MHC class I transcription is regulated by two distinct types of regulatory pathways: 1) tissue-specific pathways that establish constitutive levels of expression within a given tissue and 2) dynamically modulated pathways that increase or decrease expression within that tissue in response to hormonal or cytokine mediated stimuli. These sets of pathways target distinct upstream regulatory elements, have distinct basal transcription factor requirements, and utilize discrete sets of transcription start sites within an extended core promoter.in vitro transcription assays in HeLa, HeLa/CIITA, and tsBN462 of various promoter constructs. We have identified three novel MHC class I regulatory elements , located downstream of the major transcription start sites, that contribute to the regulation of both constitutive and activated MHC class I expression. These elements located at the 3′ end of the core promoter preferentially regulate the multiple transcription start sites clustered at the 5′ end of the core promoter.We studied regulatory elements within the MHC class I promoter by cellular transfection and Three novel downstream elements , located between +1 and +32 bp, regulate both constitutive and activated MHC class I gene expression by selectively increasing usage of transcription start sites clustered at the 5′ end of the core promoter upstream of +1 bp. Results indicate that the downstream elements preferentially regulate TAF1-dependent, relative to TAF1-independent, transcription. Transcription of genes by RNA polymerase II is a highly regulated process that requires the integration of multiple signaling pathways in order to generate a level of expression appropriate for a given set of environmental and cellular conditions. An important component of this regulation is the specific interactions between transcription factors and promoter DNA sequences that result in the assembly of the transcription initiation machinery The structures of core promoters vary but some features have contributed to our understanding of their function. In many promoters, TATA boxes and Inr elements function to establish a transcriptional start site As the site of assembly of the transcription initiation machinery, the core promoter serves as a molecular platform to integrate regulatory signals delivered by upstream silencer and enhancer elements to appropriately adjust the level of promoter activity The level of transcription for any given gene depends on the integration of its different regulatory pathways at the core promoter. Two related mechanisms exist that allow the core promoter to dictate these levels. First, some core promoters recruit distinct transcriptional machinery under different conditions. For example, IFN induced expression of MHC class I genes is mediated by CIITA, a non-DNA-binding co-activator that interacts with constitutively expressed RFX and ATF trans-acting factors already bound at the RFX/CRE site Second, in some cases, core promoter regions differ in their pattern of transcription start sites (TSS). In yeast, it has been reported that the his3 promoter initiates transcription at two distinct TATA elements that are differentially utilized under constitutive or activated conditions To delineate the role of the core promoter in these differing regulatory environments, we previously characterized elements within the core promoter of the MHC class I gene. The isolated segment between −50 bp and +1 bp retains promoter activity. Although sequences similar to canonical TATA and Inr promoter elements and an Sp1 binding site occur within this region, no single element is absolutely required for transcription In this study, we extended the analysis of the promoter to the region downstream of the major TSS at +1 to determine whether any downstream regulatory elements reside in this segment of the MHC class I promoter. We report that there are two novel downstream regulatory elements with sequence similarities to previously characterized DPE's, DPE-L1 and DPE–L2 and a third element, GLE, with sequence homology to binding sites for the transcription factor GAGA. The two DPE-L elements preferentially enhance TAF1-dependent transcription from TSS located at the 5′ end of the cluster of multiple start sites in the class I promoter, while GLE increases transcription from all TSSs. We discuss the possible mechanisms by which these downstream elements regulate transcription.Downstream promoter elements (DPE) are conserved among metazoans with a consensus sequence of (A/G)G(A/T)(T/C)(A/C) and are located between +28 to +32 bp downstream of transcription start sites. Examination of four different MHC class I gene sequences identified a consensus GAGA factor binding site at +4 and two consensus DPE sequences at +12 and +27 bp that are conserved in all four promoters , suggestTo determine whether any downstream sequences regulate either constitutive or activated MHC class I promoter activity, we compared the activities of promoter constructs that share a common 5′ extended promoter terminus but differ at their 3′ termini by the presence or absence of 32 bp downstream of +1, which contains the DPE-like and GAGA-like elements , bottom.To map these putative downstream promoter activities and to determine whether they correspond to either of the DPE-like (DPE-L) sequences or the GAGA-like (GLE) sequence within the +1–32 bp segment, we generated a series of scanning mutations between +1 to +32 bp downstream of the core promoter region . All of In native HeLa cells, mutations across the segments +4 to +6, +13 to +18, and +28 to +30 resulted in significantly reduced promoter activity, indicating the presence of a downstream element in each of these intervals . The regIn HeLa/CIITA cells, the GLE mutation resulted in significantly reduced promoter activity, relative to the wild type . Interesin vivo transcription start site at +1 in vivo . Unlike other downstream elements, the function of these downstream elements is context dependent: the GLE element is a stronger enhancer of activated than constitutive transcription. In contrast, whereas DPE-L2 markedly enhances constitutive transcription, it has a smaller effect on activated transcription.Taken together, these findings identify three novel downstream promoter elements that function to enhance MHC class I promoter activity. Two elements, DPE-L1 and DPE-L2, have sequence homology with other reported DPEs. Like other DPE elements, DPE-L2 is located approximately 30 bp downstream of an Since mutation of any one of the three elements resulted in decreased promoter activity, we next determined whether their activities were additive. To this end, a double mutant spanning both DPE-L1 and DPE-L2 and a triple mutant spanning GLE, DPE-L1, and DPE-L2 were generated  resulted in a significant decrease in CAT activity as measured in either transfected HeLa (−6) did not significantly reduce production of CAT in either HeLa or HeLa/CIITA cells, beyond the effect of the enhancer mutation itself (in vitro transcriptions of the DPE-L mutants (Out-of-frame ATG (uATG) mutations were inserted at −6 bp into the WT, the DPE-L1/2 double mutant and the GLE mutant, transiently transfected into HeLa cells and assayed for CAT activity. Consistent with previous observations that constitutive transcription largely initiates upstream of −6 bp n itself . This re mutants , these r−6 differed when measured in HeLa or in HeLa/CIITA cells. In HeLa cells, CAT activity generated by the GLE/uATG−6 construct was not significantly different from that of the GLE construct, consistent with the GLE primarily affecting upstream start sites ; two derivative constructs with mutations in the GLE and DPE-L1/2 were also generated , which is PC4 independent (data not shown). Immunodepleted extracts were first tested for their ability to support in vitro transcription of the wild-type class I promoter template. Depletion of either PC4 or CK2 resulted in a markedly reduced level of in vitro transcription  but not that of the GLE mutant, consistent with GAGA functioning through the GLE element. Paradoxically, and for reasons that remain to be determined, GAGA factor represses promoter activity through the GLE (Supplementary MHC class I expression is regulated by at least two distinct categories of pathways: 1) tissue-specific pathways that establish a baseline, or constitutive, level of transcription in any given tissue and 2) dynamically modulated pathways that increase or decrease expression in response to hormonal or cytokine mediated stimuli. The complexity of this regulatory system is reflected in the diversity of regulatory elements associated with the extended class I promoter, the complexity of the core promoter structure and the regulated use of multiple transcription start sites within the core promoter In the present study we have characterized three novel MHC class I downstream promoter elements that significantly contribute to the regulation of MHC class I expression. Two of these elements, DPE-L1 and DPE-L2, have sequence similarity to previously described downstream promoter elements, DPE. The third element, the GLE, is homologous to GAGA factor binding sequences. All three elements regulate core promoter activity and preferentially affect transcription start sites clustered at the 5′ end of the core promoter in both constitutive and activated MHC class I transcription. However, their activities appear to be context-dependent, since the relative magnitude of their effects differs in constitutive and activated transcription and they preferentially regulate TAF1-dependent, relative to TAF1-independent, transcription.DPE-L1 and DPE-L2 show superficial similarities with the canonical DPE. First, both DPE-L1 (AGACT) and DPE-L2 (GGATC) sequences are similar to the canonical DPE sequence ((A/G)G(A/T)(T/C)(A/C)). The positioning of DPE-L2 at +27 bp, relative to the major transcription start site, is similar to that of the DPE at +30 bp. The location of DPE-L1 at +12 to +16 bp places it approximately 30 bp downstream of an