tumor suppressors. For example, the co-occurrence of mutant hTERT promoter and TP53/RB1 mutations are found in bladder cancer (BC), suggesting a cooperative contribution to the BC progression . FurtherhTERT promoter mutations are tightly correlated with enhanced hTERT transcription and increased telomerase activity in tumors, which indicates that hTERT promoter mutations may be an essential mechanism of telomerase reactivation in cancers , 62–64. V600E and hTERT promoter mutations (− 124 C > T) are more aggressive compared to either mutation alone, which defined a new genetic background with the worst clinicopathologic prognosis. Therefore, combinations using BRAF and hTERT promoter mutations may redirect the risk classifications of PTC [When examining thyroid nodule specimens prior to operation, it is found that all 9 specimens from thyroid cancer patients contain mutant hTERT whereas no hTERT promoter mutations are detected in 179 benign specimens, which reveals a 100% diagnostic specificity . In addis of PTC , 70. Amos of PTC . In addis of PTC –74. Nauls of PTC . Among ms of PTC . Even ins of PTC . Taken tAs mentioned above, tumor types such as breast cancer and prostate cancer do not display high frequency of hTERT promoter mutations. Thus, there may be other regulatory mechanisms of hTERT transcription. Therefore, epigenetic mechanisms such as promoter region methylation or histone acetylation are likely to play effects. Traditionally, methylation of gene promoter region is often associated with gene expression silencing. Methylation of the first E-box CpG site (− 165 to − 160 bp) of hTERT promoter region and/or histone H3K9 hinders the binding of c-MYC and consequently decreases hTERT expression in HCC. When histone H3K9 is acetylated and the promoter region of hTERT is not methylated, c-MYC could enhance hTERT expression . BesidesOn the contrary, various studies have demonstrated that methylation within the regulatory element of the hTERT promoter region can enhance rather than weaken the expression of hTERT , 81, wheSeveral bioactive dietary, such as EGCG , SulforaHistone acetyltransferases (HATs) and HDACs induce the acetylation and deacetylation of chromatin histone or non-histone proteins respectively, thereby modulating the expression or function of their targets. Histone acetylation or deacetylation has been shown to be associated with hTERT transcription and they seem to be a general underlying characteristic of human normal cells, telomerase-negative immortal cell lines and cancer cells , 94, 95.Several agents can regulate the hTERT promoter activity via acetylation or deacetylation. The HDACi TSA enhances histone acetylation at the proximal promoter region of hTERT, and directly transactivates the hTERT gene and upregulates hTERT expression. In addition, the effect of TSA is dependent on SP1 motifs . InteresNotably, methylation and acetylation are often crossed in the regulation of hTERT expression. Distinct modifications within the chromatin of the hTERT promoter correlate with different hTERT expression levels. For example, low hTERT expression is related to hypoacetylation of histone H3 and H4 and methylation of histone H3K9 in ALT cell lines. In contrast, hyperacetylation of H3 and H4, and methylation of H3K4 are associated with high hTERT expression in telomerase-positive tumor cells. However, methylation of H4K20 is specific to hTERT promoter region but is not involved in gene expression in ALT cells . BesidesThere are a plenty of alternative splicing variants for hTERT transcripts, but the regulatory mechanisms and functions are not totally clear. Among them, the function of two hTERT splicing variants, the α-deletion and β-deletion forms have been well studied –105. TheDifferent to most alternative splicing regulatory mechanisms, the splicing of hTERT mRNA is regulated by long-range interactions rather than by the intronic/exonic elements close to the splice sites . A blockMany factors can influence hTERT splicing. TGF-β1 could downregulate c-MYC and subsequently decrease hTERT expression in human skin keratinocytes. Intriguingly, TGF-β1 still inhibits the telomerase activity without hindering hTERT transcription by decreasing the full-length (FL) hTERT transcript and retaining high levels of the inactive β-deletion variant. Upon TGF-β1 removal, the changes in the hTERT mRNA splicing pattern disappear, suggesting that alternative splicing may be a novel mechanism for TGF-β1-mediated regulation of telomerase . Besides+ T cells reduces the expression of the active FL hTERT variant and enhances the expression of the inactive β-deletion variant, which results in the decreased telomerase activity and a replicative senescent state [The alternative splicing of hTERT mRNA may provide a regulatory mechanism that could not be accomplished at the transcriptional level, and manipulating hTERT splicing may provide new insights into cancer therapeutics .Such subnt state .It has been reported that hTERT can be regulated by different microRNAs (miRNAs) in many types of cancers Table . MiRNAs Typically, miRNAs downregulate hTERT by targeting its 3′-UTR. Unconventionally, there are also miRNAs that can positively regulate the expression of hTERT. For instance, in cervical cancer cells, miR-346 and miR-138 compete with each other to bind the common 3′-UTR region of hTERT mRNA. miR-138 suppresses the expression of hTERT in an AGO2-dependent manner. In contrast, miR-346 can upregulate hTERT . The mecLong non-coding RNAs (lncRNAs) are a set of transcripts longer than 200 nt with no or limited protein coding function. LncRNAs display their functions via different manners and regulation of gene expressions by lncRNAs is emerging as a novel and critical way , 141. ThIn addition to traditional lncRNA RNAs, circular RNAs (circRNAs) represent a new direction of gene regulation. The circular RNA hsa_circ_0020397 upregulates the expression of hTERT by sequestering miR-138 (the negative regulator of hTERT). The pro-invasion effect of hsa_circ_0020397 is inhibited when hTERT is silenced, suggesting that the circRNA hsa_circ_0020397 promotes cancer cell invasion partially by regulating the expression of miR-138 target genes, such as hTERT . The lncSimilar to other molecules, the post-translational modifications (PTMs) of hTERT could influence its protein stability, subcellular localization and subsequently change its protein levels or the telomerase activity. Currently, the post-translational regulations of hTERT mainly focus on phosphorylation and ubiquitination.Several studies have revealed that hTERT can be modified by phosphorylation. A prominent effect of phosphorylation