SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors function as an endogenous age cue in regulating plant phase transition and phase-dependent morphogenesis, but the control of SPL output remains poorly understood. In Arabidopsis thaliana the spatial pattern of trichome is a hallmark of phase transition and governed by SPLs. Here, by dissecting the regulatory network controlling trichome formation on stem, we show that the miR171-targeted LOST MERISTEMS 1 (LOM1), LOM2 and LOM3, encoding GRAS family members previously known to maintain meristem cell polarity, are involved in regulating the SPL activity. Reduced LOM abundance by overexpression of miR171 led to decreased trichome density on stems and floral organs, and conversely, constitutive expression of the miR171-resistant LOM (rLOM) genes promoted trichome production, indicating that LOMs enhance trichome initiation at reproductive stage. Genetic analysis demonstrated LOMs shaping trichome distribution is dependent on SPLs, which positively regulate trichome repressor genes TRICHOMELESS 1 (TCL1) and TRIPTYCHON (TRY). Physical interaction between the N-terminus of LOMs and SPLs underpins the repression of SPL activity. Importantly, other growth and developmental events, such as flowering, are also modulated by LOM-SPL interaction, indicating a broad effect of the LOM-SPL interplay. Furthermore, we provide evidence that MIR171 gene expression is regulated by its targeted LOMs, forming a homeostatic feedback loop. Our data uncover an antagonistic interplay between the two timing miRNAs in controlling plant growth, phase transition and morphogenesis through direct interaction of their targets.The miR156-targeted  Arabidopsis the miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors play important roles as an endogenous age cue in programming phase transition and phase-dependent morphogenesis, including trichome patterning. However, how the timely increasing SPL output is modulated remains elusive. By dissecting the regulatory network controlling trichome formation on stem, we show that a group of GRAS family members, LOST MERISTEMS 1 (LOM1), LOM2 and LOM3, targeted by timing miR171, function in modulating the SPL activity through direct protein-protein interaction. LOMs promote trichome formation through attenuating the SPL (such as SPL9) activity of trichome repression. The LOM-SPL interaction affects many aspects of plant growth and development, including flowering, aging and chlorophyll biosynthesis. Interestingly, MIR171A gene expression is regulated by its own targets (LOMs), forming a feedback loop to program plant life. Our study establishes an age-dependent regulatory network composed of two timing miRNAs which act oppositely through direct interaction of their target proteins.MicroRNAs are important aging regulators in many organisms. In  Caenorhabditis elegansSQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes define an endogenous aging and flowering pathway Arabidopsis there are 17 SPL genes, 11 of which contain a miR156 target site. The miR156-targeted SPLs can be divided into four clades based on their protein structures, and the clades composed of SPL3/4/5 and SPL9/15 both promote phase transition ArabidopsisMicroRNA (miRNA) was first identified as the regulator of the juvenile-to-adult transition in Arabidopsis, trichome distribution is spatially and temporally regulated, and the distribution pattern serves as a trait to distinguish between juvenile and adult leaves Arabidopsis mutants have identified sets of regulators governing trichome formation. In brief, the trichome initiation complex of Arabidopsis comprises a WD40 protein TRANSPARENT TESTA GLABRA1 (TTG1), an R2R3 MYB protein GLABRA1 (GL1), and a basic helix-loop-helix transcription factor GL3 or its homolog, ENHANCER of GL3 (EGL3) GL2, which encodes a homeodomain leucine zipper transcription factor Trichomes are specialized epidermal cells, acting as barriers to protect plants from herbivores, UV irradiation, and excessive transpiration. In TCL1 and TRYWe previously reported that the miR156-targeted SPLs temporally repress trichome distribution on stem and inflorescence through activating Petunia hybridArabidopsis, LOM1/2/3 , also known as AtHAMs because the first functionally characterized member of this subclade is HAIRY MERISTEM (HAM) from Arabidopsis genome, there are four miR171 coding genes, MIR171A, B, C and MIR170. We found that overexpression (OE) of MIR171A/B/C under the 35S promoter reduced trichomes on stem very significantly  as reported 35S::LUC-rLOM1 plants, in which a miRNA-resistant LOM1 (rLOM1) was fused to firefly luciferase (LUC) gene and expressed constitutively. In addition to yellow-green leaves  did not show an evident change of transcript levels of GIS, GIS2, ZFP8 and GL3  and down-regulation of SPLs (35S::MIR156F) both reduced TCL1 and TRY expression and triggered ectopic trichomes, we wondered if miR171-LOMs induced trichome formation through affecting the miR156-targeted SPLs. To test this hypothesis, we crossed 35S::MIR171B to 35S::MIR156F plants. Although 35S::MIR171B repressed trichome formation on stem in wild-type plants, it did not change the ectopic trichome distribution induced by 35S::MIR156F (LOM genes caused a further trichome reduction in 35S::MIM156 plants (LOM1::Myc-rLOM1) stimulated while high level of SPL9 (SPL9::GFP-rSPL9) repressed stem trichome formation (LOM1::Myc-rLOM1×SPL9::GFP-rSPL9) showed an intermediate phenotype in terms of stem trichomes, though less than the wild-type , which was under the direct control of miR156-tageted SPLs SOC1 was up-regulated in lomt mutant along with earlier flowering  in 35S::LUC-rLOM1 but lower in lomt mutant than in wild-type plants, whereas the expression of MIR171B and C showed a less degree response to LOM1  to allow the translocation of LOM1-GR fusion protein into the nucleus. An obvious transcript increase of MIR171A and, to a less extent, of MIR171B was observed after 4 hours, whereas MIR171C was not induced during this period  which conferred the LOM1 induction  to flowering plants of both monocots and dicots Gossypium raimondii there are 15 MIR171 and seven putative LOM genes, of which five LOMs contain the miR171 recognition sites (see SU(VAR)3-9 HOMOLOG8 pair was proposed to have evolved very recently in the Arabidopsis lineage Hvu-pri-miR171a) in barley up-regulated miR156 and repressed vegetative phase transitions Arabidopsis described herein and reported by others Mining of genome data revealed that both miR156 and miR171 are highly conserved in land plants from moss , and Nicotiana benthamiana were grown at 22°C in long days (16 h light/8 h dark). The lom triple mutant 35S::LUC-rLOM135S::MIR156F35S::MIM156SPL9::GFP-rSPL9TCL1::GUSTCL1mu3/4::GUSgl1 (SALK_039478), try (SALK_029760), tcl1 (SALK_055460) and gl3 egl3 (CS66490) mutants were obtained from Arabidopsis Biological Resource Center (ABRC).Plants of MIR171A/B/C::GUS constructs, the promoters of MIR171A/B/C (∼2 kb) were PCR amplified using PrimeSTAR HS DNA polymerase (TaKaRa) and individually fused to the GUS coding region. For LOM constructs, the miRNA-resistant versions were created by two-round PCR. The resultant fragment was inserted into a vector which harbors a 35S::6×Myc cassette to generate 35S::Myc-rLOM1/2/3. Then the 35S promoter was replaced by a native promoter to generate LOM1::Myc-rLOM1 and LOM12::Myc-rLOM2. At least 30 T1 seedlings were analyzed for each construct. For BiFC constructs, coding regions of the miRNA-resistant LOMs and SPL9 were PCR amplified and cloned into JW771 and JW772 For t test.The trichome numbers were counted on each internode of stem, and the number was divided by the length of each internode to calculate the trichome density (number per centimeter). Flowering time was measured by counting the total number of leaves (rosette and cauline leaves) and the number of days to flowering under long-day condition. The data were given as mean s.d. and analyzed by β-TUBULIN2 (At5g62690). For DEX induction, 10-day-old 35S::rLOM1-GR lomt seedlings were sprayed with 10 mM DEX (Sigma-Aldrich) or alcohol (mock control). After 4 hours, rosette leaves were harvested.RNAs were extracted with Trizol reagent (Invitrogen) following the manufacturer's instructions. Total RNAs of 1 µg were used for reverse transcription in a 20 µL reaction system with M-MLV Reverse Transcriptase kit (Invitrogen). The fragments of interest were amplified by RT-PCR using sequence-specific primers . Full-length or truncated cDNAs of LOM1 were inserted into pGBKT7 and those of SPLs into pGADT7, respectively. Plasmids were transferred into yeast strain AH109 (Clontech) by the LiCl-PEG method. The interactions were tested on SD/-Leu/-Trp/-His plates supplemented with 15 mM 3-amino-1,2,4,-triazole. Three independent clones for each transformation were tested.Agrobacterium tumefaciens cells were re-suspended in infiltration buffer  at OD600 = 0.8. 