upstream start site. Second, the class I promoter, like many DPE-regulated promoters, is TATA-less and TFIID-dependent during constitutive transcription in vitroDespite their apparent similarities, the class I associated DPE-L1 and DPE-L2 are functionally and mechanistically distinct from previously described DPE elements. Although both DPE and DPE-L function to enhance transcriptional activity, the DPE-L elements differ by targeting a subset of transcription start sites in the upstream promoter region Furthermore, the TAF6/TAF9 complex, which mediates recruitment of PIC at the promoter by binding to the canonical DPE in vitro. Furthermore, mutation of GLE in the context of the ATG−6 mutant, which only monitors start sites downstream of −6 bp, reduces promoter activity. These results suggest that the GLE affects downstream start site usage. However, paradoxically, the GLE mutation has no effect on activity of the drop-out promoter which contains only downstream start sites. As detailed below and schematized in The function of the GLE is more complex than that of the DPE-L elements. Mutation of GLE leads to decreases in both upstream and downstream transcription start site usage The core promoter serves as the molecular platform where regulatory signals delivered by upstream silencer and enhancer elements are integrated −6 mutant, but not in the −50 to +3 bp drop-out.Based on these observations, we propose a model in which upstream sequences, located between −50 and +3 bp, negatively regulate downstream promoter activity and the downstream GLE, DPE-l and DPE-L2 elements regulate the activity of the upstream sequences . SpecifiRegulatory mechanisms governing transcriptional activation are generally thought to be limited to the recruitment of transcription factors to upstream enhancer and silencer DNA binding sites, which in turn target a set of general transcription factors and co-activators to a core promoter which serves only as a scaffold for transcriptional machinery recruitment. However, it is now evident that the core promoter plays an active role in integrating signaling pathways 2, except tsBN462 which were maintained at 32°C in 7.5% CO2. HeLa-CIITA cells were provided by Drs. Paul Roche (NIH) and Dr. Peter Cresswell .The HeLa epithelial, baby hamster kidney (BHK) and tsBN462 cell lines were grown in Dulbecco modified Eagles medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 20 mM HEPES (pH 7.2) and gentamicin sulfate (10 ug/mL). Cell lines were maintained in a humidified incubator at 37°C in 7.5% CO−6 constructs , a translational out-of-frame ATG (uATG) was inserted at the −6 bp position (CTG −> ATG) of the extended core promoter (WT+32 CAT) as previously described The MHC class I promoter used in these studies was derived from the swine class I gene, PD1 6 HeLa, HeLa/CIITA or tsBN462 cells were seeded in 100-mm tissue culture dishes. Transfections utilized standard calcium phosphate precipitation as previously described Transient transfections were performed by using a constant amount of DNA (5 ug). At 24 hr prior to transfection 10In vitro transcription reaction mixtures contained 2 ug of class I CAT reporter construct, 6 mM MgCl2, 0.8 mM ribonucleoside triphosphates and 30 U HeLa nuclear extract (Promega) in 20 mM HEPES (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 20% glycerol in a total of 25 uL was incubated at 23°C for 60 min. Analysis of the in vitro -transcribed RNA was done by primer extension as previously described In vitro transcriptions were as described above using equal amounts of either the parent HeLa extract or its CK2/PC4 depleted derivatives.250 uL of the Santa Cruz anti-CK2b Mab (SC-12739) or anti-PC4 rabbit serum were conjugated to protein A-agarose beads (Boehringer) as described PC4 and CK2β proteins in HeLa nuclear extract (150 µg) were analyzed by sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gel electrophoresis (PAGE) followed by electrophoretic transfer to nitrocellulose membranes. Membranes were blocked in Blotto A  for 12 hr at 4°C. Subsequently, an antiserum directed against either PC4 or CK2b (Santa Cruz Biotechnology) was added and incubated in Blotto A-0.05% Tween 20 for 60 min at room temperature. Blots were washed twice in Tris-buffered saline -0.05% Tween 20. 20 uL of a secondary antibody  was added to Blotto A-0.05% Tween 20 and incubated for a further 60 min. Blots were then extensively washed in Tris-buffered saline-0.05% Tween 20; specific proteins were detected by chemiluminescence with SuperSignal substrate (Pierce).Figure S1PC4 and CK2 contribute to constitutive transcription, but do not mediate the activity of DPE-Ls.A) Immunodepletion of HeLa nuclear extract with anti-PC4 and anti-CK2 antibodies effectively deplete PC4 and CK2. HeLa nuclear extracts (HeLa NE) were depleted with either anti-PC4 (PC4depNE) or anti- CK2β (CK2βNE). Extracts were probed Western blots with either anti-PC4 or anti- CK2β antibodies. B) DPE-L mutations do not rescue the requirement for PC4 and CK2. To examine the class I promoter requirement for PC4 and CK2, in vitro transcription assays with class I promoter templates  in either HeLa nuclear extract, extracts depleted of CK2 , or extracts depleted of PC4 . Arrows indicate major in vivo transcription start sites. C) Depletion of PC4 reduces the activity of a wild type promoter template (WT+32). Addition of exogenous rPC4 to a PC4-depleted HeLa nuclear extract restores promoter activity in vitro. In vitro transcription reactions were performed with HeLa nuclear extract depleted of PC4 and reconstituted with increasing amounts of exogenous PC4, as indicated. rPC4, recombinant PC4 added to depleted HeLa nuclear extract; WT+32-PC4-dep: in vitro transcription of wild type promoter in PC4-depleted HeLa nuclear extract; WT+32, in vitro transcription of wild type promoter in HeLa nuclear extract; HeLa NE, background transcription of extract in the absence of exogenous DNA.(TIF)Click here for additional data file.Figure S2Effect of CIITA on absolute level of MHC class I promoter activity. HeLa cells were co-transfected with a CIITA-expression vector, or control vector, and either the −416/+32 CAT (WT+32) or −416/+1 CAT (WT+1) constructs. Promoter activity was assessed as described in (TIF)Click here for additional data file.Figure S3The promoter activity of the GLE/DPE1/2 triple mutant is indistinguishable from that of GLE/DPE-L double mutants. The two double GLE/DPE-L mutant constructs and the triple GLE/DPE-L1/2 mutant construct were transfected into HeLa cells (upper panel) or HeLa/CIITA cells (lower panel) and the promoter activity was determined relative to wild type promoter (WT+32), as described in (TIF)Click here for additional data file.Figure S4Transcription from the MHC class I promoter predominantly initiates downstream of −50 bp. HeLa cells were transfected with either the wild type construct (WT+32), the dropout construct, which has a deletion in the region between −50 and +3 in the context of −416/+32 construct ligated to the luciferase (luc) reporter (Dropout), a 3′ truncation construct deleted of the region −50 to +32(WT-50) or a control vector (pGL3basic) . Promoter activity was assessed as described in (TIF)Click here for additional data file.Figure S5Effect of GAGA factor on MHC class I promoter activity. HeLa cells were co-transfected with a GAGA factor-expression vector, or control vector, and either the −416/+32 CAT (WT+32) or GLE constructs. Promoter activity was assessed as described in (TIF)Click here for additional data file.


ESR1 gene, nucleates tissue-specific long-range chromosomal interactions in collaboration with the cohesin complex. Furthermore, the Mediator complex not only regulates ERα activity, but also interacts with the cohesin complex to facilitate long-range chromosomal interactions. However, whether the cohesin and Mediator complexes function together to contribute to estrogen-regulated gene transcription remains unknown.In conjunction with posttranslational chromatin modifications, proper arrangement of higher order chromatin structure appears to be important for controlling transcription in the nucleus. Recent genome-wide studies have shown that the Estrogen Receptor-alpha (ERα), encoded by the ESR1 transcription and ERα protein levels. Moreover, we provide evidence that both SMC3 and MED12 colocalize on the ESR1 gene and are mutually required for their own occupancy as well as for RNAPII occupancy across the ESR1 gene. Finally, we show that extended proteasome inhibition decreases the mRNA expression of cohesin subunits which accompanies a decrease in ESR1 mRNA and ERα protein levels as well as estrogen-regulated transcription.In this study we show that depletion of the cohesin subunit SMC3 or the Mediator subunit MED12 significantly impairs the ERα-regulated transcriptome. Surprisingly, SMC3 depletion appears to elicit this effect indirectly by rapidly decreasing ESR1 gene as a cohesin/Mediator-dependent gene and indicate that this regulation may potentially be exploited for the treatment of estrogen-dependent breast cancer.These results identify the  Transcriptional control is a highly ordered and complex process involving interactions between various transcription factors and the transcriptional apparatus . ImportaThe estrogen receptor-alpha (ERα) is a ligand-activated transcription factor which plays an essential role in directing tissue-specific gene expression . ImportaNIPBL) gene .Total RNA was isolated from cells using the Qiazol reagent  according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed using random nonamers . Real-time PCR analysis was performed as previously described  using thCXCL12 (forward′), GREB1 , PGR  (forward3′), PKIB , cohesin binding site 1 , cohesin binding site 2 , 3′ end . ChIP DNA samples were normalized to input DNA from the same sample and expressed as percent input. Chromatin conformation capture studies were performed as described previously [CXCL12 positive interaction , CXCL12 negative control , GREB1 positive interaction , GREB1 negative control , normalization control (within the GREB1 gene) . All interactions were quantified by real-time PCR using standard curves containing digested and re-ligated DNA from BAC clones  covering the investigated regions of the CXCL12 (RP-13309I17) and GREB1 (RPCIB753E0150Q) genes and normalized using a probe-primer pair detecting an amplicon between BtgI sites as described previously [Chromatin immunoprecipitation was performed and analyzed by quantitative real-time PCR as previously described  using thviously  using thviously .BrdU: Bromodeoxyuridine; CdLS: Cornelia de Lange Syndrome; ChIP: Chromatin immunoprecipitation; CTD: C-terminal domain; ERα: Estrogen receptor-α; ESR1: Estrogen receptor 1; FC: Fold change; FDR: False discovery rate; HDACi: Histone deacetylase inhibitor; MED12: Mediator complex subunit 12; qRT-PCR: Quantitative real-time reverse transcription polymerase chain reaction; RNAPII: RNA Polymerase II; siRNA: Small interfering ribonucleic acid; SMC: Structural maintenance of chromosomes 3; TSS: Transcriptional start site.Frank Kramer and Upasana Bedi contributed equally to this work.The authors declare that they have no competing interests.TP and SAJ designed the experiments. TP, UB and SN performed the experiments. FK and TB performed bioinformatic analyses of the microarray data. SJ wrote the manuscript. All authors read and approve the final manuscript.Figure S1. RAD21 depletion significantly reduces the estrogen-induced expression of ERα target genes. MCF7 cells transfected with control or RAD21 siRNA were grown in normal growth medium for 24 h and then for another 42 h in hormone-depleted medium before treating with 10 nM 17β-Estradiol (E2) for 6 h as indicated. Total mRNA was extracted, reverse-transcribed and analyzed by quantitative real-time PCR (qRT-PCR). (A) The expression levels of the estrogen-regulated genes CXCL12, GREB1, PGR, and PKIB were normalized to 28S ribosomal mRNA, graphed relative to the control sample and expressed as relative mRNA expression; mean values + SD, n = 2. (B) Efficient knockdown of RAD21 and its effects on ESR1 mRNA levels was verified by qRT-PCR in the same samples used in (A). RAD21 and ESR1 were normalized and expressed as in (A). Mean values + SD, n = 2. Figure S2. SMC3 or MED12 knockdown enhance hormone-withdrawal induced G1 cell cycle arrest. MCF7 cells transfected with control, SMC3 or MED12 siRNA were grown in normal growth medium for 24 h and then for another 42 h in hormone-depleted medium before flow cytometric analyses. Shown are representative profiles and the respective quantitation for duplicate samples (± SD). Click here for file


Hes7, which is regulated by the Notch and Fgf/Mapk pathways, but its relation to other pathways is unknown. In addition, chemical alteration of the Wnt pathway changes the segmentation clock period but the mechanism is unclear.The mouse segmentation is established from somites, which are iteratively induced every two hours from the presomitic mesoderm (PSM) by a system known as the segmentation clock. A crucial component of the segmentation clock is the gene Hes7 promoter analysis in transgenic mouse embryos and have identified an essential 400 bp region, which contains binding sites of Tbx6 and the Wnt signaling effector Lef1. We have found that the Hes7 promoter is activated by Tbx6, and normal activity of the Hes7 promoter in the mouse PSM requires Tbx6 binding sites. Our results demonstrate that Wnt pathway molecules activate the Hes7 promoter cooperatively with Tbx6 in cell culture and are necessary for its proper expression in the mouse PSM. Furthermore, it is shown that the chemical Gsk3 inhibitor LiCl lengthens the oscillatory period of Hes7 promoter activity.To clarify these questions, we have carried out Hes7 expression. Furthermore, proper Hes7 promoter activity and expression is important for the normal pace of oscillation.Our data suggest that Tbx6 and the Wnt pathway cooperatively regulate proper  Vertebrate embryos are divided along the longitudinal axis into somites in a process called segmentation. During the vertebrate segmentation, a new pair of bilateral somites arises every two hours from the presomitic mesoderm (PSM). The pace of somite formation correlates with the periodic expression of genes of the Notch, Fgf and Wnt pathways Hes7 gene, a crucial component of the segmentation clock, is downstream of the Notch and Fgf pathways and drives the oscillation of several cyclic genes of these pathways Hes7 expression is specified.The The oscillation period of the segmentation clock in vertebrates is changed after perturbation of the Notch and Wnt pathways Hes7 promoter and have found evidence that Tbx6 and the Wnt pathway regulate Hes7 expression in the PSM. Our results suggest that Tbx6 and the Wnt pathway are necessary for proper Hes7 expression. We have also found that treatment with the chemical Gsk3 inhibitor LiCl activates the Wnt pathway and lengthens the oscillatory period of Hes7 expression.To investigate these questions, we have analyzed the Hes7 promoter  www.Genomatix.de). For the H7p2.6dR reporter, we introduced point mutations in the Rbpj binding sites at −124 bp (CTTaCaACAT), −425 bp (TTTaCaACAG), −467 bp (TTGTtGtAAT), and −1463 bp (GTGTtGtAAA) to generate the H7p2.6dR construct. Then different promoter sizes (H7p1.9dR: −1939 bp (tggaagac…) to +76; H7p1.4dR: −1410 (ttctctcc…) to +76; H7p1.0dR: −998 (cccaggcc…) to +76) were amplified by PCR, sequenced and subcloned into the pGL3-basic (Promega) vector to obtain the luciferase reporter. Two point mutations were introduced in the T-box binding sites at −1306 (TGCcGGTtAG) and −1350 (CCCAaAaCCG) by PCR for H7p1.4dRdT. For the lacZ reporters, the luciferase gene was replaced with the lacZ gene. The expression plasmids for NICD The luciferase reporters were created by inserting the 2.6 kb Transgenic embryos were generated as previously described in situ hybridization assays, wild-type embryos were transferred to culture medium  in the presence of solvent or inhibitor and cultured for the indicated times at 37°C with 5% CO2. For timelapse imaging experiments, tails of embryos were cut up to the second somite, transferred to 1 µM luciferin-containing culture medium and genotyped by observation of luminescence with a CCD camera. One positive tail was selected, transferred to inhibitor containing medium and immediately imaged . The oscillation period was measured by two different methods by analyzing movies that spanned at least two oscillation cycles. In the first method, we counted the number of oscillation cycles and divided it by their duration. In the second method, we created a spatiotemporal plot of the oscillations with time in the x-axis and measured the distance between peaks (Embryos were taken out and freed from extraembryonic structures in prewarmed PBS. For en peaks . InhibitThe luciferase assay was carried out as previously described using C3H10T1/2 cells in 10% FBS and 1% P/S in DMEM and Lipofectamin LTX/Plus (Invitrogen) transfection reagent in vitro translation kit (Promega). The in vitro translation was carried out following the manufacturer's protocol. Oligonucleotides (15 bp) containing the wild-type and mutant T-box binding sites were annealed and labeled with gamma-32P-ATP (GE Heathcare) using T4 polynucleotide kinase. The sense-strand sequences of the wild-type and mutant probes for T-box −1306 are: agc ctc acg tgc agg tga gaa aaa ctc aac and agc ctc acg tgc Cgg tTa gaa aaa ctc aac, respectively and for T-box −1350: cag ggg cgg ccc cac acc cgg gtg caa act g and cag ggg cgg ccc caA aAc cgg gtg caa act g, respectively. Purified proteins and labeled probes were incubated for 30 min at room temperature in 5× binding buffer . Samples were then electrophoresed on a 5% non-denaturing polyacrylamide gel .We subcloned the Tbx6 cDNA from pCS2-3xFlag-Tbx6 In situ hybridization was performed as previously described in situ hybridizations after inhibitor culture, we classified control and treated embryos into categories of high and low staining and plotted the percentage of control and treated embryos with high staining. For real-time PCR, the PSM was cut until the first visible somite border, RNA was isolated using the NucleoSpin RNA II kit (Macherey-Nagel) and reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen). Two biological replicates were carried out. Real-time PCR was performed with the LightCycler 480 SYBR Green I Master mix and the Real-time PCR system (Roche). Primer sequences (forward and reverse) are Gapdh: atc ttc ttg tgc agt gcc agc ctc gtc ccg and agt tga ggt caa tga agg ggt cgt tga tgg; Axin2  and Msgn1: cgg ctt agt cga gct gga tta and ctc cgc tgg aca gac atc ttg.For immunofluorescence detection, embryos were fixed in PFA incubated overnight at 4°C, paraffin embedded and sectioned (10 µm). Antigen retrieval was carried out in citrate buffer (pH 6) placed in an autoclave at 105°C for 15 min. After 1 h blocking at room temperature, primary antibodies were diluted in Can Get Signal immunostain Solution B  to the lacZ gene. Transgenic embryos showed X-gal staining in the endogenous Hes7 expression region and somites . This construct also showed X-gal staining in the posterior PSM  with Notch intracellular domain (NICD) and Tbx6 expression plasmids. NICD upregulated the H7p2.6wt promoter reporter (Hes7 promoter in cultured cells. This is similar to the reported enhancement of Mesp2 reporter activity by NICD and Tbx6 Two T-box binding sites caught our attention (T-1306 and T-1350), because such binding sites are important in other PSM gene promoters, namely nd Msgn1  and 2A [reporter . Tbx6 inreporter . These din vitro synthesized Tbx6 can bind to oligos containing the two T-box binding sites by electrophoresis mobility shift assay (EMSA). The EMSA showed that in vitro synthesized Tbx6 protein can bind to WT  and Lef1 in C3H10T1/2 cells and analyzed the luciferase luminescence. Our results show that the Hes7 promoter is activated by Ctnnb1 and Lef1 Wnt3a mutant embryos at embryonic stage