is the cellular localization change of hTERT. The most reported kinases that phosphorylate hTERT are AKT, PKC and SRC kinases.817AVRIRGKSYV826) could be phosphorylated by the activated AKT kinase in vitro, which plays a crucial role in maintaining the telomere length and heightening telomerase activity [227 phosphorylation of hTERT is equivalently necessary with the bipartite nuclear localization signal (NLS) motifs for efficient hTERT nuclear localization and immortalization of human foreskin fibroblast cells [227 phosphorylation by AKT increases the binding affinity of hTERT with the nuclear import receptors importin-α, which promotes nuclear import of hTERT, thereby increasing telomerase activity [707) phosphorylation by the SRC kinase family. The specific SRC kinase inhibitor PP1 weakens hTERT nuclear export and decreases the antiapoptotic function of hTERT [2O2 stimulation, AKT1 is inactivated and the SRC kinase is activated. In the meantime, mitochondrial hTERT is decreased, which depends on SRC kinase-mediated Tyr707 phosphorylation [2O2-induced hTERT nuclear export via regulating Tyr707 phosphorylation of hTERT [2O2-induced hTERT tyrosine phosphorylation [+ T lymphocytes activation, CD4+ T cells regulate telomerase function independent of the hTERT protein levels, nevertheless, hTERT phosphorylation and subsequent nuclear translocation may play an important role in regulating telomerase function [As a multifunctional kinase, AKT kinase phosphorylates and regulates hTERT in various ways. The hTERT peptide  mediates the ubiquitination of hTERT. Overexpression of MKRN1 could promote hTERT degradation and downregulate telomerase activity and telomere length . In addiAs the core subunit of telomerase, hTERT is normally expressed in highly self-renewal cells and absent in normal somatic cells. Deregulations of hTERT significantly promote cancer initiation and development. Up to now, a large range of mechanisms for hTERT regulation in both normal and cancer cells have been revealed, which have provided significant insights into the hTERT-based therapeutics. Our review has summarized different layers of hTERT regulation, including transcriptional , post-transcriptional  and post-translational levels (phosphorylation and ubiquitination), which may provide comprehensive perspectives for further basic and translational investigations regarding hTERT. Interestingly, these mechanisms are not isolated, but interact with each other. For instance, binding of the transcription factors to the hTERT promoter region could influence the alternative splicing of hTERT transcripts , 119. BeA plenty of studies have illustrated the significant roles of hTERT regulation in human cancers. However, numerous questions still remain to be clarified. For example, what causes the significantly different frequencies of hTERT promoter mutation between distinct cancer types. In addition, although the mutant hTERT promoter sometimes coexists with other mutations, the detailed relationships (upstream or downstream) and the corresponding regulatory mechanisms are still unknown. Furthermore, whether mutant hTERT promoter targeted strategies are sufficient to suppress telomerase activity in cancer therapeutics also needs further investigations. At another layer, the post-translational modifications of hTERT dramatically influence the protein stabilization and localization of hTERT. Notably, there are few studies about the methylation, acetylation or SUMOylation modifications of the hTERT protein, and studies in these fields require further deep investigations.Due to its critical roles in carcinogenesis and cancer progression, hTERT-based diagnosis and therapies have emerged and are worthy of expectations. The mutant hTERT promoter is a relatively unique feature in cancer mutations. Whether it can be a diagnostic biomarker used in liquid biopsies, such as detecting in exosomes or in circulating tumor cells, needs further explorations. In addition, hTERT-based agents , 165, pe


Genome is a complex hierarchical structure, and its spatial organization plays an important role in its function. Chromatin loops and topological domains form the basic structural units of this multiscale organization and are essential to orchestrate complex regulatory networks and transcription mechanisms. They also form higher-order structures such as chromosomal compartments and chromosome territories. Each level of this intrinsic architecture is governed by principles and mechanisms that we only start to understand. In this review, we summarize the current view of the genome architecture on the scales ranging from chromatin loops to the whole genome. We describe cell-to-cell variability, links between genome reorganization and various genomic processes, such as chromosome X inactivation and cell differentiation, and the interplay between different experimental techniques. One of the greatest milestones of the modern biology was the discovery of the DNA double helix structure by Watson and Crick in 1953. This discovery explained how the genomic material can be replicated and passed to further generations and laid the groundwork for future genome research. In the 1970s, as the electron microscopy techniques had matured, they eventually became capable of capturing chromatin fibers and nucleosomes. Another microscopy-based technique, fluorescence in situ hybridization (FISH) was instrumental in many discoveries related to genome organization  colocalizes with the Sonic hedgehog (Shh) gene in developing mouse limb bud cells, the expression only occurs in posterior cells, in which Shh loops out from the chromosomal territory , 25% of promoters did not interact with any enhancer, 40% interacted with several (2–10), and 12% with more than 10 enhancers, with the level of expression being correlated with the number of enhancers. Seventy percent of enhancer-like elements were contacted by 1–5 promoters and 2–4% by more than 5 promoters. Additionally, super-enhancers do not enter contacts with more promoters than highly connected enhancers, but they tend to regulate highly expressed genes  , compared to ~ 20% of CRUs in which PIRs had mixed states (PIRs in the remaining CRUs were in the background state). Interestingly, in general, the prevalent CRU chromatin state determined the chromatin state of a corresponding promoter.It was shown that enhancers generally have an additive effect on the expression of the target genes  with their associated genes, which lead to establishment of gene clusters. Such clusters were observed for example for Klf1 in mouse fetal liver cells (FLCs) or Klf4 and pluripotency factors such as