35S::P19-HA and the suspension was co-infiltrated to inhibit gene silencing BiFC assays were performed as described Total RNAs were extracted using Trizol reagent (Invitrogen), and 5–20 µg of the total RNA were resolved on 17% polyacrylamide gels under denaturing conditions (7 M urea). RNAs were then transferred to HyBond-N+ membranes  by semidry blotting, and membranes were hybridized with oligonucleotide DNA probes labeled with digoxigenin using the DIG Oligonucleotide 3-End Labeling Kit, Second Generation (Roche). Oligonucleotide sequences are listed in Supplementary 35S::Myc-rLOM1 or wild-type seedlings were harvested and then cross-linked in formaldehyde solution (1%) under a vacuum. The material was washed and ground in liquid nitrogen, the resultant powder was re-suspended in extraction buffer  and lysis buffer , successively, followed by sonification . An anti-Myc antibody (Abmart) was added for precipitation. After several washes, DNA samples were reversely cross-linked and then purified using a PCR purification kit (Qiagen). The relative amounts of the DNA amplicons were analyzed by quantitative PCR using the β-TUBULIN2 gene promoter as a reference. Relative enrichment was calculated by normalizing the value in 35S::Myc-rLOM1 against the value in wild-type.Chromatin immunoprecipitation (ChIP) experiments were performed as described N. benthamiana plants was used 35S::rLOM1 or empty vector, and the reporter plasmid, pGreen-0800-LUC, harbors two luciferases: the firefly luciferase (LUC) controlled by the MIR171 promoter, and the Renilla (REN) luciferase controlled by the constitutive 35S promoter. The Agrobacterium strain containing the reporter was mixed with the effector strain (at the reporter:effector ratio of 1∶3). The mixture was infiltrated into leaves of N. benthamiana. Three days later, leaf samples were collected for the dual-luc assay using commercial Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instruction. The LUC activity was normalized to REN. Three biological repeats were measured for each sample.A dual-luc method using GUS activity was assayed by staining. Plant materials were submerged in 0.5 mg/mL X-Gluc solution , vacuumized and kept at 37°C. Subsequent materials were decolorized in 70% ethanol.LOM1 (At2G45160), LOM2 (At3G60630), LOM3 (At4G00150), MIR171A (At3G51375), MIR171B (At1G11735), MIR171C (At1G62035), SPL9 (At2g42200), SPL2 (At5G43270), MIR156F (At5G26147), SOC1 (At2g45660), GL1 (At3G27920), GL3 (At5G41315), EGL3 (At1G63650), TCL1 (At2g30432), TRY (At5G53200), GIS (At3g58070), GIS2 (At5g06650 ), ZFP8 (At2g41940), β-TUBULIN-2 (At5g62690) and Ph-HAM (AY112704).Figure S1Expression of trichome regulator genes. (A) qRT-PCR analysis of expression of trichome promoting genes in wild-type, lomt mutant and LUC-rLOM1OE plants. Expression of GIS, GIS2, ZFP8 and GL3 did not change evidently. (B and C) Expression of trichome repressor genes in first internode (B) and inflorescence (C). Expression of CPC, ETC1 and TCL2 did not change in lomt and LUC-rLOM1OE plants in first internode; CPC was higher in LUC-rLOM1OE inflorescence.(TIF)Click here for additional data file.Figure S2LOM1OE and MIR156FOE plants show similar phenotypes. View of 49-day-old plants of the indicated genotypes. Plants were grown under short-day condition (8 h light/16 h dark). Note that LOM1OE (35S::LUC-rLOM1) and MIR156FOE (35S::MIR156F) plants were both yellow-green; on the contrary, lomt mutant and SPL accumulation plants (35S::MIM156 and SPL9::GFP-rSPL9) were dark-green. 35S::LUC-rLOM1 and 35S::MIR156F plants produced more rosette leaves than wild-type. Bar = 2 cm.(TIF)Click here for additional data file.Figure S3LOMs accumulation delays flowering. (A) SPL2 interacts with three LOMs in yeast. (B–D) Flowering time of wild-type (B), lomt (C) and 35S::LUC-rLOM1 (D) plants under long-day conditions. The x axis indicates the number of days after germination and the y axis indicates percentage of plants that flowered on a given day. (E) Flowering time of the plants indicated in (B–D). The number of days to flowering was counted when the first floral bud opens. Data are given as mean s.d. and analyzed by t test. **P<0.01, relative to the wild-type; n, number of plants analyzed.(TIF)Click here for additional data file.Figure S4Alignment of N-terminus of three LOMs of Arabidopsis thaliana. LOM1N and LOM2N are more similar in length and amino acid sequence identity.(TIF)Click here for additional data file.Figure S5Expression of SPL9 in seedling and inflorescence. Analysis by qRT-PCR shows that expression of SPL9 did not change evidently in lomt and 35S::LUC-rLOM1 plants in either seedling or inflorescence.(TIF)Click here for additional data file.Figure S6Identification of promoter regions of MIR171 genes responsible for LOM1 induction. (A) LUC activities driven by different upstream fragments of the MIR171A gene. Nucleotides are numbered from the stem-loop of the pri-miRNA. The Agrobacterium strain containing the MIR171::LUC reporter combined with the effector strain containing the 35S::rLOM1 or the empty vector were infiltrated into N. benthamiana leaves. After 3 days, luciferin was infiltrated into the same region and the LUC activities were monitored. Note that LUC activities were increased when MIR171A::LUC was combined with 35S::rLOM1, in comparison with the empty vector control. Because A-P3 fragment still responded to LOM1 while A-P4 did not, the LOM1-binding motifs are located in the region between −343 and −201. (B and C) LUC activities driven by truncated promoters of MIR171B (B) and MIR171C (C) in response to LOM1.(TIF)Click here for additional data file.Figure S7SPL9, LOMs and MIR171 show similar temporal expression pattern. (A) GUS staining of LOM1::rLOM1-GUS plants. GUS activity in newly emerged leaves was stronger than in elder ones. (B) Relative expression level of SPL9 and three LOMs in stem and inflorescence. Based on qRT-PCR, expression of SPL9 and three LOMs increased gradually along the stem from bottom to top (Int.1 to Int.3) and reached the highest level in inflorescence (Inf.). The expression level of each gene in Int.1 was set to 1. (C) MIR171 expression in main stem and inflorescence. Transcript abundance of pri-MIR171A, B and C increased gradually along the main stem from basal to apex and reached the highest levels in inflorescence. The expression level of each gene in Int.1 was set to 1. (D) The mature miR171 level was increasing with age, and the miR156 showed an opposite accumulation pattern. U6 was used as an internal reference.(TIF)Click here for additional data file.Figure S8LOM-SPL module plays a role in chlorophyll synthesis. (A) View of wild-type, 35S::MIR156F, 35S::MIR171B and 35S::MIR156F×35S::MIR171B plants. Plants were grown in long-day condition for 20 days. Bar = 1 cm. (B) Chlorophyll content of the genotypes shown in (A). FW, fresh weight. **P<0.01 compared with wild-type. Chlorophyll was measured as described previously 35S::Myc-rLOM2 transgenic Arabidopsis at indicated age under long day condition. This transgenic line showed severe development defects, including up-curly yellowish leaves, soft stem, late flowering and infertility. Arrows in (D) indicate one leaf exhibiting different colors. After the transition shown in (D), the newly emerged leaves showed a wild-type shape and color, suggesting normal chlorophyll content (E). Stars in (E) indicate the elder up-curly yellow-green rosette leaves. Scale bars, 1 cm.(TIF)Click here for additional data file.Figure S9MiR171s and LOMs in Gossypium raimondii. (A) Secondary structure of cotton miR171 predicted precursors. Sequences (80–200 bp) of 15 potential pri-miR171s derived from G. raimondii genome were analyzed using RNAfold program. (B) Phylogeny of LOM proteins. This unrooted phylogenetic tree of 12 LOM proteins was generated using Neighbour-joining method without distance corrections in Clustalw2 web service of EMBL-EBI. Sequences include three LOMs, At-HAM4 (At4G36710) from Arabidopsis thaliana, Ph-HAM (AY112704) from Petunia hybrida and seven cotton LOMs from Gossypium raimondii.(TIF)Click here for additional data file.Table S1Oligonucleotide primers used in the study.(DOCX)Click here for additional data file.