E10.5, Hes7 intronic expression was downregulated  of XAV939 did not cause apparent effects the reporter activity, but high concentration (5 µM) downregulated promoter activity  of CKI-7, whereas the longer period was found with a different lower concentration (100 µM) of CKI-7 Hes7 promoter in the presence of 100 µM CKI-7. Measurement of the oscillation period showed an increase of the period from 2.5 h to 3.3 h (in situ hybridization of embryos cultured with 100 µM CKI-7 for 3 h did not show a clear change of Hes7 expression levels (data not shown). These results suggest that CKI-7 inhibitor lengthens the oscillatory period although the mechanism for this effect requires further investigation.A previous study found a correlation between lower to 3.3 h , which aAxin2 and Msgn1 and the levels of Ctnnb1 suggest that the effects of LiCl on the period is mediated by the Wnt pathway. However, a mutant mouse where Ctnnb1 is stabilized does not change the oscillation period Hes7 expression Our results suggest that the Wnt related chemicals LiCl and CKI-7 lengthen the period, whereas another Wnt related chemical, XAV939, does not. Our data that LiCl upregulates the expression of the Wnt target genes Hes7 expression, in an effort to better understand the mechanism of the mouse segmentation clock. We found evidence that Tbx6 binding sites are required for normal Hes7 promoter activity  with human (Bottom) and transcription factor binding sites (TFBS). Several conserved binding sites of transcription factors expressed in the PSM can be found.(TIF)Click here for additional data file.Figure S2In situ hybridization of Tbx6 mRNA in E10.5 Wnt3a mutant embryos. Tbx6 expression is downregulated in the Wnt3a hypomorph embryos .(TIF)Click here for additional data file.Figure S3LiCl activates the Wnt pathway in the PSM.  E9.5 embryos were cultured with LiCl for 6 h and the expression of Axin2 and Msgn1 was measured by real-time PCR. The expression of both Axin2 (A) and Msgn1 (B) increases in the presence of 40 mM LiCl . (C) E10.5 embryos were cultured with 40 mM LiCl for 6 h and immunohistochemistry was carried with a Ctnnb1 antibody. Ctnnb1 protein is stabilized by 40 mM LiCl treatment .(TIF)Click here for additional data file.Figure S4Hes7 in situ hybridization of embryos cultured in the presence of Gsk3 inhibitors. E10.5 embryos were cultured with 20 mM LiCl for 6 h  (A), E9.5 embryos with 100 mM LiCl for 2 h  (B) and E9.5 embryos with 10 µM Gsk3 Inhibitor IX for 6 h  (C). Our results show a non-significant increase of the Hes7 expression levels under these culture conditions.(TIF)Click here for additional data file.Figure S5Timelapse imaging of E10.5 Hes7 promoter luciferase reporter embryos in the presence of the 40 mM LiCl. Some embryos treated with this concentration of LiCl showed stabilization of the Hes7 promoter reporter activity and arrest of oscillations (Red arrowheads).(TIF)Click here for additional data file.Figure S6Period quantification method and duration of oscillation cycles after addition of LiCl. To measure the oscillatory period, we created a spatiotemporal plot of the timelapse activity with time in the x-axis and measured the distance between peaks in the posterior PSM (A). This allows us to measure the duration of individual cycles after addition of the chemical LiCl. We started to observe a period difference during the second oscillation cycle (B).(TIF)Click here for additional data file.Figure S7Timelapse imaging of E10.5 Hes7 promoter luciferase reporter embryos in the presence of the Wnt inhibitor XAV939 and period quantification. (A) The control and 1 µM XAV939 samples show the normal reporter activity, while 5 µM XAV939 treatment rapidly downregulates Hes7 promoter activity. (B) XAV939 treatment does not change the period between the control (n = 11) and the 1 µM XAV939 samples (n = 2).(TIF)Click here for additional data file.Figure S8Period quantification of the oscillations of the E10.5 Hes7 promoter reporter embryos in the presence of the chemical agent CKI-7 observed by timelapse imaging. Treatment with 100 µM CKI-7 lengthens the period from 2.5 h (n = 11) to 3.3 h (n = 3).(TIF)Click here for additional data file.Movie S1Control timelapse imaging of a transgenic embryo at E10.5 carrying a Hes7 promoter luciferase reporter.(AVI)Click here for additional data file.Movie S2Timelapse imaging of a transgenic embryo at E10.5 carrying a Hes7 promoter luciferase reporter in the presence of 20 mM LiCl.(AVI)Click here for additional data file.Movie S3Timelapse imaging of a transgenic embryo at E10.5 carrying a Hes7 promoter luciferase reporter in the presence of 40 mM LiCl.(AVI)Click here for additional data file.Movie S4Timelapse imaging of a transgenic embryo at E10.5 carrying a Hes7 promoter luciferase reporter in the presence of 40 mM LiCl that shows constant promoter activity.(AVI)Click here for additional data file.Movie S5Timelapse imaging of a transgenic embryo at E10.5 carrying a Hes7 promoter luciferase reporter in the presence of 1 µM XAV939.(AVI)Click here for additional data file.Movie S6Timelapse imaging of a transgenic embryo at E10.5 carrying a Hes7 promoter luciferase reporter in the presence of 5 µM XAV939 that shows inactivation of the promoter activity.(AVI)Click here for additional data file.Movie S7Timelapse imaging of a transgenic embryo at E10.5 carrying a Hes7 promoter luciferase reporter in the presence of 100 µM CKI-7 inhibitor.(AVI)Click here for additional data file.


K. pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. Although elevated blood glucose levels cause various immune problems, its effects on K. pneumoniae virulence are unknown. This study investigated the regulation of capsular polysaccharide (CPS) biosynthesis, a major determinant for K. pneumoniae virulence, in response to exogenous glucose. We found that K. pneumoniae produce more CPS in glucose-rich medium via reduction in cyclic AMP (cAMP) levels. Individual deletion of cyaA or crp, which respectively encode adenylate cyclase and cAMP receptor protein in K. pneumoniae, markedly increased CPS production, while deletion of cpdA, which encodes cAMP phosphodiesterase, decreased CPS production. These results indicate that K. pneumoniae CPS biosynthesis is controlled by the cAMP-dependent carbon catabolite repression (CCR). To investigate the underlying mechanism, quantitative real-time PCR and promoter-reporter assays were used to verify that the transcription of CPS biosynthesis genes, which are organized into 3 transcription units , were activated by the deletion of crp. Sequence analysis revealed putative CRP binding sites located on Porf3-15 and Porf16-17, suggesting direct CRP-cAMP regulation on the promoters. These results were then confirmed by electrophoretic mobility shift assay. In addition, we found putative CRP binding sites located in the promoter region of rcsA, which encodes a cps transcriptional activator, demonstrating a direct repression of CRP-cAMP and PrcsA. The deletion of rcsA in mutation of crp partially reduced CPS biosynthesis and the transcription of orf1-2 but not of orf3-15 or orf16-17. These results suggest that RcsA participates in the CRP-cAMP regulation of orf1-2 transcription and influences CPS biosynthesis. Finally, the effect of glucose and CCR proteins on CPS biosynthesis also reflects bacterial resistance to serum killing. We here provide evidence that K. pneumoniae increases CPS biosynthesis for successful infection in response to exogenous glucose via cAMP-dependent CCR. Klebsiella pneumoniae is a Gram-negative pathogen which causes suppurative lesions, bacteremia, and urinary as well as respiratory tract infections mostly in patients with underlying diseases K. pneumoniae is the predominant pathogen responsible for pyogenic liver abscess in diabetic patients Klebsiella liver abscess (KLA) in western countries have also been accumulating K. pneumoniae, capsular polysaccharide (CPS) is considered as the major determinant for K. pneumoniae virulence. Pyogenic liver abscess isolates often carry heavy CPS loads that could protect the bacteria from phagocytosis and killing by serum factors K. pneumoniae have been classified into more than 77 known types K. pneumoniae was documented in liver abscess of diabetes mellitus patients K. pneumoniae, liver abscess, and diabetes mellitus remains unclear.K. pneumoniae strains are more virulent in diabetic than in normal mice Escherichia coli but highly conserved across bacteria, the carbon catabolite repression (CCR) regulates uptake of glucose and repression of genes required for utilization of less preferred carbon sources E. coli, CRP-cAMP acts as a global regulator of gene expression by controlling the expression of almost 200 operons Diabetic patients have been reported to be more susceptible to infections E. coli and K. pneumoniae, suggesting a conserved regulatory mechanism. In the previous study, CRP-cAMP has been demonstrated to regulate the expression of citrate fermentation genes in K. pneumoniae under fermentative conditions K. pneumoniae pathogenesis is large uncharacterized. In this study, we aimed to examine the effect of glucose levels and CCR proteins on the regulation of K. pneumoniae CPS biosynthesis. Individual strains of K. pneumoniae CG43, a highly virulent liver abscess isolate of the K2 serotype, in which cyaA, cpdA, and crp had been deleted were constructed for the assessment of CPS production, and the regulatory mechanism of cAMP-dependent CCR in cps transcription was analysed.Sequence analysis revealed a high similarity between CCR proteins  in K. pneumoniae CPS biosynthesis, CG43S3 was grown in LB broth supplemented with increasing amount of glucose for the quantification of CPS. As shown in K. pneumoniae CPS biosynthesis through a reduction of cAMP level.To analyse if exogenous glucose affects K. pneumoniae CPS biosynthesis is regulated by cAMP, individual strains with deletion of cyaA and cpdA, which respectively encodes adenylate cyclase and cAMP phosphodiesterase from CG43S3, were constructed, and