Oct4, Sox2, and Nanog in mESC , and cortical neurons (CNs) cells has shown that, as opposed to enhancer-promoter loops, these interactions are not mediated by CTCF and that they often span across domain boundaries, suggesting that different mechanisms are responsible for formation of these two types of loops  was absent in FLC, and in its place, the cell cycle and DNA replication-related subnetworks appeared , mostly related to early developmental transcription factors linked to body plan specification, morphogenesis, and organogenesis regulation. Polycomb removal led to widespread enhancer activation and upregulation of previously repressed genes, based on which the authors hypothesized that the selective release of genes from this silencing network may be an important mechanism of cell fate specification  to be required for establishment and/or maintenance of these long-range interactions. While in the EED absence, the contacting loci remained spatially collocalized, the frequency of interactions significantly decreased , which take a very characteristic form of squares positioned along the contact map diagonal Fig. d, indicaSimilarly as in the case of loops, it is difficult to uniquely determine the number of domains and their size. Dixon et al. reported discovery of 2200 domains with a median size of 880 kb in mice , active transcription marks such as H3K4me3 and H3K36me3, nascent transcripts, repeat elements, housekeeping genes (present in ~ 34% of TAD boundaries), and transfer RNA  Hi-C data to discover that the compartments A and B can be further divided into subcompartments with distinctive features based solely on the chromatin interaction patterns. The compartment A comprises subcompartments A1 and A2, which are both gene dense, possess highly expressed genes and chromatin marks characteristic for active chromatin, such as H3K36me3, H3K79me2, H3K27ac, and H3K4me1, and have early replication times. The difference between subcompartments A1 and A2 is that the latter contains longer genes, has lower GC content, and finishes replication later than the former . Similarly, compartment B can be partitioned into subcompartments B1 (with epigenetic marks indicative of facultative heterochromatin), B2 (containing mostly pericentromeric heterochromatin), B3 (enriched at nuclear lamina and depleted at the nucleolus-associated domains), and B4   permeated by an interchromatin compartment (IC), with an intermediate layer of decondensed chromatin called perichromatin region (PR). In this model, IC forms a contiguous network of channels anchored at the nuclear pores and containing various factors related to transcription, splicing, DNA replication, and repair  of the cis and trans. The contact networks varied significantly between cells. As in the case of intrachromosomal contacts, interchromosomal interactions preferentially took place between regions belonging to the same compartment. Interestingly, the number of chromosomes that a particular chromosome is interacting with was relatively unchanged and did not depend on the chromosome size  will be required to fully assess its source, importance, and biological consequences , another type of NB, are preferentially located at the CT boundaries near the inter-chromosomal interfaces . Interestingly, it was shown that sperm genome organization shares many features with other cell lines, such as mESC. Mainly, it contains a similar number  of CTCF and cohesin bound sites, and the patterns of TADs and compartments it exhibits mostly overlap with the ones observed in mESC and fibroblasts  may explain a well-studied fact that during the cell development, genes change their nuclear position  . In turn, 3C techniques use crosslinking and proximity-based ligation to merge DNA fragments that were in spatial vicinity. For two fragments to be ligated, they need to be sufficiently close to each other. This capture distance depends on multiple factors, such as restriction frequency and efficiency. Thus, 3C measures the frequency with which a pair of loci is located closer than the capture distance. While interaction frequencies from 3C are often used as a proxy of spatial distance, these two quantities substantially differ, and using them interchangeably may lead to incorrect conclusions , a method that extracts thin nuclear sections from individual cells using cryosectioning and laser microdissection, and create corresponding genomic profiles by sequencing their DNA content   and microscopy  providing independent verification of the existence and importance of these features and the corresponding structures. Moreover, they are particularly useful for detecting multi-way interactions and other features of genome organization, such as association with nuclear bodies, and may be helpful to reconcile 3C and microscopy techniques.For a long time, genome was treated mostly as a one-dimensional sequence of DNA, but in order to understand how it functions, it should be regarded as a complex, multi-level, three-dimensional system. Recently, a lot of effort was put to further investigate the inner workings of the DNA organization. While these studies brought out many new insights about processes and mechanisms involved in the genome functioning, there are still many unanswered questions., enabling us to precisely observe the small-scale genome topology readjustments in response to various stimuli. Theoretical models and considerations such as polymer physics simulations played an important role in verification of the biological hypotheses, and also helped to formulate new models that can explain the phenomena observed in experiments.Many of the discoveries described above were made using population-based methods, in which gathered data represent an average over millions of cells. Recent explosion and further improvement of single-cell techniques will allow to assess the structural dynamics of the genome topology during the cell cycle, and to better understand the genome organization variability across the cells. Technological developments in high-resolution and high-throughput microscopy made it possible to track the regions-of-interest motion in vivoCurrently, we possess a wide arsenal of experimental and computational techniques, and while the new ones are developed, the existing ones are continuously perfected. It is now important to properly assess their strengths, but also their technical and biological limitations. Integration of all these types different of data will lead to new insights providing a better understanding of the genome as a whole. In perspective, they may also contribute to the development of new diagnostic tools and therapies.