To further understand transcriptional regulation in chloroplasts, we here established a ChIP-based method in Arabidopsis thaliana and analyzed the binding pattern of a chloroplast sigma factor, SIG1. We found that SIG1 specifically binds to newly identified target promoters as well as to a set of promoters of genes whose mRNA expression is dependent on OsSIG1 in rice and that this binding changed in response to high-light stress. These results suggested that the ChIP-based approach is very useful in understanding transcriptional regulation of chloroplast genes and can overcome several problems posed by conventional methods.Chloroplasts have their own DNA and gene expression systems. Transcription in chloroplasts is regulated by two types of RNA polymerase, nuclear-encoded plastid RNA polymerase (NEP) and plastid-encoded plastid RNA polymerase (PEP), and multiple sigma factors for PEP. To study transcriptional regulation in chloroplasts, a molecular genetic approach has extensively been used. However, this method may include indirect effects, and it cannot be applied to the analysis of factors essential to survival. These limitations make understanding specific regulation by transcription factors difficult. Chromatin immunoprecipitation (ChIP) is a powerful and useful tool for obtaining information on transcription-factor binding sites; it can directly detect dynamic changes in their interaction patterns  Chloroplasts are plant organelles that originated from an endosymbiotic event involving an ancestral oxygen-evolving photosynthetic cyanobacterium. Chloroplasts thus have their own genomes and transcription-translation machineries of cyanobacterial origin. The chloroplast genomes of most higher plants are circular, double-stranded DNA molecules containing about 120 genes required for photosynthesis, gene expression, and some metabolic functions . Thus, crpoA, B, C1 and C2) are located in the chloroplast genome, but those encoding sigma factors, which are required for promoter recognition and transcription initiation, are located in the nuclear genome [Arabidopsis thaliana, six genes encode PEP sigma factors (SIG1–SIG6) and specific roles for each sigma factor in response to various developmental and/or environmental conditions have been clarified [In the chloroplasts of higher plants, there are at least two types of plastid RNA polymerase, called NEP (nuclear-encoded plastid RNA polymerase) and PEP (plastid-encoded plastid RNA polymerase) ,6. The Nr genome . In Arablarified –16.in vitro transcription assays, have clearly demonstrated specific interactions between promoter DNA and sigma factor protein(s) [psbD BLRP (blue-light responsive promoter) [To study the function of each sigma factor in transcriptional regulation of chloroplast genes, biochemical as well as molecular genetic approaches have mainly been used. Biochemical studies, such as gel-shift analyses and otein(s) ,18. In aotein(s) –23. For otein(s) –26. On tromoter) ,27. Howein vivo [Chromatin immunoprecipitation (ChIP) is a recently-established and convenient tool for obtaining information on target regions of DNA-binding proteins. This method can directly detect dynamic changes in interaction patterns in vivo . Initialin vivo  and was A. thaliana. We analyzed the binding pattern of SIG1, one of the sigma factors for PEP. We identified specific interactions between SIG1 and several chloroplast promoters and found that binding decreased in response to high-light stress. We concluded that ChIP analysis is useful to understand transcriptional regulation of chloroplast genes and can detect dynamic changes in DNA-protein interactions in chloroplasts in vivo.In this work, we used a ChIP-based method to further investigate the transcriptional regulation of chloroplast genes in E.coli  that was expressed in E.coli  and puriE.coli , and we Arabidopsis thaliana wild-type plants were cultivated under standard conditions and sampled after 1, 2, 3, and 4 weeks. As shown in Before we performed ChIP analysis to identify SIG1-dependent promoters, the accumulation of SIG1 during seedling development was examined. A. thaliana, we performed ChIP-qPCR analysis to detect the binding of SIG1 with its target promoters in vivo. Rosette leaves of wild-type A. thaliana plants grown in normal-light conditions for four weeks were treated with formaldehyde to fix DNA-protein interactions, whole-cell extracts containing total DNA-protein complexes were used for immunoprecipitation with the SIG1 antibody, then DNA was unlinked from the proteins. Purified DNA amounts were determined by quantitative PCR. To identify SIG1-dependent promoters across the chloroplast genome of A. thaliana, we designed 23 primer sets to amplify each upstream region of various chloroplast genes and quantified the binding levels of SIG1. In this method, the amount of purified DNA after immunoprecipitation reflected specific binding of a protein to target promoters. The amount of purified DNA was expressed as percent recovery against several dilutions of input DNA, which was plotted on standard curve obtained from qPCR analysis. Of 23 promoter regions, immunoprecipitated DNA could be detected for several specific promoters, rbcL, psbBT, clpP, psbEFLJ, and psaAB  for 1 h, and ChIP-qPCR analyses were performed. As shown in An advantage of the ChIP method is to detect changes in binding between DNA and a protein of interest. Therefore, we examined dynamic changes in the SIG1 binding level to target promoters under high-light stress conditions. Whole-cell extracts were prepared from rosette leaves treated with high light , it still has an advantage in that it can monitor in vivo characteristics of the protein of interest. In this study, we introduced the ChIP system to the research of chloroplasts in the model plant A. thaliana to further extend the field’s approaches for understanding transcriptional regulation. Transcriptional regulation by SIG1, a sigma factor in A. thaliana, was demonstrated, and both specific binding of SIG1 to target promoters and dynamic changes in its binding pattern could be clearly detected. Based on our results and recent reports applying the ChIP-based method to maize, tobacco and wheat [Many papers have examined transcriptional regulation in chloroplasts, but ChIP analysis has not yet contributed substantially to this field, in spite of its powerful ability to detect nd wheat –32, we eA. thaliana seedlings gradually increased over the course of leaf development (Zea mays) [The previous report demonstrated that SIG1 could accumulate cotyledon and true leaves of 12 days-old seedlings . In thiselopment , suggestea mays) . Expressea mays) –26, someOryza sativa), OsSIG1 could be disrupted by insertion of the Tos17 transposon, and the expressions of several chloroplast genes were markedly reduced [psaAB, psbBT, and psbEFLJ, which were also identified in this work as SIG1-bound promoters in A. thaliana. This consistency suggests that the role of SIG1 in chloroplast transcriptional regulation is similar in both monocot and dicot plants and that our ChIP-based method could successfully detect SIG1-target genes in vivo. In addition to the conserved genes discussed above, we identified other target genes, such as rbcL and clpP, which might be caused by evolutionary divergence. Previous studies demonstrated that transcription of rbcL could be mediated by SIG1 in vitro [clpP promoter was recognized by PEP, especially in later stages of leaf development [rbcL codes for a subunit of RUBISCO, and the essential role of the ClpP protein, a proteolytic subunit of the ATP-dependent Clp protease, has been confirmed in tobacco [A. thaliana might have specific and significant roles related to transcription of these genes at any stage. Although a T-DNA insertion line for sig1 was reported in A. thaliana [In a previous study in rice  were sterilized with 70% ethanol and 3% sodium hypochlorite before sowing on MS plates containing 0.4% Gelrite  or Jiffy 7 . After stratification at 4 °C for 24 h in the dark, the seeds were grown at 23 °C under continuous white light (50 μmol photons m−2 s−1) and harvested after 1, 2, 3, or 4 weeks. For high-light stress treatment, the plants grown for four weeks were exposed to 1,200 μmol photons m−2 s−1 at 23 °C for 1 h. For light-dark treatment, the plants grown for four weeks were dark-adapted for 18 h and re-illuminated under same light conditions (50 μmol photons m−2 s−1) for 1 h.Seeds of SIG1 full-length cDNA clone [NdeI and BamHI sites (underlined), respectively and was cloned into NdeI-BamHI-digested pET15b  to generate an in-frame fusion with the 6xHis-tag under the control of the T7 promoter. The recombinant SIG1 protein was expressed and purified in denatured condition using Ni-NTA agarose  following the manufacturer’s protocol. The rabbit polyclonal antibody was prepared at RIKEN Bioresource Center. Extraction of total proteins and immunoblotting were performed as previously reported [The coding region for SIG1 was amplified from the NA clone  with a preported .A. thaliana leaves were obtained as previously reported, with a few modifications [2 pieces in ~100 mL of ice-cold TBS . Then, Silwet L-77  was added to a final concentration of 0.01% (v/v) and mixed well. To crosslink DNA-protein complexes, formaldehyde was added immediately to a final concentration of 1% (v/v). Leaves were fixed by vacuum-infiltration (15 min × 2) at 700–800 mm Hg and washed (2 h × 2) with 150 mL of fresh TBS solution containing 0.3 M Glycine at 4 °C.Extracts from ications . BrieflyLeaves were filtered through two layers of Miracloth  into a test tube and frozen in liquid nitrogen. They were ground to a fine powder in liquid nitrogen with a Multibeads Shocker  at 1800 rpm (30 s × 2). The powder was resuspended in 3 mL of lysis buffer , and 10% glycerol) and homogenized by vortexing. The resultant slurry was filtered through two layers of Miracloth, and residual powder was washed with ~1 mL of lysis buffer and pooled. Crude extracts in the filtrate were sonicated on ice with a sonifier 250  on setting 3 and 60% duty cycle (15 s × 10) with cooling on ice (>1 min) between sonication cycles.g for 20 min at 4 °C. The supernatant was further purified by centrifugation once more. Final (combined) supernatants were divided into aliquots (~400 μL), flash-frozen in liquid nitrogen, and stored at −80 °C. Protein extract concentrations were determined with the BCA protein assay system .The sample was then aliquoted into five eppendorf tubes (~800 μL each) and centrifuged at 15,000ChIP and qPCR analyses were performed as described previously , except A. thaliana chloroplasts is very useful for the identification of direct-target genes for proteins of interest as well as dynamic changes in in vivo binding patterns. In particular, this method could be a powerful tool for the identification of target promoters for essential transcription factors. The results of this study also suggested that SIG1 could be released from target promoters in response to high-light stress, whereas the SIG1 protein still accumulated under the same condition, implying a complicated regulatory system in chloroplasts. Together with other approaches, ChIP methods could clarify these regulations.Our results indicated that the ChIP-based method established here for


Here, we show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of the adjacent gene, TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. When Homie is deleted in the context of a large transgene that mimics both eve and TER94 regulation, TER94 is repressed. This repression depends on the eve PRE. Ubiquitous TER94 expression is “replaced” by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed. Repression of TER94 is attributable to spreading of the eve Pc domain into the TER94 locus, accompanied by an increase in histone H3 trimethylation at lysine 27. Other PREs can functionally replace the eve PRE, and other insulators can block PRE-dependent repression in this context. The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication. Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions. Thus, insulator activities in a native context include enhancer blocking and enhancer-promoter facilitation, as well as preventing the spread of repressive chromatin.Insulators can block the action of enhancers on promoters and the spreading of repressive chromatin, as well as facilitating specific enhancer-promoter interactions. However, recent studies have called into question whether the activities ascribed to insulators in model transgene assays actually reflect their functions in the genome. The Drosophila  eve and TER94, which are expressed in very different patterns. Also, the eve gene is a Polycomb (Pc) domain, a specialized type of chromatin that is found in many places throughout the genome. We show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. Each of these activities are consistent with those seen with model transgenes, and other known insulators can provide these functions in this context. This work provides a novel and convincing example of the normal role of insulators in regulating the eukaryotic genome, as well as providing insights into their mechanisms of action.Insulators are specialized DNA elements that can separate the genome into functional units. Most of the current thinking about these elements comes from studies done with model transgenes. Studies of insulators within the specialized Hox gene complexes have suggested that model transgenes can reflect the normal functions of these elements in their native context. However, recent genome-wide studies have called this into question. This work analyzes the native function of an insulator that resides between the Drosophila genes  Cis-regulatory modules  are bound by DNA-binding transcription factors that coordinately recruit coactivators and corepressors. Enhancers communicate with basal promoters at least in part through a looping out of intervening DNA, allowing them to act over large distances along a chromosome, or even in Insulators typically show “barrier” function that prevents the spread of heterochromatin, as well as enhancer blocking activity, in model transgene assays transRepressive chromatin structures include heterochromatin and Polycomb (Pc) chromatin, which constitutes a form of epigenetic transcriptional memory, stabilizing developmental fate choices, among other functions. Pc chromatin is maintained through the recruitment of Pc-group (PcG) gene products to PREs The functions of PREs and insulators have been studied within Drosophila Hox genes Drosophila, insulator proteins include dCTCF (CCCTC-binding factor), Mod(mdg4)67.2, Su(Hw) (Suppressor of hairy wing), CP190 , BEAF32 (boundary element-associated factor 32), and Zw5 (Zeste-white-5). Recent genome-wide studies also implicate the mitotic spindle protein Chromator β-globinIgf2 and H19 loci Genome-wide chromatin immunoprecipitation (ChIP) analysis of the locations of insulator binding proteins show a wide range of binding patterns even skipped (eve) locus is a well-defined Pc domain based on genome-wide analysis eve PRE at its 3′ end eve regulatory elements from the constitutively expressed neighboring gene TER94, and/or to prevent ectopic activation of eve by TER94 enhancers. This insulator was shown to have 3 distinct activities in model transgene assays. In addition to enhancer blocking, it causes homing of P-element transgenes to the endogenous eve neighborhood, for which it was nicknamed Homie (Homing insulator at eve). Furthermore, from within a several megabase region flanking endogenous eve, it causes long-range interactions of transgenic promoters with endogenous eve enhancers The eve-TER94 locus that mimics the normal regulation of both genes. Using this artificial locus, we show that Homie functions as a PRE blocker to protect TER94 from repression due to spreading of the eve Pc domain. Heterologous insulators and PREs can substitute for Homie and the eve PRE, suggesting that limiting the range of PRE action is an important function of insulators generally. Homie also prevents the eve enhancers from activating TER94 in specific