the effects of the deletions on CPS production were analysed. As shown in cyaA, and introduction of pcyaA, but not the empty vector control (pACYC184), into ΔcyaA could reverse the effect of cyaA mutation. In contrast, the deletion of cpdA caused a decreased in CPS levels, which could be complemented by introducing a plasmid-carried cpdA (pETQ-cpdA) into the ΔcpdA strain. These results confirmed that cAMP can act as a signalling molecule for regulation of CPS biosynthesis. In addition, since cAMP affects gene transcription through its effector protein CRP, we assessed the effect of deletion of crp on CPS levels. As shown in crp produced higher levels of CPS. crp, but not the empty vector control (pACYC184), into Δcrp reversed the effect of the deletion. This result indicates that the CRP-cAMP signalling pathway is involved in the regulation of CPS biosynthesis, and that CRP acts as a negative regulator of CPS biosynthesis. In addition, since the functions of CyaA, CpdA, and CRP in controlling the cAMP level in K. pneumoniae have not yet been demonstrated, enzyme-linked immunosorbent assays were performed to determine the intracellular cAMP level upon the deletion of cyaA, cpdA, or crp. Compared to WT (9.75±0.35 nM), the intracellular cAMP level was almost undetectable in ΔcyaA (<1 nM), whereas a higher cAMP level was found in ΔcpdA (38±2.8 nM) (crp (14.5±0.7 nM). These result confirmed that CyaA and CpdA are responsible for cAMP biosynthesis and degradation, respectively, in K. pneumoniae.To confirm that ±2.8 nM) . In addicps gene cluster of K. pneumoniae Chedid contains 19 open reading frames (ORFs) organised into 3 transcription units, namely, orf1–2, orf3–15, and orf16–17cps gene clusters, the mRNA level of orf1 , orf3 (named wzi), and orf16 (named manC) were measured by qRT-PCR in WT grown in LB medium containing 0.5% glucose with or without 1 mM cAMP. In addition, the effect of cyaA, cpdA, andcrp mutation strains on the mRNA levels of galF, wzi, and manC were also determined. As shown in galF, wzi and manC was increased in glucose-rich medium (LB+0.5% glucose), whereas addition of 1 mM cAMP to the glucose-rich medium could restore the galF, wzi and manC expression, similar to the trends observed in the WT strain. Furthermore, the mRNA levels of galF, wzi, and manC revealed an apparent increase in the ΔcyaA and Δcrp strains as compared to that observed in the WT strain. In contrast, a slight reduction in the mRNA level of cps genes was found in the ΔcpdA strain. This result indicates that cps mRNA expression is regulated by cAMP-dependent CCR, and CRP may acts a repressor of cps expression.The K2 galF, wzi, and manC, the reporter plasmids pOrf12 (Porf1-2::lacZ), pOrf315 (Porf3-15::lacZ), and pOrf1617 (Porf16-17::lacZ), each carrying a lacZ reporter gene transcriptionally fused to the putative promoter region of the K2 cps gene cluster K. pneumoniae strains CG43S3ΔlacZ and ΔlacZΔcrp. The promoter activity measurements shown in crp in the ΔlacZ strain apparently increased the promoter activities of galF, wzi, and manC. These results verify that CRP represses cps expression at the transcriptional level.To further confirm whether CRP acts as a transcriptional repressor of the promoter activity of cps gene cluster had not been characterized. 5′ rapid amplification of cDNA ends (5′ RACE) was first performed to determine the transcriptional start sites of the 3 transcription units in the K2 cps gene cluster. A single DNA band was obtained for galF, wzi, and manC from the 5′ RACE analysis using either primer pair (data not shown). As shown in galF, wzi, and manC revealed a transcriptional start site at the A nucleotide at position −61 relative to the translational start site of galF, at the G nucleotide at position −470 relative to the translational start site of wzi, and at the G nucleotide at position −56 relative to the translational start site of manC. The conserved −10 and −35 promoter sequence of σ70 could be readily identified and is shown in Until now, the transcriptional start sites of the cps gene transcription, the sequences of the E. coli CRP binding sites (TGTGA-N6-TCACA and TGCGA-N6-TCGCA) were used in searching the promoter sequence of the K2 cps gene cluster of K. pneumoniae Chedid. The maximum number of possible mismatched nucleotides was set at 3, and only the intergenic regions of the 3 transcriptional units were analysed in this search. Using these criteria, no typical CRP binding sites were located the upstream of galF , the other 2 CRP binding sites were found to be located at position −29 to −14 and −87 to −72 relative to the transcriptional start site of wzi  was performed using the recombinant His6-CRP protein and different DNA fragments containing truncated forms of Pwzi  and PmanC (PmanC-1 and PmanC-2), as described in the Materials and Methods. Using Pwzi fragments of different lengths, binding of His6-CRP could be observed for Pwzi-1, Pwzi-2, and Pwzi-3 but not for Pwzi-4  are located at −192 to −177 and −40 to −25 relative to the translation start site of RcsA  in PrcsA to repress rcsA transcription.To further investigate whether CRP binds directly to the rcsA, Δcrp, and ΔcrpΔrcsA strains. As shown in rcsA resulted in a slight reduction in CPS level as compared to WT strain. However, the deletion of rcsA partially restored CPS production in the Δcrp strain. In addition, introducing the complementary plasmid prcsA into the ΔcrpΔrcsA strain increased CPS levels as compared to the strain carrying the empty vector control (pRK415). These results indicate that RcsA participates in CRP regulation of CPS biosynthesis.To understand whether RcsA participates in CRP regulation of CPS biosynthesis, the level of CPS was determined in WT, Δcps transcriptions, the promoter activity of galF (pOrf12), wzi (pOrf315), and manC (pOrf1617) was measured in ΔlacZ, ΔlacZΔcrp, ΔlacZΔrcsA, and ΔlacZΔcrpΔrcsA strains. As shown in rcsA in ΔlacZ caused an apparent reduction on the promoter activity of galF, but no effect on the promoter activity of wzi and manC was observed, indicating that RcsA plays a positive role in galF transcription. In addition, the deletion of rcsA in the ΔlacZΔcrp strain caused a slight reduction in the promoter activity of galF, unlike that in the Δcrp strain, but still showed higher promoter activity than the ΔlacZ strain. The result implies that RcsA participates in CRP regulation of galF transcription. However, we suggest that other unknown transcriptional regulator(s) are also involved in this regulation.To further understand the role of RcsA in CRP regulation of K. pneumoniae from killing by serum factors, we suggest that glucose, cAMP, and cAMP-related proteins may also affect the ability of K. pneumoniae, through modulation of CPS levels, to resist the bactericidal effects of serum. To test the hypothesis, the effects of exogenous glucose and cAMP on survival rate were first determined in treatment with 75% normal human serum. We found that the survival rate of WT grown in LB supplemented with 0.5% glucose (43.2±3.9%) increased about 2-fold as compared to the survival rate of WT in LB alone (21.2±2.3%). Addition of 1 mM cAMP diminished the higher survival rate of WT when grown in LB with 0.5% glucose (34.3±0.1%). This implies that the intracellular cAMP level decreased in K. pneumoniae grown in LB with 0.5% glucose, which then increased the serum resistance in K. pneumoniae. In addition, the deletion of cyaA in WT apparently increased the survival rate (40.7±0.7%), while the deletion of cpdA reduced the survival rate (12.7±0.6%). It also confirmed that the intracellular cAMP level could affect the ability of K. pneumoniae to resist the bactericidal effects of serum. Finally, the deletion of crp in WT had a higher survival rate (28.5±1.9%) in treatments with 75% normal human serum as compared to WT strain. Thus, the results imply that K. pneumoniae could resist serum killing in response to higher glucose levels via the trigger of cAMP-dependent CCR.Since CPS has been demonstrated to protect K. pneumoniae strains usually produce a large amount of CPS, which confers not only a mucoid phenotype to the bacteria but also resistance to engulfment by phagocytes or to serum bactericidal factors K. pneumoniae infections and multiple CPS regulators have been found, the environmental stimuli that modulate CPS biosynthesis has remained largely unknown. Our previous studies have reported that extracellular ferric ion could repress K. pneumoniae CPS production through Fur regulation K. pneumoniae could produce more CPS to facilitate its persistence in the blood.Clinically isolated E. coli, the expression of cps::lacZ was activated when cells were grown at a low temperature (20°C) in the presence of glucose (0.4%) as a carbon source K. pneumoniae CG43, a K2 serotype strain, we found that higher CPS production in glucose-rich medium is dependent on reducing the intracellular cAMP concentration. In addition, the activation of CPS in response to exogenous glucose was also found in K. pneumoniae NTUH-K2044, a highly virulent liver abscess isolate of K1 serotype, and addition of increasing amounts of exogenous cAMP in glucose-rich medium could completely reverse the effect of glucose on CPS production  was located at −144 to −129 relative to the translation start site of cyaA. In addition, the mRNA level of cyaA increased more than 16-fold in the Δcrp stain, unlike that in the WT strain (data not shown), indicating that the deletion of crp could increase cyaA expression to elevate the cAMP level in K. pneumoniae, consistent with the findings for E. coli.In bacteria, CyaA and CpdA are responsible for cAMP production and degradation ynthesis . Althougcps transcription, the transcriptional start sites of 3 transcriptional units in the cps gene cluster were first determined    and 4D. K. pneumoniae CPS biosynthesis genes cps transcription is regulated by cAMP-dependent CCR. In E. coli, CRP has been reported to regulate the expression of fur at the transcriptional as well as at the