TEF1 promoter and determined the minimal length needed for sufficient protein expression. Furthermore, this minimal core promoter sequence was used for the creation of a promoter library covering different expression strengths. This resulted in a group of short, 69 bp promoters with an 8.0-fold expression range. One exemplar had a two and four times higher expression compared to the native CYC1 and ADH1 promoter, respectively. Additionally, as it was described that the protein expression range could be broadened by upstream activating sequences (UASs), we integrated earlier described single and multiple short, synthetic UASs in front of the strongest yeast core promoter. This approach resulted to further variation in protein expression and an overall promoter library spanning a 20-fold activity range and covering a length from 69 bp to maximally 129 bp. Furthermore, the robustness of this library was assessed on three alternative carbon sources besides glucose. As such, the suitability of short yeast core promoters for metabolic engineering applications on different media, either in an individual context or combined with UAS elements, was demonstrated.Altering gene expression regulation by promoter engineering is a very effective way to fine-tune heterologous pathways in eukaryotic hosts. Typically, pathway building approaches in yeast still use a limited set of long, native promoters. With the today’s introduction of longer and more complex pathways, an expansion of this synthetic biology toolbox is necessary. In this study we elucidated the core promoter structure of the well-characterized yeast  Saccharomyces cerevisiae serves as an ideal platform organism for the economically viable production of bulk and fine chemicals . Th. Th11]. greed on . Generalgreed on ,17,22–24activity . Promoteactivity .TEF1 promoter [i.e. orthogonality [e.g. LexA-ER-AD TFs [Many approaches for promoter engineering in yeast have already been developed ,11. Wellpromoter ,28. In tpromoter ,29. Lastpromoter . Besidespromoter –37. Prompromoter ,39. TherR-AD TFs ,33 and sR-AD TFs ), ensureR-AD TFs ,37.S. cerevisiae is still hampered by the big length of its promoters. Indeed, native promoters in yeast typically span ranges of hundreds of nucleotides, needed for the recruitment of the large RNA polymerase II. Even when only the core promoter regions are considered in the construction of yeast synthetic circuits, the lengths still vary between 150 and 200 base pairs [in vivo production of RNA transcripts with this orthogonal system has been demonstrated in S. cerevisiae [Despite these promising reports though, the synthetic biology field of se pairs ,37. Togerevisiae ,41, it hrevisiae , the occrevisiae ,34. It irevisiae –34 couldet al. [TEF1 core promoter. Next, a library of semi-synthetic yeast core promoters (< 70 bp) was constructed by randomization of this TEF1 minimal core promoter. Finally, this expression range was further expanded by the insertion of existing synthetic upstream activating elements in front of the strongest core promoter [These hurdles could be tackled by the design of short, constitutive synthetic yeast promoters with a range of different strengths comparable to those of broadly used native constitutive promoters . Currentet al. , with a promoter . More spUnless otherwise stated, all products were purchased from Sigma-Aldrich , all fragments were PCR purified using the innuPREP PCRpure Kit , Circular Polymerase Extension Cloning (CPEC)  was usedS. cerevisiae SY992  was use™ EC100™ Electrocompetent Escherichia coli  was used for cloning procedures and for maintaining plasmids. E. coli strains were cultured in Lysogeny Broth (LB) consisting of 1% tryptone-peptone (Difco), 0.5% yeast extract (Difco), 1% sodium chloride  and 100 μg/ml ampicillin. For solid growth medium, 1% agar  was added.TransformaxTEF1 [ADH1 [CYC1 [PGK1 [TDH3 [ADH1 terminator [CEN6/ARS4 origin of replication (ori) and a URA3 auxotrophic marker (p2a backbone), resulting in the vectors pRef-pTEF1 . In addition, vector pRef-cpRedden1 containing the extra short synthetic core promoter 1 defined by Redden et al. [TEF1 core promoter library plasmids  TU  under tTEF1 , ADH1 [1F1 [ADH1 , CYC1 [1H1 [CYC1 , PGK1 [7K1 [TDH3  promoterrminator  were conrminator . The TUsef-pTEF1 , pRef-pAef-pTEF1 . All pron et al. , was conplasmids . The cor  each containing 18 degeneracies , interesting candidates covering a wide range of protein expression were Sanger sequenced  to elucidate their sequence. Further, UASs were inserted in front of the strongest obtained core promoter by CPEC. UASA, UASC and UASFEC [After screening of the randomized d UASFEC  were allTaq DNA polymerase . For library evaluation, single colonies were randomly picked from agar plate and grown in 96-well microtiter plates (MTPs). An overview of all plasmids used and constructed in this study can be found in Table B in All plasmids and libraries were transformed in yeast SY992 via the lithium-acetate method . After tFluorescence was used here as a measure of protein levels. Fluorescent proteins are optimized and generally known to fold very well in bacteria and yeasts. As such, it is supposed that these proteins are synthesized in a fully active form and that higher fluorescence levels correspond to more production of the protein. Fluorescence as measure of protein abundance is also widely accepted in the field of synthetic biology ,42,48–50® sealing membrane (Sigma-Aldrich) containing 150 μl selective SD–URA medium with the respective carbon source. These plates were incubated on a Compact Digital Microplate Shaker  at 800 rpm and 30°C for 24h. Subsequently, these pre-cultures were diluted 1:150 in 150 μl fresh selective SD–URA medium with the respective carbon source and grown in sterile polystyrene black μclear flat-bottomed 96 well plates (Greiner Bio-One) for evaluation. Except for the randomized TEF1 core promoter library, the μclear flat-bottomed plate cultures were evaluated in continuous growth experiments performed in a TECAN Infinite® 200 PRO MTP reader (Tecan). Optical density  and fluorescence  were measured every 15 min for minimum 50 hours at 30°C . For every strain, the endpoint OD was determined as the OD value after which three descending OD values were observed. This endpoint OD with its corresponding FP value were used for further data analysis. For