tissues. Furthermore, Homie facilitates normal eve expression by augmenting communication between the eve promoter and its 3′ enhancers, likely through a chromosomal looping mechanism.Homie shares properties with other insulators based on model transgene assays and, like many other putative insulators, is situated close to both a transcription start site (TSS) and a PRE. Thus, understanding Homie's function in its native context can illuminate many of the mysteries that surround this enigmatic group of regulatory elements. In order to investigate its native function, we constructed a transgenic TER94 gene and the highly patterned eve gene. We find that blocking the spread of repressive Polycomb chromatin by Homie is critical for normal TER94 promoter expression. In addition to exhibiting the canonical insulator activities in a near-native context, we find that Homie facilitates certain aspects of normal eve expression, and we present a model for how this occurs.Insulators are generally considered to have two major functions. First, they can shield promoters from the effects of distal enhancers. Second, they can block the spread of repressive chromatin. Here, we investigate the roles that these activities play in the normal functions of an insulator (Homie) located between the ubiquitously expressed eve 3′ insulator Homie, we employed a pseudo-locus that contains all the regulatory DNA necessary for normal expression of both eveTER94eve TSS, from the 5′-most enhancer of eve to the 3rd exon of TER94. In addition to all of the eve enhancers, this region contains a characterized PRE eve side) of Homie TER94 promoter and TSS, which are sufficient for ubiquitous expression, augmented by enhancers in the TER94 introns (data not shown). The eve coding region was replaced with lacZ coding DNA, and the 3rd exon of TER94 was fused with the EGFP coding region . At stage 10 and later, strong expression is also observed throughout the brain and central nervous system (CNS) TER94-GFP expression from our transgene simulates endogenous TER94 expression  female flies are crossed with transgene-carrying males, so that there is no maternal GFP RNA in the progeny. Two chromosomal locations were analyzed. In both cases, GFP expression was reduced when Homie was deleted  for the eve 3′ PRE. In fact, the eve promoter-proximal region has PRE-like properties Because of the relatively low level of expression, we quantified GFP RNA using RT-PCR. Embryos from three timed collections were analyzed: 2–3 hr. (stages 5–6) and 4–6 hr. (stages 9–11) after egg deposition, and stages 13–15. The effect of Homie deletion paralleled those described above for both ovaries and embryos, in that expression was repressed. However, unlike in ovaries, when both Homie and the PRE were deleted, TER94 promoter by the eve PRE , it did work in the opposite orientation (scs(inv)”). Fab-8 and gypsy showed a minor directionality in their effectiveness (not shown). Restoration of GFP expression is somewhat weaker for scs' and scs(inv) than for gypsy and Fab-7, indicating that they only partially block eve PRE action. Despite differences in efficiency, blocking of PRE action in this context is a shared property of insulators.Are the functions of Homie seen in our assays unique, or are they shared among insulators? In order to test this, we replaced Homie with other known insulators. As a negative control, a >500 bp stretch of λ phage DNA was tested. It had no effect on repression of the  eve PRE  “ΔHomie”E  expression seems clearly to be caused by deletion of Homie , CP190, Mod(mdg4)67.2, BEAF32, CTCF, and GAF eve locus eve, which led to a decrease in H3K27me3. In contrast, we found that deletion of Homie did not cause a significant reduction in H3K27me3 levels in the eve-lacZ region of our pseudo-locus, either in embryos or in ovaries , active enhancers prevent the establishment of repressive Pc-dependent chromatin in early embryos. Conversely, in tissues where such repressive chromatin has been established, such as in parts of the CNS and imaginal discs, later-acting enhancers are repressed eve locus? Extensive dissection of eve regulatory DNA has not identified enhancers that can drive expression outside the normal eve pattern, arguing against such a close analogy with the BX-C. However, in PcG mutants, eve is ectopically expressed throughout the late-stage embryonic CNS eve, as they do the Hox complexes.It is intriguing that the eve PRE activity, we found that in a transgenic context, both the 3′ PRE and the PRE-like eve promoter region could facilitate positive maintenance of an eve CNS enhancer from embryonic to larval stages, as well as prevent ectopic expression in cells that normally do not express eveeve 3′ PRE was found to require the DNA binding PcG protein Pleiohomeotic rather than Trithorax-group members for positive maintenance eve 3′ PRE on enhancer activity. In this case, it facilitates TER94-GFP expression in an eve pattern when Homie is removed . One possible explanation for this is that eve enhancers have evolved to function within a Pc domain, and they may be better able to activate the TER94 promoter when the Pc domain spreads over it. In this view, PREs facilitate both the on state and the off state, yet the chromatin may be differently modified in the two cases. This model is similar to the “integration model” proposed for how heterochromatin can have a positive effect on the expression of genes that normally reside within it In our previous studies of eve 3′ PRE, an arrangement that is reminiscent of some boundaries in the BX-C. The mammalian homologs of eve, evx1 and evx2, are located at the 3′ end of the HOX-A and HOX-D clusters, respectively, suggesting that the ancestral eve locus was part of a Hox cluster eve insulator-PRE relationship, recent studies identified an enhancer-blocking activity between evx2 and Hoxd13Homie sits adjacent to the scs nor gypsy could functionally replace Fab-7eve Pc domain, reminiscent of the shielding of transgenic reporter genes from repressive effects at some insertion sites scs' showed slightly weaker activity than either gypsy, Fab-7, or Fab-8, while the activity of scs was highly orientation-dependent  conformation that allows the eve promoter to better access the 3′ enhancers is stabilized by the presence of Homie. One possible conformation is a loop between the eve promoter region and Homie  was created as follows  in situ hybridization using DIG-labeled anti-sense RNA probes against either lacZ or GFP. Expression patterns were visualized by alkaline phosphatase-conjugated anti-DIG with BCIP and NBT as substrates (Roche Applied Science).Embryos were collected at time points described in figure legends, and subjected to GFP expression was detected by fluorescence microscopy in ovaries dissected from 1–2 day-old females. In some cases, expression was also detected using anti-GFP antibody staining (Roche Applied Science), analyzed by confocal microscopy (Zeiss) of material in DAPI-containing mounting medium.Ovaries were dissected from 2–3 day-old females. Fifty ovaries were cross-linked in 1.8% formaldehyde in PBS for 10 min. After sonication so as to produce a peak near 500 bp in the DNA fragment size distribution, isolated chromatin was immunoprecipitated with anti-H3K27me3 (EMD Millipore), and with rabbit IgG (Jackson ImmunoResearch) as a negative control. Precipitated chromatin samples were collected using ProteinG magnetic beads (EMD Millipore). Immunoprecipitated DNA samples were dissolved in 20–50 µl TE, and 1 µl was used for each PCR reaction. Either duplicate or triplicate samples were analyzed by real-time PCR , using SYBR Green Master Mix with ROX dye (Roche Applied Science). Data were analyzed with StepOne software (Life Technologies), using the standard curve method. Standard deviations were calculated using Excel software (Microsoft). Embryo ChIP analysis was described previously eve were determined by adding the relative errors in quadrature; that is, by taking the sum of the squares of the relative standard deviations (the standard deviations divided