posttranscriptional level K. pneumoniae, CPS biosynthesis was modulated by iron availability and Fur has been shown to repress the transcription of rmpA/A2 and rcsA to control CPS biosynthesis rcsA is also regulated by glucose and cAMP-dependent CCR  was found to be located at position −119 to −107 relative to the transcriptional start site of galF, indicating that RcsA could directly activate galF transcription. In addition, we noted that the deletion of rcsA in the ΔlacZΔcrp strain retains a higher galF promoter activity compared to the ΔlacZ strain, implying that CRP could directly or indirectly regulate galF expression. Although no obvious CRP binding site was found in the promoter sequence of galF, an EMSA was performed to investigate whether CRP could directly bind to PgalF. As shown in 6-CRP with 10 ng PgalF, implying that CRP-cAMP could directly repress the galF transcription. However, the exact binding site of CRP in PgalF needs to be further investigated. In addition, we also found that introduction of a plasmid carrying His6-CRP encoding gene into Δcrp strain could complement the effect of crp mutation on CPS biosynthesis  containing hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) K. pneumoniae not only regulates CPS production to protect the bacteria from serum killing and phagocytosis, but also alters bacterial resistance to oxidative stress to enhance the survival rate in the phagosome, and we are currently working to demonstrate this possibility. In addition, CPS and adherence factors, such as type 1 and type 3 fimbriae, have been demonstrated to play important roles in biofilm formation and pathogenesis, and their expression could be co-regulated E. coli and Serratia marcescens, glucose/CRP-cAMP has been described to regulate the expression of type 1 fimbriae and bacterial biofilm formation K. pneumoniae. Since glucose has also been described to repress K. pneumoniae biofilm formation In K. pneumoniae to protect the bacteria from serum killing, and cAMP-dependent CCR plays a profound regulatory role in CPS expression in response to glucose levels in the environment. In diabetes mellitus patients, the higher glucose level in the bloodstream is thought to have a major impact on bacterial virulence. We suggest that K. pneumoniae could evade the immune response via the regulation of cAMP-dependent CCR on CPS biosynthesis, especially during infection of diabetes mellitus patients. Future studies will include determining the role of cAMP-dependent CCR in modulating CPS biosynthesis, fimbria production, and biofilm formation in response to environmental stimuli.In this study, we provide important evidence that glucose stimulates CPS biosynthesis in Bacterial strains and plasmids used in this study are listed in 7 colony forming units (c.f.u.)/ml and washed twice in phosphate-buffered saline (PBS). Then, the bacteria were resuspended in 300 µl of 1X lysis buffer and lysated by sonication. The lysate was centrifuged briefly at 14,000 rpm for 10 min, and the supernatant was tested for cAMP levels by using cAMP XP™ Assay Kit  and according to manufacturer’s recommendations.Bacteria was adjusted to 1×10crp, cyaA, and cpdA was introduced into K. pneumoniae CG43S3 using an allelic exchange strategy as previously described respectively rpsL, which allows positive selection with streptomycin for vector loss. The resulting plasmid was then mobilized from E. coli S17-1λpirK. pneumoniae CG43S3, K. pneumoniae CG43S3ΔlacZ, K. pneumoniae CG43S3ΔrcsA or CG43S3-derived strains, by conjugation. The transconjugants, with the plasmid integrated into the chromosome via homologous recombination, were selected with ampicillin and kanamycin on M9 agar plates. Several of the colonies were grown in LB broth supplemented with 500 µg/mL of streptomycin to log phase at 37°C and then spread onto an LB agar plate containing 500 µg/mL of streptomycin. The streptomycin-resistant and kanamycin-sensitive colonies were selected, and the deletion was verified by PCR and Southern hybridization (data not shown). The resulting K. pneumoniae mutants are listed in Specific gene deletion containing crp, cyaA, and rcsA were individually PCR-amplified with primer pairs GT131/GT132, GT196/GT197, and HY001/GT145  and expressed as micrograms per 109 c.f.u. CPS was extracted and quantified as previously described -ΔΔCT method with 23S rRNA as the endogenous reference.Total RNAs were isolated from early-exponential-phase grown bacteria cells by use of the RNeasy midi-column (QIAGEN) according to the manufacturer’s instructions. RNA was DNase-treated with RNase-free DNase I (MoBioPlus) to eliminate DNA contamination. RNA of 100 ng was reverse-transcribed with the Transcriptor First Strand cDNA Synthesis Kit (Roche) using random primers. qRT-PCR was performed in a Roche LightCycler® 1.5 Instrument using LightCycler TaqMan Master (Roche). Primers and probes were designed for selected target sequences using Universal ProbeLibrary Assay Design Center (Roche-applied science) and listed in K. pneumoniae strains by conjugation from E. coli S17-1 λpir. The bacteria were grown to logarithmic phase in LB broth, and the β-galactosidase activity was measured as previously described The promoter-reporter plasmids, pRcsAZ15, pOrf12, pOrf315, and pOrf1617, were individually mobilized into cps gene cluster, a rapid amplification of PCR was performed using 5′ RACE kit (Clontech) according to the manufacturer’s instruction as previously described galF, wzi, and manC, respectively, were subjected to sequence analysis, and the transcriptional start sites of galF, wzi, and manC were determined. All the sequencing results indicated the same nucleotide as the transcriptional start site of galF, wzi, and manC.For the determination of 5′ mRNA ends in the three transcriptional units in the K2 crp was PCR amplified with primer sets GT132/GT137 . The resulting plasmid pET30b-CRP was then transformed into E. coli BL21(DE3) (New England Biolabs), and overproduction of the recombinant protein was induced by the addition of 0.1 mM IPTG for 4 h at 37°C. The recombinant proteins were then purified from the soluble fraction of the total cell lysate by affinity chromatography using His-Bind resin . Finally, the purified proteins were dialyzed against 1X TE buffer  containing 10% glycerol at 4°C overnight, and the purity was determined by SDS-PAGE.The coding region of 32/GT137  and clonorf1-2, orf3-15, orf16-17, and rcsA were respectively PCR amplified by using specific primer sets (6-CRP was incubated with 10 ng DNA in a 15 µl solution containing 4 mM Tris-HCl (pH 7.4), 10 mM KCl, 100 mM dithiothreitol, 200 µM cAMP, and 10 µg/ml BSA at room temperature for 30 min. The samples were then loaded onto a native gel of 5% nondenaturing polyacrylamide containing 200 µM cAMP in 0.5X TB buffer . Gels were electrophoresed with a 20-mA current at 4°C and then stained with SYBR Green I dye (Invitrogen).The DNA fragments of the putative promoter region of mer sets . The pur6 c.f.u./ml. The reaction mixture containing 250 µl of the cell suspension and 750 µl of pooled human serum was incubated at 37°C for 15 min. The number of viable bacteria was then determined by plate counting. The survival rate was expressed as the number of viable bacteria treated with human serum compared to the number of pre-treatment. The assay was performed triple, each with triplicate samples. The data from one of the representative experiments are shown and expressed as the mean and standard deviation from the three samples. A CPS-deficient mutant strain, K. pneumoniae CG43S3ΔgalU  is served as a control.Normal human serum, pooled from healthy volunteers, was divided into equal volumes and stored at −70°C before use. Bacterial survival in serum was determined with minor modifications t-test was used to determine the statistical significance and values of P<0.05 and P<0.01 were considered significant. The results of CPS quantification, qRT-PCR analysis, and promoter activity measurement were derived from a single experiment representative of three independent experiments. Each sample was assayed in triplicate and the mean activity and standard deviation are presented.An unpaired For isolation of normal human serum from healthy volunteers, the procedure and the respective consent documents were approved by the Ethics Committee of the China Medical University Hospital, Taichung, Taiwan. All healthy volunteers provided written informed consent.Figure S1Glucose and cAMP affects the CPS levels of K. pneumoniae NTUH-K2044. CPS levels of K. pneumoniae NTUH-K2044 were activated by increasing environmental glucose. Bacterial strains were grown in LB broth supplemented with glucose and cAMP as indicated at 37°C with agitation. After 16 h of growth, the bacterial glucuronic acid content was determined. *P<0.05 and **P<0.01 compared with no addition. #P<0.05 and $ P<0.01 compared to the indicated group.(TIF)Click here for additional data file.Figure S2CRP directly binds to PgalF. Diagrammatic representation of the galF loci. The large arrows represent the open reading frames. The relative positions of the primer set used in PCR-amplification of the DNA probes are indicated, and the numbers denote the positions relative to the translational start site. Name of the DNA probes are shown on the left. Different concentrations of purified His6-CRP were incubated with 10 ng of the upstream regions of galF. Following incubation at room temperature for 30 min, the mixtures were analyzed on a 5% non-denaturing polyacrylamide gel containing 200 µM cAMP. The gel was stained with SYBR Green I dye and photographed.(TIF)Click here for additional data file.Figure S36-CRP complements the effect of Induced expression of Hiscrp mutation on CPS biosynthesis. CPS levels of K. pneumoniae strains carrying the IPTG inducible vector pETQ or pETQ-His-crp, as shown in the left panel, were determined. Bacteria were grown in LB medium with 100 µM IPTG at 37°C with agitation. **P<0.01 compared to the indicated group.(TIF)Click here for additional data file.