the evaluation of the randomized TEF1 core promoter library, an endpoint OD and FP measurement was taken after 26h of growth (stationary phase) at 30°C while shaking at 800 rpm .Four biological replicates were inoculated from agar plate in sterile 96-well flat-bottomed, black microtiter plates  enclosed by a Breathe-Easyi.e. SD–URA medium) was used for the correction of background absorbance of the medium (ODbg). sRef-bl lacking fluorescent protein expression and containing p2a_empty was used to correct for the background fluorescence of yeast (FPbg). Fluorescence corrected for OD was used as measure for fluorescent protein expression and calculated as follows:For analysis of fluorescence measurements, two types of controls were included in every single MTP. A medium blank . One-way ANOVA, executed in MS Excel, was used to determine intervariability between promoters in the different growth experiments on glucose . Error bars represent the standard error of the mean (n = 4). Pairwise comparisons between different strains were done by a two-sided T-test using the scipy.stats package in Python. In all cases, a significance level of 0.05 was applied.Saccharomyces cerevisiae TEF1 promoter, which is well described in literature [TEF1 promoter has no canonical TATA box, the distinction between UAS and core promoter is not straightforward. However, based on the categorization of Blazeck et al. [TEF1 promoter was divided in an UAS of 203 bp and a 176 bp long core promoter which starts with the weaker TATA-like sequence AATAAAAA differing in only 1 nucleotide with the TATA box consensus sequence TATA(A/T)A(A/T)(A/G) [For the development of minimal yeast core promoters, a yECitrine based fluorescent reporter system to evaluate the altered promoter influence on gene expression was constructed. As a starting point, the terature ,28 and iterature –55, was k et al. , the TEF/T)(A/G) . Further/T)(A/G) ,57. In tTEF1 core promoter led to an overall decrease in protein expression for both libraries  core promoters as key determinants of gene expression levels [Truncating the ibraries , which wactivity –62. In aactivity . It was activity . In genen levels .TEF1 in front of a truncated LEU promoter [CIT1 or CLB2 UAS in front of some synthetic core promoter elements also did not improve protein expression [TEF1 does not influence expression levels, presumably by the inability of RNA polymerase II to bind, leading to complete failure of proper transcription initiation.Pairwise comparisons of the UAS-cpTEF_x and cpTEF_x strains showed only a significant positive influence of the UAS on protein expression for strain s_UAS-cpTEF_6 (p = 6.73E-4) and a significant negative effect for strains s_UAS-cpTEF_3 and s_UAS-cpTEF_9 . This was somewhat surprising since the addition of an UASpromoter  significpression , our resi.e. ca. 0.66-fold lower than the weak ADH1 and CYC1 promoters) and showing significant activation by its UAS, this core promoter element was chosen for random library construction.Altogether, since cpTEF_6 is the shortest core promoter giving rise to detectable transcription . sRef-bl and s_cpTEF_6 were taken along as references in every MTP. Histograms of the four libraries revealed a small shift toward higher fluorescence levels for libraries cpTEF_6-libA and D  and variation in this region alters core promoter activity [From each library 94 colonies were randomly picked and evaluated for fluorescence A(A/T)(A/G) [et al. (2015) where it was observed that core promoters having a TATA box or a TATA-like sequences gave rise to higher gene expression levels [P. pastoris, a profound effect in promoter functionality was observed by mutating the TATA box [CYC1 (287 bp) promoter. Also, these three promoters are stronger than the yeast ADH1 promoter, having a length of 1461 bp. Compared to the core promoter reported by Redden et al. (sRef_cpRedden1), having a length of around 65 bp, our truncated cpTEF_6 had equal yECitrine levels. Since the lengths of both core promoters are very similar, this could indicate the minimal stretch of DNA needed for proper transcription initiation in yeast. Furthermore, after randomization in cpTEF_6, core promoters transcending the strength of cpRedden1 were retrieved, even without upstream elements. Despite the fact that the native pTEF1 is still four times stronger than the short cpTEF_6-I, the latter does have a reduction of 82% in sequence length. As such, the reported set of promoters ..e.g. pro/T)(A/G) ) is visin levels . Also inTATA box . In addiromoters  shows thi.e. 69 bp, that are stronger than for example the native CYC1 promoter (up to 2 times) without the need of UASs. In earlier observations by Redden et al., an extra UAS had to be added to the core promoters to reach higher expression levels than the native CYC1 promoter, leading to promoters over the 100 bp in length [Although a set of nine minimal core promoters has been described before , the setn length . Addition length , this imTEF1 promoter library [TEF1 promoter (i.e. core promoter inclusive its UAS). As such, not only the sequence to bind RNA polymerase II is altered, but also the sequence to recruit transcription factors which could consequently lead to extra variation in gene expression. Therefore, in the view to further broaden our expression range, the influence of introducing extra UAS sequences in front of the strongest yeast core promoter cpTEF_6-I was evaluated.Still, the obtained promoter library of short core promoters shows a less broader expression range compared to the earlier reported 15-fold  library . The maiA, UASC and UASFEC with the latter in fact an array of three synthetic UAS elements  which was also seen in earlier observations [CLB2 [C and UASFEC both significantly increased the promoter activity for cpRedden1 by a factor 3.9 and 4.8 and for cpTEF_6-I by a factor 1.5 and 2.5, respectively . In all cases, stronger promoters than the reference promoters with cpRedden1 were achieved, however the impact of UASs on the increase in promoter activity was smaller for cpTEF_6-I. These results reveal that the degree of change in transcription levels is strongly dependent on the combination of a specific UAS type with a specific core promoter. Furthermore, multiple UAS elements, i.e. UASFEC, can certainly be used as modular building blocks to amplify transcription, but the