by their respective averages) to give the square of the relative standard deviation of the ratio.Specific ChIP signals were determined by subtracting the average non-specific IgG signal from the average α-H3K27me3 signal, with standard deviations combined by adding. Errors bars for specific signals relative to that of endogenous TCCAGTCCGGATAACTCCTTGAAC and TGTAGAACTCCTTCTCCAAGCGAC for the endogenous eve coding region, TGAAGCCACCGCGTGGTATTCTTA and TTTGGACATGATCTCCGGTCCGTT for the endogenous TER94 coding region, GCTGTGCCGAAATGGTCCATCAAA and TACTGACGAAACGCCTGCCAGTAT for the transgenic eve-lacZ coding region, and GGGCACAAGCTGGAGTACAACTACAA and TGGCGGATCTTGAAGTTCACCTTG for the transgenic TER94-GFP coding region.The following primers were used: RpL32 (a.k.a. RP49), was used to normalize GFP RNA levels. The primers listed above for TER94-GFP were used for GFP, and AAGCCCAAGGGTATCGACAACAGA and TGCACCAGGAACTTCTTGAATCCG were used for RpL32.Total RNA was purified from either five pairs of ovaries from 2–3 day-old females or 10–20 µl of dechorionated embryos for each data point, using an RNA purification kit (Roche Applied Science). RNA was eluted in 50–100 µl elution buffer and stored at −80°C. cDNA was synthesized using the Transcriptor first strand cDNA synthesis kit (Roche Applied Science), and quantified by real-time PCR as described above. A constitutively expressed RNA, Figure S1TER94 promoter from eve PRE activity. A, B: similar effects on TER94 promoter activity are seen at two chromosomal landing sites (different from that used in eve-like expression (visible in the other lines when Homie is deleted) difficult to detect. The attP sites used are at cytological locations 74A2 (A) and 30B5 (B). A longer staining reaction time than that used in B, “ΔHomie”, showed both expression in stripes and in eve-expressing cells of the mesoderm (not shown).Homie shields the (TIF)Click here for additional data file.Figure S2eve-TER94 locus is heterozygous and paternally derived, embryonic TER94-GFP expression is repressed in the absence of Homie as it is in ovaries, but this repression is not dependent on the 3′ PRE. Expression of GFP RNA in the transgenic lines indicated on the left from two different transgene landing sites (in A and B) is visualized at 3 different embryonic stages  by in situ hybridization. The levels of GFP RNA (extracted from 2–3 hr-old embryos), quantified in triplicate, are show on the right . The quantitation shown in the graph suggests that ubiquitous zygotic expression is repressed when Homie is deleted. However, unlike for maternally derived expression . Note that at this landing site, eve-like stripe expression is present from the intact transgene, indicating that some eve enhancers can work on the TER94 promoter when Homie is present, at least at some landing sites. Quantitation, shown in the graph, indicates that overall zygotic expression is reduced when Homie is deleted, consistent with repression of ubiquitous expression driven by TER94 enhancers present in the transgene. As with the other landing site, this repression is not dependent on the PRE, but may be due to redundant PRE activity.When the transgenic ocus see . B: zygo(TIF)Click here for additional data file.Figure S3TER94-GFP is expressed throughout oogenesis in both germ cells and somatic epithelial follicle cells. Ovarioles dissected from female flies (carrying transgenic TER94-GFP in the context of the transgene diagrammed in (TIF)Click here for additional data file.Figure S4TER94-GFP, while additional deletion of the PRE causes restoration of TER94 promoter activity. The graph at the bottom shows, on a log scale, the results of quantitation, as in Homie blocks PRE action in ovaries. Fluorescence in ovarioles from transgenic lines carrying the indicated transgenes, at two different chromosomal insertion sites , distinct from that shown in (TIF)Click here for additional data file.Figure S5eve 3′ enhancer action on the eve promoter. Expression of lacZ RNA driven by the eve promoter from the transgene diagrammed in in situ hybridization. A, B: results from two different transgene landing sites , distinct from that shown in C: results from two of the Homie-replacement lines shown in gypsy, but not scs, facilitates 3′ enhancer activity.Homie facilitates (TIF)Click here for additional data file.Figure S6A are listed the details of construction of the eve-TER94 pseudo-locus transgene and it primary derivatives, as described in B are listed the details of modifications to that construct, as described in Construct details. In (TIF)Click here for additional data file.


Shigella flexneri, upon contact of the bacteria with host cells, and its deregulation, as in ipaB mutants, specifically increases transcription of a set of effector-encoding genes controlled by MxiE, an activator of the AraC family, and IpgC, the chaperone of the IpaB and IpaC translocators. Thirteen genes carried by the virulence plasmid  and five genes carried by the chromosome (ipaHa-e) are regulated by the T3SA activity. A conserved 17-bp MxiE box is present 5′ of most of these genes. To characterize the promoter activity of these MxiE box-containing regions, similar ∼67-bp DNA fragments encompassing the MxiE box of 14 MxiE-regulated genes were cloned 5′ of lacZ in a promoter probe plasmid; β-galactosidase activity detected in wild-type and ipaB strains harboring these plasmids indicated that most MxiE box-carrying regions contain a promoter regulated by the T3SA activity and that the relative strengths of these promoters cover an eight-fold range. The various MxiE boxes exhibiting up to three differences as compared to the MxiE box consensus sequence were introduced into the ipaH9.8 promoter without affecting its activity, suggesting that they are equally efficient in promoter activation. In contrast, all nucleotides conserved among MxiE boxes were found to be involved in MxiE-dependent promoter activity. In addition, we present evidence that the 5′ UTRs of four MxiE-regulated genes enhance expression of the downstream gene, presumably by preventing degradation of the mRNA, and the 5′ UTRs of two other genes carry an ancillary promoter.Activation of the type III secretion apparatus (T3SA) of  The type III secretion (T3S) pathway is used by numerous Gram-negative bacteria to deliver virulence proteins to the membrane or the cytoplasm of cells of their host, where they interfere with cellular signaling pathways. T3S systems comprise (i) a secretion apparatus (T3SA) spanning the bacterial envelope, (ii) translocators transiting through the T3SA that are inserted into the membrane of the host cell in which they form a pore, (iii) effectors injected into host cells via the T3SA and the translocator pore, (iv) molecular chaperones associating with translocators and some effectors within the bacterial cytoplasm, (iv) and transcription regulators Shigella use a T3S system to invade the colonic epithelium in humans and cause bacillary dysentery S. flexneri T3S system is composed of ∼50 genes, most of which are carried by a virulence plasmid i.e. constitutively active, in ipaB and ipaD mutants Members of the genus Escherichia coliTranscription of genes in response to the activation of the T3SA is dependent upon MxiE and IpgC that are produced independently of the TS3A activity ospB, ospC1, ospD2, ospD3, ospE1, ospE2, ospF, ospG, virA, ipaH1.4, ipaH4.5, ipaH7.8 and ipaH9.8 and some chromosomal ipaH genes ipaB mutant was abolished in an ipaB mxiE mutant ospC1, virA and ipaH9.8 mRNA ospB, ospE1, ospE2, ospF, ipaH4.5, ipaH7.8 and the five different ipaH genes carried by the chromosome ospG  of four MxiE-regulated genes enhance expression of the downstream gene and the 5′ UTRs of two other genes contain an ancillary promoter.Genes under the control of MxiE include the virulence plasmid genes ome ospG , the traS. flexneri strains are derivatives of M90T-Sm (wild-type) ipaB4), SF1070 (ipaB4 mxiE2) −1.E. coli and S. flexneri strains were performed according to standard methods. DNA fragments were amplified from the virulence plasmid and the chromosome