magnitude of their effect is difficult to predict. This confirms an important interplay between UASs and core promoters where both must be compatible for the enhancement of transcription [The effect of the different UAS elements on transcription initiation was evaluated by measuring yECitrine fluorescence on SDG medium . To starrvations . Conversns [CLB2 . On the cription ,68.FEC_cpTEF_6-I led overall to the highest expression, which was even a factor 10 larger than the cpTEF_6 we initially started from. Even, this promoter, with a total promoter length of only 129 bp, showed almost a comparable activity to the native and strong yeast TEF1 and PGK1 promoters. Considering these are bulky promoters with lengths of respectively 379 [FEC_cpTEF_6-I stranded at roughly 33%, yet it has a reduction of 80% in DNA load. These observations are partially in agreement with the study of Redden et al. since their final promoter also contributed to strong gene expression in only 20% of the pTDH3 DNA space [A_cpTEF_6-I, all hybrid promoters obtained were stronger than the cpTEF_6-I promoter. In general, this study delivered a semi-synthetic promoter library that spans an overall expression range on glucose medium between about 53% and 1000% of the original 69 bp cpTEF_6 promoter.Nevertheless, UASNA space . DespiteNA space  could fui.e. pCYC1, pPGK1 and pTDH3) showed a lower chance of equal activities under the same conditions . In this regard, pTEF1 and pADH1 are trustworthy promoters on glucose for strong and weak gene expression respectively and are preferred above pPGK1 and pCYC1 having comparable strengths.With view on the accuracy of promoters across the different growth experiments on SDG medium , the varSince it is known that the activity of yeast promoters can change on different carbon sources ,70, synte.g. 1500% compared to 3000% for pTDH3 on glucose, A and UASC, the amplifying effect on promoter strength for cpTEF_6-I and cpRedden1 disappeared on pyruvate  had in general an equal behavior on the alternative media . For exaTEF1 promoter was truncated to elucidate its minimal core promoter and to enable the design of short functional yeast promoters. Six of the nine truncated promoters remained functional, even without the addition of an UAS and a 69 bp long TEF1 minimal core promoter, cpTEF_6, was determined. Randomization of this core promoter sequence revealed influential regions at its 5’ and 3’ ends, respectively suggesting a location of the PIC region and confirming the importance of the region around the TSS. This randomization approach led to short semi-synthetic yeast promoters with a very reproducible output (cfr. cpTEF_6-I) compared to commonly used native promoters. Furthermore, an increasing number of UASs in front of this core promoter gradually enhanced transcription, however, no stronger protein expression could be achieved as with the pTDH3 which is less good compared to similar work by Redden et al. where 70% was reached of the total pTDH3 strength [et al. [In this study, the well-characterized strength . On the  [et al. , even wh [et al. , also af [et al. –15, whic [et al. . By doinAltogether, this study demonstrated the possibility of short yeast promoter libraries, of which the expression range can be expanded with UASs, to function as full transcriptional regulators. As a result, a usable promoter library validated in four different media with lengths not exceeding 130 bp and having a 20-fold expression range is available for yeast metabolic engineering purposes.S1 FigTEF1 5’UTR is indicated in bold and underlined. The UASTEF1 is underlined and the TEF1 core promoter is indicated in bold. The respective sequences of the truncated TEF1 core promoter are represented in Table D in The (PDF)Click here for additional data file.S2 FigThe horizontal line represents the mean fluorescence corrected for OD of the native cpTEF_6 which was grown as biological triplicate. Error bars representing the standard error are a consequence of OD correction with biological triplicates of sRef-bl and the medium.(PDF)Click here for additional data file.S3 FigThe horizontal line represents the mean fluorescence corrected for OD of the native cpTEF_6 which was grown as biological triplicate. Error bars representing the standard error are a consequence of OD correction with biological triplicates of sRef-bl and the medium.(PDF)Click here for additional data file.S4 FigThe horizontal line represents the mean fluorescence corrected for OD of the native cpTEF_6 which was grown as biological triplicate. Error bars representing the standard error are a consequence of OD correction with biological triplicates of sRef-bl and the medium.(PDF)Click here for additional data file.S5 FigThe horizontal line represents the mean fluorescence corrected for OD of the native cpTEF_6 which was grown as biological triplicate. Error bars representing the standard error are a consequence of OD correction with biological triplicates of sRef-bl and the medium.(PDF)Click here for additional data file.S6 FigThe horizontal line represents the mean fluorescence corrected for OD of the native cpTEF_6 which was grown as biological triplicate. Error bars representing the standard error are a consequence of OD correction with biological triplicates of sRef-bl and the medium.(PDF)Click here for additional data file.S7 FigProtein expression levels were normalized against the native cpTEF_6 promoter . Error bars represent the standard error of the mean . All strains are listed in Table A in (PDF)Click here for additional data file.S8 FigProtein expression levels were normalized against the native cpTEF_6 promoter . Error bars represent the standard error of the mean . All strains are listed in Table A in (PDF)Click here for additional data file.S9 FigProtein expression levels were normalized against the native cpTEF_6 promoter . Error bars represent the standard error of the mean . All strains are listed in Table A in (PDF)Click here for additional data file.S1 FileTable A. Strains used in this study. Table B. Plasmids used in this study. Table C. Primers used in this study. Table D. Sequences of the core promoters. Table E. Synthetic upstream activating sequences. Table F. p-values obtained after one-way ANOVA to investigate the yECitrine variability of the native and synthetic promoters.(PDF)Click here for additional data file.