of the S. flexneri strain M90T-Sm (serotype 2a) using primers derived from the sequence of the virulence plasmid pWR100 of M90T S. flexneri strain 8401 (serotype 5b) ipaH genes from M90T were identical to those of 8401. There are five different chromosomal ipaH genes, some of which are duplicated in different strains; the same genes are annotated with different names in different genomes and, for the sake of clarity, we used the letter-based nomenclature ipaH genes are designated by the same letter , plasmid constructions and transformations of e letter . The proipaH9.8 promoter was also inserted between the NcoI and HindIII sites of pQF50.For the analysis of minimal promoter regions, DNA fragments extending from 8 bp 5′ of the MxiE box to 11 bp 3′ of the proposed transcription start site of 14 MxiE-regulated genes were inserted between the NcoI and KpnI sites of pQF50 . The fraipaH9.8 promoter was first inserted between the BamHI and HindIII sites of pQF50 to construct pCB23. Double-stranded oligonucleotides were then inserted between the NcoI and BamHI sites of pCB23 to reconstruct a complete ipaH9.8 promoter region and its 5′ UTR (from −57 to +85) inserted between the NcoI and HindIII sites of pQF50 and carrying a BamHI site between the MxiE and −10 boxes. The presence of the sequence 5′-GGATCC-3′ (BamHI) instead of 5′-TTATCC-3′ in positions −22 to −17 did not affect regulation of the promoter. Double-stranded oligonucleotides carrying variations or mutations in the MxiE box were inserted between the NcoI and BamHI sites of pCB23 to construct plasmids carrying a reconstituted ipaH9.8 promoter (from −57 to +85) harboring different MxiE boxes.For the analysis of specific nucleotides within the MxiE box, the region extending from positions −16 to +85 of the For the analysis of the promoter activity of 5′ UTRs, DNA fragments encompassing the region located between position +2, with respect to the transcription start site of each promoter, and position −13, with respect to the translation start site of each gene, were inserted between the BamHI and HindIII sites of pQF50.lacZ transcribed from the lac promoter, a DNA fragment corresponding to nucleotides −39 to +1 of the lac promoter (5′-TTATTTGCTTTGTGAGCGGATAACAATTATAATAGATTCA-3′) was first inserted between the NcoI and BamHI sites of pQF50 to construct pCB55. DNA fragments encompassing the region encoding the 5′ UTR (as defined above) of each gene were then inserted between the BamHI and HindIII sites of pCB55, thereby placing each 5′ UTR between the lac promoter and lacZ. Coordinates of the 3′ end of the cloned 5′ UTR with respect to the original promoter are: ipaH7.8, +260; ipaH9.8, +85; ipaHb, +331; ipaHc, +85; ipaHd, +153; ipaHe, +74; ospB, +45; ospC1, +218; virA, +46. In the 5′ UTR of ipaHe, the C at position +70 was changed to a G to prevent pairing of the 3′ region of the 5′ UTR with the ribosome binding site of lacZ.For the analysis of the role of 5′ UTRs on expression of ipaH9.8 5′ UTR, DNA fragments were inserted between the BamHI and HindIII sites of pCB24 to construct plasmids carrying an ipaH9.8 promoter and various portions of the ipaH9.8 5′ UTR.For the deletion analysis of the 3′ part of the o-nitrophenyl-β-D-galactopyranoside as described The β-galactosidase activity present in bacteria grown for 16 h at 37°C in TCS medium was assayed by using the substrate http://mobyle.pasteur.fr/cgi-bin/portal.py#forms::mfold.Prediction of potential secondary structures of 5′ UTRs were performed with the program mfold lacZ in the promoter-probe vector pQF50 i.e. not taking into account duplicated copies of chromosomal ipaH genes (ipaHe was not obtained in pQF50. Recombinant plasmids were introduced into the S. flexneri strains M90T-Sm (wt) and SF1076 (ipaB4); the T3SA is constitutively active in ipaB mutants, leading to the increased transcription of MxiE-regulated genes in these mutants as compared to the wild-type strain ipaB strain contained increased activities, from 50 to 700 Miller units, except for the strain harboring the plasmid carrying the MxiE box-containing region of ospG (ipaB (58 Miller units) strains suggest that the MxiE box containing region 5′ of ospG does not contain a promoter regulated by the T3SA activity, consistent with previous observations made with a plasmid carrying a fragment encompassing 230 bp 5′ and 100 bp 3′ of the ospG translation start site ipaB strain harboring each plasmid suggest that the relative strengths of MxiE-regulated promoters cover an eight-fold range, with ipaH7.8>ospB, ospE1, ospF, ipaH9.8, ipaHa, ipaHd, ipaHc>virA, ospC1, ospE2>ipaH4.5, ipaHb.Transcriptional fusions to some MxiE-regulated genes constructed previously carried DNA fragments of different sizes and varying in their respective 5′ and 3′ ends aH genes , only th of ospG . In the ipaB strains harboring plasmids carrying these minimal promoter regions inserted at the KpnI site of pQF50 were lower than the ones obtained previously with derivatives of pQF50 carrying 300 to 500-bp fragments encompassing the MxiE box inserted at the HindIII site (located 12 bp downstream from the KpnI site) lacZ ribosome-binding site  in pQF50  is functional in the context of the ipaH9.8 promoter. Furthermore, β-galactosidase activities detected in the ipaB strain harboring plasmids carrying different MxiE boxes (other than that of ospG) were in the same range, from 10,000 to 16,000 Miller units, suggesting that all these MxiE boxes have the same efficiency for the MxiE-dependent activation of the ipaH9.8 promoter. The lack of activity of the ipaH9.8 promoter carrying the ospG MxiE box suggests that this box, which exhibits five base substitutions as compared to the consensus sequence, is not functional; this result is consistent with the weak and unregulated promoter activity observed for the MxiE box-containing region of ospG  and β-galactosidase activity was assayed in derivatives of the wild-type and ipaB strains harboring each plasmid. Mutations G1T and T7A led to a two-fold decrease in β-galactosidase activity, mutations T8A, T9A and A14C to a three-fold decrease and mutations G6C, T10A, T12A and A16C to a six-fold decrease, abolishing almost completely the activity of the promoter in the ipaB mutant , an A (six occurrences) or a T (one occurrence) (ipaH9.8 promoter (from −57 to +85) was changed to A, G or T. The C15A mutation had no effects on the promoter activity in the wild-type and ipaB strains, whereas the C15G mutation drastically reduced the promoter activity in the ipaB strain . The increased activity observed upon introducing a T at position 15 in the ipaH9.8 promoter might be due to the creation of an artificial −35 region. Overall, these results on the effect of the nucleotide at position 15 on the activity of the ipaH9.8 promoter are consistent with the frequent occurrence of a C and an A and the exclusion of a G and a T (with the exception of the MxiE box of ospC1) at this position in MxiE boxes.Among functional MxiE boxes, urrence) . To inveB strain . UnexpecB strain . With thipaB strains harboring plasmids carrying the ipaH9.8 promoter either alone (from −57 to +11) or together with its 5′ UTR (from −57 to +85) inserted 5′ of the HindIII site in pQF50 contained 1,700 and 15,000 Miller units of β-galactosidase activity, respectively. This observation led us to investigate the role of the 5′ UTR of ipaH9.8. The increased β-galactosidase activity observed with the plasmid carrying the 5′ UTR could potentially be due to either an ancillary promoter carried by the DNA region corresponding to the 5′ UTR or an effect of the 5′ UTR mRNA on expression of the downstream gene. The study of the ipaH9.8 5′ UTR was extended to the 5′ UTRs of other MxiE-regulated genes displaying a wide heterogeneity in length  β-galactosidase activities were detected in the wild-type strain harboring plasmids carrying the ipaHb, ipaHc, ipaHd, ipaH9.8 and virA 5′ UTRs, suggesting that these regions do not contain an active promoter  was first cloned 5′ of lacZ in pQF50 and DNA fragments encoding the 5′ UTRs were inserted between this lac