Pseudomonas aeruginosa possesses multiple quorum sensing systems that regulate and coordinate production of virulence factors and adaptation to different environments. Despite extensive research, the regulatory elements that play a role in this complex network are still not fully understood. By using several RNA sequencing techniques, we were able to identify a small regulatory RNA we named RhlS. RhlS increases translation of RhlI, a key enzyme in the quorum sensing pathway, and represses the fpvA mRNA encoding one of the siderophore pyoverdine receptors. Our results highlight a new regulatory layer of P. aeruginosa quorum sensing and contribute to the growing understanding of the role regulatory RNAs play in bacterial physiology.The opportunistic human pathogen  N-Acyl homoserine lactone (AHL) quorum sensing (QS) controls expression of over 200 genes in Pseudomonas aeruginosa. There are two AHL regulatory systems: the LasR-LasI circuit and the RhlR-RhlI system. We mapped transcription termination sites affected by AHL QS in P. aeruginosa, and in doing so we identified AHL-regulated small RNAs (sRNAs). Of interest, we noted that one particular sRNA was located within the rhlI locus. We found that rhlI, which encodes the enzyme that produces the AHL N-butanoyl-homoserine lactone (C4-HSL), is controlled by a 5′ untranslated region (UTR)-derived sRNA we name RhlS. We also identified an antisense RNA encoded opposite the beginning of the rhlI open reading frame, which we name asRhlS. RhlS accumulates as wild-type cells enter stationary phase and is required for the production of normal levels of C4-HSL through activation of rhlI translation. RhlS also directly posttranscriptionally regulates at least one other unlinked gene, fpvA. The asRhlS appears to be expressed at maximal levels during logarithmic growth, and we suggest RhlS may act antagonistically to the asRhlS to regulate rhlI translation. The rhlI-encoded sRNAs represent a novel aspect of RNA-mediated tuning of P. aeruginosa QS. Pseudomonas aeruginosa, like many bacteria, has the ability to sense its population density and respond to environmental changes by initiating a gene regulatory system termed quorum sensing (QS). In proteobacteria like P. aeruginosa, QS commonly involves diffusible N-acylhomoserine lactone (AHL) signaling molecules that are recognized by corresponding transcription factors. When the population density, and thus signal concentration, reaches a critical threshold, a coordinated, population-wide shift in gene expression occurs. This facilitates P. aeruginosa adaptation to its environment , and the 3OC12-HSL-dependent transcription factor LasR. The Rhl system consists of RhlI, the N-butanoyl-homoserine lactone (C4-HSL) synthase, and the C4-HSL-dependent transcription factor RhlR. Together these two QS circuits activate over 200 genes  is known to occur by two mechanisms: by direct base pairing to target mRNAs or by binding to proteins. In the class of base-pairing sRNAs, P. aeruginosa the sRNA PhrS regulates expression of pqsR, which codes for a transcriptional activator, by binding the RBS of an upstream open reading frame (uORF). The binding alters the RNA structure to activate translation of the uORF, which by translational coupling leads to pqsR translation  of some genes or operons. They control gene expression or translation by directly binding metabolites or signaling molecules, by sensing pH, or by sensing changes in temperature , 12. In nslation . Recent nslation , 15.P. aeruginosa Hfq mutants grow abnormally and are attenuated for virulence  mutant incubated with or without added 3OC12-HSL and C4-HSL by using term-seq as diagrammed in P. aeruginosa genes or operons  in a 10.1128/mBio.02253-19.1TEXT S1Text S1, PDF file, 0.3 MB.Supplemental materials and methods. Download Copyright © 2019 Thomason et al.2019Thomason et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02253-19.9TABLE S1P. aeruginosa PAO1 genes. (Tab B) Differential expression analysis for reproducible term-seq sites with and without AHLs. Base mean, fold change, and adjusted P value were calculated using DESeq2. (Tab C) Predicted targets of RhlS according to TargetRNA2 analysis. (Tab D) Strains, plasmids, and oligonucleotides used in this study. Download Table S1, XLSX file, 0.6 MB.(Tab A) Transcription termination sites (TTS) associated with Copyright © 2019 Thomason et al.2019Thomason et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the lasB, which codes for the QS-induced elastase enzyme, were much more abundant in cells incubated with AHLs than in cells without AHLs  were diluted into 100 ml fresh LB plus 50 mM MOPS in 500-ml baffled flasks and grown at 37°C with shaking. At an OD600 of ∼0.4, the cultures were split and either only 10 μM C4-HSL, only 0.2% arabinose, both, or neither were added to each flask. At the indicated times, samples were collected and β-galactosidase levels were determined. Download FIG S1, PDF file, 0.2 MB.The 5′ UTR of Copyright © 2019 Thomason et al.2019Thomason et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the rhlI 5′ UTR with added AHLs could generate a stable sRNA and investigated this using Northern blot analysis. As shown in -rhlI deletion mutant, consistent with the conclusion that the sRNA is specific to the rhlI locus (RhlI-associated sRNA). The location and size of RhlS are consistent with a previously identified 70-nucleotide P. aeruginosa sRNA (SPA104) that was not known to be AHL induced (We hypothesized the premature termination of the lI locus . We have induced .rhlI with 5′ RACE (rapid amplification of cDNA ends)  . We founreported . We alsosequence . By usinsequence . Our date 5′ UTR .10.1128/mBio.02253-19.3FIG S2rhlI. (A) Primer extension analysis. To detect the 5′ end of RhlS, primer VA0001 (corresponding to nucleotides −61 to −42 relative to the rhlI start of translation) was end labeled with [γ-32P]ATP and incubated with the primer extension enzyme mixture. The rhlI-rhlR fragment was amplified from PAO1 to generate the sequencing ladder. To resolve the 5′ end of RhlS, primer extension reactions and the DNA sequencing ladder were run on an 8% polyacrylamide–6 M urea gel. * corresponds to the +1 site of RhlS indicated in rhlI. Total RNA isolated from WT PAO1 was subjected to 5’ RACE analysis using the indicated primers. Each row represents an independent clone (and RNA transcript) that was sequenced. Download FIG S2, PDF file, 0.2 MB.Analysis of transcription start sites identifies a single transcription start for RhlS and Copyright © 2019 Thomason et al.2019Thomason et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa. Northern blotting showed that RhlS levels in early-logarithmic-phase cells were relatively low, increased in late logarithmic phase, and were at maximal levels in stationary-phase cells   .rhII promoter, with full-length rhlI mRNA accounting for between 3 and 15% of the total at steady state. RNA structure prediction using Mfold (rhlI transcript and thus elevated levels of C4-HSL. We tested this hypothesis by constructing a strain where the Rho-independent terminator is deleted (RhlS-Δterm). Surprisingly, the amount of rhlI mRNA in this mutant was similar to that in wild-type cells  were grown for 24 h in 10 ml LB plus 50 mM MOPS in 50-ml flasks at 37°C with shaking. Total RNA was extracted, cDNA was generated, and mRNA levels were measured as in FIG S3, PDF file, 0.3 MB.Levels of the Copyright © 2019 Thomason et al.2019Thomason et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the rhlI mRNA but produced low levels of C4-HSL and low levels of RhlS  in the RhlS terminator structure mutation strain (P. aeruginosa (pRhlS) had RhlS transcript levels similar to those of wild-type cells indicating expression from the pRhlS plasmid is physiologically relevant  containing pRhlS or the vector pMKT1 are similar to WT PAO1. For the mutants, overnight cultures were diluted to an OD600 of ∼0.005 in LB plus 50 mM MOPS, and at an OD600 of ∼0.4, the cultures were split and either no or 1% l-arabinose was added. Samples were collected at the indicated times after addition of arabinose. For wild-type PAO1 cells were grown similarly but arabinose was not added and culture densities at sampling times are shown. For all samples, RNA was extracted and analyzed by Northern analysis as in E. coli with a chromosomal insertion of rhlI compared to levels produced by wild-type P. aeruginosa PAO1. Single colonies of PAO1 or E. coli PM1205-PBAD-rhlS-rhlI (MPK0603) were used to inoculate 10 ml LB plus 50 mM MOPS in 50-ml flasks and grown at 37°C in the presence of 1% l-arabinose for 18 h, C4-HSL was extracted and measured as in FIG S4, PDF file, 0.1 MB.Ectopic expression of RhlS is physiologically relevant. (A) RhlS levels in the Copyright © 2019 Thomason et al.2019Thomason et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the rhlI mRNA but low levels of RhlS produce low levels of C4-HSL, we hypothesized that RhlS might stimulate translation of rhlI mRNA. To test this hypothesis, we incorporated a C-terminal vesicular stomatitis virus glycoprotein G (VSV-G) epitope tag at the native rhlI locus such that it encoded an RhlI-VSV-G polypeptide. We examined levels of this tagged RhlI in our RhlS mutant strains by Western blotting , RhlS Mut A-VSV-G (MPK0689), RhlS MutA+B VSV-G (MPK0697) and the asRhlS-1-VSV-G (MPK0687) strains were grown for 24 h in 10 ml LB plus 50 mM MOPS in 50-ml flasks at 37°C with shaking. C4-HSL was measured as described in Copyright © 2019 Thomason et al.2019Thomason et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the rhlI. To assess this possibility, we used the base-pairing prediction algorithm TargetRNA2  or included the empty vector (pMKT1). Levels of β-galactosidase in arabinose-grown cells containing pRhlS were about 25% of the levels in cells without RhlS  expressing the mutant RhlS in place of wild-type RhlS, fpvA-lacZ expression was not repressed , in the AHL− condition. The RNA-seq reads of RhlS  and asRhlS  and the asRhlS term-seq position (green arrow) are indicated. The gap in RNA-seq coverage within rhlI is due to the deletion of the ORF. Download FIG S6, PDF file, 0.2 MB.Expression of the asRhlS. term-seq and strand-specific RNA-seq data  σE consensus  restores C4-HSL levels almost to those of wild-type PAO1 . What is unclear at this point is how RhlS mediates this positive regulation of rhlI. We have two models for how this RhlS regulation may occur. First RhlS could act directly on rhlI to relieve translational repression mediated by a highly structured 5′ UTR. Mfold predicts the secondary structure of the rhlI 5′ UTR occludes the primary RBS by a stem-loop structure  where an sRNA base pairs to a highly structured 5′ UTR to relieve translation repression mediated by RBS occlusion ..50).32P]ATP end-labeled oligonucleotides specific to the desired transcript  bioassay (C4-HSL was ethyl acetate extracted from 24-h LB plus 50 mM MOPS (morpholinepropanesulfonic acid) culture supernatant as described previously . The amobioassay , 53 and PRJEB31965.RNA-seq and term-seq data sets have been deposited in the European Nucleotide Database (ENA) under study accession no.