promoter and lacZ. The lac promoter is constitutively active in S. flexneri, due to the absence of the lacI gene. The wild-type strain harboring the plasmid carrying the lac promoter alone contained approximately 600 Miller units of β-galactosidase activity and this activity was not increased upon insertion of the 5′ UTR of ipaH7.8, ipaHb, ipaHe, ospB and ospC1 , which also indicated that the increased expression of lacZ observed in the presence of these 5′ UTRs is not controlled by the T3SA activity.To investigate the potential role of the 5′ UTR mRNA on expression of the downstream gene, we placed the 5′ UTRs of MxiE-regulated genes under the control of a different promoter. A minimal nd ospC1 . In cont ipaH9.8 , even th ipaH9.8 . As expeipaH9.8 5′ UTR responsible for the increased expression of the reporter gene, we constructed plasmids carrying the ipaH9.8 promoter with shortened versions of its 5′ UTR, by truncating the 5′ UTR from its 3′ extremity. These plasmids were introduced into the wild-type and ipaB strains. Derivatives of the ipaB strain harboring plasmids encoding a 5′ UTR ending at positions +85, +66, +56, +39 and +32 all produced high amounts of β-galactosidase  the ipaH9.8 and ipaHc promoters differ mostly by an insertion or a deletion of one bp 5′ of the −10 box and exhibit similar activities; (ii) deleting one bp 3′ of the ipaH9.8 MxiE box did not affect the activity of the promoter (data not shown); (iii) and, among MxiE-regulated promoters tested, there are no correlations between the strength of the promoter and the size of the region located between the MxiE box and the −10 box. Thus, elements controlling the relative strength of each promoter remain to be identified. In the case of the almost identical ospE1 and ospE2 promoters, the lower activity of the ospE2 promoter might be due to the presence of nucleotides TA at positions +2 and +3 in ospE2 instead of AT in ospE1. The weak activity of the ipaH4.5 and ipaHb promoters might be due to an altered −10 box in the ipaH4.5 promoter and the presence of a C at the expected position for the transcription start site of the ipaHb promoter, which remains to be tested.β-galactosidase activities detected in the ospG, ipaH4.5 and ipaHb MxiE box-containing regions and, in the case of ospG, this promoter activity was not regulated by the T3SA activity. The ipaH4.5 and ospG genes are located downstream from ipaH7.8 and ipaH9.8, respectively, and the MxiE-regulated expression of ipaH4.5 and ospGipaH7.8 and ipaH9.8 promoters. No other MxiE boxes were detected upstream from ipaHb, suggesting that this gene is transcribed much less than other MxiE-regulated genes.Only weak promoter activity was exhibited by the ipaH9.8 promoter was also observed upon replacement of T8, T9 and A14 (three-fold decrease) and G1 and T7 (two-fold decrease). Thus, changes of any of the strictly conserved nucleotides among MxiE boxes decreased the promoter activity. There is a remarkable stretch of seven consecutive Ts (from T7 to T13) in the MxiE box consensus sequence and this stretch of Ts is conserved in 12 MxiE boxes (ospF (harboring T11A and T13A) and virA (harboring T11C), both of which were shown here to be as functional as MxiE boxes containing an uninterrupted stretch of Ts. Furthermore, introducing the mutations T11A or T13A in the ipaH9.8 promoter did not affect its activity (data not shown). Similar β-galactosidase activities were detected when position 15 of the MxiE box of the ipaH9.8 promoter was occupied by either a C or an A, consistent with the preferential occurrence of these nucleotides at this position in MxiE boxes. Introducing a G at position 15 abolished promoter activation, which is also consistent with the observation that a G is absent at this position in MxiE boxes. The C15T mutation increased the ipaH9.8 promoter activity in a manner that was not dependent upon MxiE; because a T is present at position 15 in the ospC1 MxiE box and the ospC1 promoter did not exhibit a high activity in the wild-type strain, the increased basal activity of the ipaH9.8 promoter carrying a T at position 15 is tentatively interpreted as the result of the creation of an artificial promoter −35 region.Among positions conserved between MxiE boxes, G6, T10, T12 and A16 appear to be essential for promoter activity. A decreased activity of the iE boxes . HoweveripaH9.8 induced to a marked increased in the production of β-galactosidase led us to investigate the potential role of the 5′ UTR of MxiE-regulated genes. The DNA regions encoding the ospC1 and ipaH7.8 5′ UTRs were found to carry ancillary promoters that are not controlled by the T3SA activity. Comparison of β-galactosidase activities produced by bacteria harboring plasmids containing either the MxiE-regulated promoters or the 5′ UTR-carried promoters must take into account that MxiE-regulated promoters were inserted at the KpnI site whereas 5′ UTR were inserted at the HindIII site of pQF50. Indeed, in the course of this study, we found that the presence of the KpnI-HindIII fragment of pQF50 was responsible for a six-fold decrease in β-galactosidase production, presumably due to the formation of a secondary structure in the mRNA masking the lacZ ribosome-binding site. By using this six-fold factor to standardize β-galactosidase activities, calculated values for promoters carried by the opsC1 and ipaH7.8 5′ UTRs cloned at the KpnI site are ∼50 Miller units (in both the wild-type and ipaB strains). This estimate is in the same range as the β-galactosidase activities detected for ospC1 and ipaH7.8 MxiE-regulated promoters in the wild-type strain and is much lower than the activities detected for these promoters in the ipaB strain (150 Miller units for the opsC1 promoter and 750 Miller units for the ipaH7.8 promoter). These considerations suggest that the activity of 5′ UTR-carried promoters is similar to the basal activity of MxiE-regulated promoters when the T3SA is not active; however, the contribution of these 5′ UTR-carried promoters to the overall transcription of ospC1 and ipaH7.8 is very weak once the T3SA is activated.The observation that the presence of the 5′ UTR of virA, ipaHd, ipaHc and ipaH9.8 between the lac promoter and lacZ led to three- to twelve-fold increases in β-galactosidase activity. For ipaH9.8, the region responsible for this effect was mapped to the first 32 nucleotides of the 5′ UTR that are predicted to fold into a stem and loop structure. The same sequence is present in the ipaHc 5′ UTR and is probably involved in the increased expression of lacZ also observed with this 5′ UTR. A stem and loop structure formed at the 5′ extremity of the E. coli ompA and papA 5′ UTRs was demonstrated to function as a potent mRNA stabilizer virA and ipaHd 5′ UTRs between the lac promoter and lacZ have not been characterized. Various potential secondary structures are predicted in the mRNA corresponding to the ipaHd 5′ UTR, however, they are not located exactly at the 5′ extremity of the 5′ UTR and many secondary structures are possible in the 46-nucleotide virA 5′ UTR containing 40 As and Ts. In Listeria monocytogenes, the inlA, actA and hly 5′ UTRs have been shown to have a positive effect on expression of downstream genes, although specific sequences or mRNA secondary structures responsible for this effect have not been identified Insertion of the 5′ UTRs of cis-acting elements, including MxiE boxes of different sequences, promoters of different strengths and 5′ UTRs of different lengths and functions. Combination of these elements might permit the control of gene expression over a wider range. All natural MxiE boxes appear to have the same efficiency for activation of the ipaH9.8 promoter, whereas mutations introduced in any of the positions conserved among MxiE boxes decreased the ipaH9.8 promoter activity in the ipaB strain. The 5′ UTRs of ipaH9.8, ipaHc and ipaHd were found to increase expression of the downstream gene by an approximately ten-fold factor, most likely by functioning as mRNA stabilizers.In conclusion, expression of MxiE-regulated genes involves an array of