Arabidopsis. We showed that miR172b and its target SNZ were co-expressed during early seedling development. Expression of miR172b and SNZ was low after radicle emergence and sharply increased at the checkpoint to autotrophic development under normal conditions. Interestingly, activation of miR172b and SNZ was completely abolished by ABA and osmotic stress. miR172b overexpression and snz-1 exhibited increased sensitivity to ABA and osmotic stress during specific post-germinative stage, and resulted in higher expression of ABI3, ABI5 and downstream genes, such as Em6 and RAB18, than wild type under ABA treatment. Our results revealed that miR172b is a critical regulator specifically controlling cotyledon greening during post-germinative growth by directly targeting SNZ under ABA treatment and osmotic stress.Seedling establishment is a critical phase in the life of plants when they are the most vulnerable to environment. Growth arrest at post-germinative stage under stress is the major adaptive strategy to help germinating seedlings to survive a spectrum of stressful conditions. ABA signaling is the key pathway to control stress-induced developmental arrest. However, mechanisms controlling the phase transition under abiotic stress are not fully understood. Here, we described miR172b as a new key regulator controlling transition of germinating seedlings from heterotrophic to autotrophic growth under osmotic stress in  ArabidopsisSeed germination, post-germinative growth and subsequent seedling establishment are central to successful stand establishment and population maintenance in plants, which are of great importance to agriculture and ecology ABI1 and ABI2) ABI3 and ABI5ABI3 and ABI5 are expressed in a narrow developmental window and control the post-germination developmental arrest checkpoint. For example, the ABI5 level was undetectable on day 1 after stratification, but progressively increased thereafter and reached the highest level on day 4 Compelling evidence has shown that the checkpoint from heterotrophic to autotrophic development at the post-germinative stage is genetically controlled, and that this phase transition is also highly vulnerable to various stresses including osmotic stress Arabidopsis is miR156, which regulates post-germinative development through modulating the mRNA of its target SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE13 (SPL13) SCHNARCHZAPFEN (SNZ), an AP2-like gene targeted by miR172 in ArabidopsisABI3 and ABI5 at post-germination stage. Further, we show that miR172b mediates its function by partially targeting SNZ. Together, these data identify miR172b as a master regulator in stress-induced developmental arrest and adaptation to stress.microRNAs (miRNAs) are small, single-stranded, non-coding RNAs, which down-regulate their target genes at the post-transcriptional level through miRNA cleavage or translational repression 35S: miR172bsnz-1abi5-8 (SALK_013163) snz-1 overexpressing SNZ and snz-1abi5-8 double mutants (Seeds of Columbia ecotype (Col-0),  mutants  were surSNZ under the control of cauliflower mosaic virus 35S promoter in snz-1 mutant background, we cloned the coding region of SNZ by polymerase chain reaction (PCR) which was then inserted into the pTF101 vector containing a bar gene. The primers used for the construction are listed in To generate transgenic plants overexpressing snz-1 was crossed with abi5-8 to generate the snz-1abi5-8 double mutant. Because both mutants contain T-DNA insertions, we performed PCR to identify the homozygous lines containing both mutations in SNZ and ABI5 using the specific primers . The primers used for genotyping are listed in Homozygous 35S: miR172bsnz-1−1 sucrose, 8 gL−1 agar, and different concentrations of ABA , NaCl , Mannitol . After 2-day stratification at 4°C in darkness, the plates were incubated in a growth chamber at 22±2°C under long-day conditions (16 h light/8 h dark). Germination was defined as the emergence of 1 mm or more of the radicle from the seed coat as described previously For germination assays, at least 60 seeds from All experiments were repeated three times with three replicates (n>60) in each. The data reported in the figures are means of the values with standard deviation (SD).GAPC gene, as a reference gene expressed stably. All the experiments were performed three times, each with three replicates. Error bars denote SD.Seeds were kept in darkness at 4°C for 2 days with or without ABA (0.4 µM or 5 µM) and then transferred to constant light at 22°C. Total RNA was extracted from plant tissue using Trizol reagent (Invitrogen) according to the manufacturer's instructions. 2 mg of total RNA was DNase I-treated and single-stranded cDNA was synthesized using oligo (dT) and the RevertAid First Strand cDNA Synthesis Kit (Invitrogen). The sequences of primers that were used for gene expression analysis are listed in 6-3′ (wRAV1-A), labeled with biotin at the 3′ end . An oligonucleotide containing mutated RAV1-A motif, 5′-CACCTG(CGGTA) 6-3′ (mRAV1-A), labeled with biotin at the 3′ end  was used as a control. The specific probes containing RAV1-A motifs corresponding to the fragment of ABI5 promoter were synthesized and labeled with biotin at the 3′ end as mentioned above. The sequences of probes used for EMSA are listed in ABI5 probes). EMSA reaction solutions were prepared by adding the following components in order according to the manufacturer's protocol (LightShift Chemilu minescent EMSA kit): binding buffer, 50 ng poly (dI-dC), 2.5% glycerol, 0.06% NP-40, 5 mM MgCl2, 19 mg BSA, proteins (28 µg), competitor and biotin-labeled probes. Reaction solutions were incubated for 30 min at room temperature. The protein-probes mixture was separated in a 8% poly-acrylamide gel and transferred to a Biodyne B Nylon membrane. Migration of biotin-labeled probes was detected by using streptavidin-horseradish peroxidase conjugates that bind to biotin and chemiluminescent substrate according to the manufacturer's protocol. This experiment was performed three times.Recombinant MBP-SNZ fusion protein was expressed and purified using Amylose Resin (NEB)according to the manufacturer's introduction. The binding activity of the protein was analyzed using an oligonucleotide containing 6 copies of RAV1-A motif, 5′-CACCTG(CAACA)All the data were analyzed using software GraphPad Prism 5. The Student's t-test was performed and the statistically significant treatments were marked with ‘*’ (P<0.05), ‘**’ (P<0.01) and ‘***’ (P<0.001).Arabidopsis grown under normal conditions After seed germination, germinating seedlings proceed through a critical developmental stage referred to as the transition from heterotrophic to autotrophic growth that can be tracked by cotyledon greening. Thus, cotyledon greening is thought to be the critical checkpoint in seedling establishment. When plant seeds are germinated under stress conditions, the developmental program to autotrophic growth is interrupted and stress reinstates the embryonic stage ArabidopsisBecause ABA is previously shown to play a prominent role in regulation of post-germinative growth arrest under osmotic stress in As the levels of miR172 expression are critical for regulating phase transition during plant development, and overexpression of miR172b results in post-germinative developmental arrest under osmotic stress and ABA , we therWhen wild type seeds were germinated on the medium containing 5 µM ABA, levels and patterns of miR172b expression were strikingly different . In the cis elements responsive to osmotic stress and/or ABA. Bioinformatic analysis of the miR172b promoter identified several ABA responsive cis elements, such as ABRE, Couple element 3 and cis elements related to stress response  in the promoter region of miR172b  is also directly regulated by ABI3 and ABI5 during seed germination MFT was induced by ABA at dramatically higher level in 35S:miR172b germinating seedlings  transcription factors: AP2, TOE1, TOE2, TOE3, SMZ, SNZhttp://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi). We found that among the targets, SNZ and TOE3 were responsive to ABA and osmotic stress/drought in 7 and 18 day-old seedlings  cis regulatory elements ABI3, ABI5, and their downstream genes Em1, Em6, RAB18 and MFT were identified  promoter fragments of ABI5, which contain the RAV1-A motif, as probes. The results showed that SNZ directly interacted with ABI5 promoter fragment  detected in fragment . By contsnz-1 and abi5-8. We generated a snz-1abi5-8 double mutant and performed phenotypic analysis. As shown in snz-1abi5-8 double mutant exhibited ABA insensitive phenotypes similar to the abi5-8 mutant during both germination  or ABA . The photographs were taken 7 days after transfer to NaCl (A), and the primary root length was estimated (B). Photographs were taken 5 days after transfer to ABA (C), and the primary root length was estimated (D). (E) Identification of overexpression of miR172b in 35S:miR172b transgenic lines. Bar = 250 µm. All the experiments were performed three times with three replicates. Bars represent mean±SD.(TIF)Click here for additional data file.Figure S2Post-geminative developmental arrest of 35S:: miR172b on ABA was restored after removing ABA. Seeds of 35::miR172b and Col-0 were germinated on MS medium containing 0.8 µM ABA, the seedlings which turned green were counted at 12 days after stratification (A); the arrested seedlings were transferred onto MS medium (B) or MS plus 0.8 µM ABA (C) for another 3 days, and the number of seedlings turning green were recorded. Bar =  5mm. All the experiments were performed three times with three replicates. Bars represent mean±SD.(TIF)Click here for additional data file.Figure S3Phenotype of 35::miR172b transgenic plants and snz-1 under LiCl and KCl treatment. (A) KCl and LiCl dose–response analysis of seed germination for Col-0, 35S:: miR172b and snz-1 at 3 days after stratification. (B) and (C) Greening of 35:: miR172b transgenic plants and snz-1 was not affected under KCl and LiCl treatment. Photographs were taken 7 days after stratification. Bar = 5 mm. All the experiments were performed three times with three replicates. Error bars denote±SD.(TIF)Click here for additional data file.Figure S4SNZ and TOE3 were up-regulated by ABA and osmotic stress/drought. Plant materials from 7 and 18 day old wild-type Col-0 was treated by ABA and osmotic stress, respectively. (A) and (B) Expression of SNZ (A) and TOE3 (B) in response to ABA and osmotic stress/drought; The colors from yellow to red indicate the increased absolute signal values of SNZ and TOE3 expression retrieved from microarray data. (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi).(TIF)Click here for additional data file.Figure S5Expression of SNZ and TOE3 in response to ABA. (A) Expression analysis of TOE3 in response to ABA. Wild-type seeds were germinated on MS with or without 5 µM, and materials were collected at 2 to 4 days after stratification. (B) SNZ expression in wild-type (Col-0) and 35S:: miR172b plants on day 3 and 4 after stratification with or without 0.4 µM ABA. Student's t test was performed, and the statistically significant treatments are marked with asterisks. (*) P<0.05, (**) P<0.01 and (***) P<0.001. All the experiments were performed three times with three replicates. Error bars denote±SD.(TIF)Click here for additional data file.Figure S6SNZ regulates post-germinative developmental arrest induced by abiotic stress through the ABA dependent pathway.SNZ cDNA under control of 35S promoter was expressed in snz-1 mutant, and the transgenic lines were characterized and used for phenotypic analysis. (A) Transcript abundance of SNZ in wild-type, snz-1 and 35S:: SNZ/snz-1 transgenic lines was monitored using RT-PCR. Shown are RT-PCR products amplified after 30 cycles, and the transcript expression levels were normalized against the reference gene GAPC. The red boxes indicate the line which was used in the phenotypic analysis. (B) ABA dose–response analysis of seed germination for Col-0 and snz-1 at 5 days after stratification. (C) A greening time course on medium containing 0.4 µM ABA. All the experiments were performed three times with three replicates. Error bars denote±SD.(TIF)Click here for additional data file.Figure S7Genotyping of snz-1/abi5-8. (A) Schematics showing the location of the T-DNA insertion in abi5-8(SALK_013163) and snz-1(SALK_030031). Black boxes indicate exons, green box indicate 5′UTR, yellow box indicates 3′UTR, and the horizontal lines indicate introns. (B) Confirmation of the homologous T-DNA insertion in snz-1/abi5-8 by PCR. Col-0 was used as positive control. The red boxes indicate the lines with the homologous T-DNA insertions both snz-1 and abi5-8 mutations, which were used in the phenotypic analysis.(TIF)Click here for additional data file.Table S1cis-acting regulatory elements analysis of promoter sequence of miR172b by PLACE and PROMO.(DOC)Click here for additional data file.Table S2cis-acting regulatory elements analysis of the SNZ promoter sequence by PLACE and AtcisDB.(DOC)Click here for additional data file.Table S3Primers pairs used for real-time RT-PCR (Sequence 5′→3′).(DOC)Click here for additional data file.Table S4Primers pairs used for construction and genotyping, probes used for EMSA (Sequence 5′→3′).(DOC)Click here for additional data file.Table S5Information of the plant materials used in this work.(DOC)Click here for additional data file.Table S6cis-acting regulatory elements RAV1 analysis of promoter sequence of ABA response genes by PLACE and AtcisDB.(DOC)Click here for additional data file.


A novel class of transcripts, long non coding RNAs (lncRNAs), has recently emerged as key players in several biological processes, including dosage compensation, genomic imprinting, chromatin regulation, embryonic development and segmentation, stem cell pluripotency, cell fate determination and potentially many other biological processes, which still are to be elucidated. LncRNAs are pervasively transcribed in the genome and several lines of evidence correlate dysregulation of different lncRNAs to human diseases including neurological disorders. Although their mechanisms of action are yet to be fully elucidated, evidence suggests lncRNA contributions to the pathogenesis of a number of diseases. In this review, the current state of knowledge linking lncRNAs to different neurological disorders is discussed and potential future directions are considered. A few exceptions to this paradigm were known . Non-coding RNAs (ncRNAs) include the familial “housekeeping” RNAs and the thousands of regulatory RNAs that are the subject of recent intense investigation. NcRNAs come in many different sizes and for this reason have been divided into small and long classes: small ncRNAs (sncRNA) being less than 200 nucleotides (nt), and long ncRNAs (lncRNA) greater than 200 nt to over 100 kb in length [An estimation of the number of lncRNA loci from transcriptional surveys in mammals suggests that they are at least as numerous as protein-coding genes  with manThe lncRNAs that have been studied in detail were found to be involved in different biological processes including X chromosome inactivation, nuclear structure, genomic imprinting and development.In the following paragraphs some of the known functions of lncRNAs will be discussed with particular regard to their role in neurological disorders.2.Data derived from massive cloning and traditional sequencing methods have demonstrated that mammalian genomes produce thousands of RNA transcripts in addition to protein-coding genes . These sRecent advances in RNA sequencing technology (RNA-Seq) addressed further interrogation of the total cellular RNA, or, transcriptome, at a much higher resolution . Thousan3.Many of the lncRNAs seem to be involved in epigenetic mechanisms of gene regulation. Although the field of epigenetics earned its name over 50 years ago, just in this past decade the significance of epigenetics has been recognized in human health and disease . The terN-termini of histones such as lysine, arginine and serine residues that can be acetylated or methylated (lysine and arginine) and phosphorylated (serine). Histone modifications are placed on and removed from histone residues by numerous enzymes that usually work as part of multi-protein complexes [The modification of histone proteins has also been found in epigenetic regulation . Histoneomplexes .A relevant question in biology is how chromatin-modifying complexes are targeted to specific genomic loci since many of these enzymes lack DNA binding capacity. Recent studies suggest a potential role for ncRNA in driving chromatin-modifying complexes to genomic loci –13, howe4.cis and trans processes.Basing on our current knowledge of lncRNAs, it appears that such molecules are involved in many different aspects of cellular functions . The4.1.cis. Several imprinted clusters contain protein-coding genes and lncRNAs that are reciprocally expressed, such as IGf2r/Air [Dlk1/Gtl2 [Nesp/Nespas/Gnas [While the function of parental gene imprinting is still unclear, lncRNAs have been found to participate in imprinting processes. These are referred to as the events that influence the monoallelic expression of a gene according to its parents of origin. Imprinting control regions (ICRs) are DNA regions that are differentially methylated depending on their parental origins. Unmethylated ICRs cause specific expression of nearby lncRNAs, which then suppress neighboring genes in Gf2r/Air , Dlk1/Gtlk1/Gtl2 ,19, and pas/Gnas . Some ofpas/Gnas ,22.cis through interaction with repressive chromatin modifying complexes [Airn and Kcnq1ot1 are examples of lncRNAs that cause suppression of paternally inherited genes. Kcnq1ot1, in particular, is involved in the repression of several protein-coding genes in omplexes ,24.Igf2.H19 was one of the first mammalian lncRNAs to be identified and is highly expressed in the embryo ,26. Thou4.2.Xic) [Xist) is highly expressed from Xi during the onset of XCI but not from Xa, contributing a defining moment for the realization that lncRNAs can have profound roles in the control of gene expression even though the exact mechanism of action is still not completely understood. Some evidence suggests that Xist mediates the chromosome X silencing effects by interacting with repressive chromatin-modifying complexes such as PRC2 [The X chromosome inactivation (XCI) indicates the mechanisms by which the difference in X-linked gene dosage between XX females and XY males is exerted in therian mammals in which one of the two X chromosomes in females is silenced (the inactive X or Xi) so that only one X remains active and is expressed in each female cell  . It is wXic) . The X  and this activity recruits dnmt3a to suppress Xist from being transcribed on Xa [Xi, it is Tsix that is suppressed, potentially through another lncRNA that is part of the X inactivation center, Jpx [Tsix suppressed, the protein PRC2 is recruited to induce histone modification marks at the 5′ end of Xist. This upregulates Xist expression on Xi and causes further propagation of these silencing marks throughout Xi, which are maintained across the lifetime of the organism [ed on Xa . Converster, Jpx . With Tsorganism .4.3.Hox genes encode homeodomain TFs that are crucial for anterior-posterior pattern formation in bilateral metazoans [Hox genes are structured in linear clusters along the chromosome, and mammals have four paralogous clusters, HoxA, -B, -C, and -D. Several lncRNAs are encoded within these clusters, including HOTAIR (Hox antisense intergenic RNA) from HoxC, and HOTTIP and Mistral from HoxA [Hox genes is also regulated by lncRNAs [cis, having either enhancing or repressive effects. However, some like the human HOTAIR works in trans, and it is expressed from the Hox locus marking a boundary of active and inactive chromatin [Xist, interacts with chromatin-modifying complexes such as PRC2 and the corepressor complex CoREST [LncRNAs are likely implicated in processes involving animal development. The etazoans . Hox genrom HoxA –37. The  lncRNAs . Some Hohromatin . Furtherx CoREST  and may x CoREST .Recently HOTAIR was found to be crucial for cell growth and viability and that its knockdown induced apoptosis in breast cancer cells Moreover it was found that HOTAIR is transcriptionally induced by estradiol . It is p4.4.et al. recently [The promoters of more than 100 lncRNAs are bound by stem cell factors. Disruption of these lncRNAs can alter cell differentiation. The human lncRNA-RoR (RoR) is a recently identified lncRNA that is capable of reprogramming differentiated cells to induce pluripotent stem cells ,40. RoR recently  demonstret al. [Recently, a refined analysis from Guttman et al.  performeTUG1) is an lncRNA expressed in the developing retina and brain, as well as in adult tissues. It has been found that in the newborn retina, loss of TUG1 resulted in malformed or non-existent outer segments of transfected photoreceptors, thus suggesting that TUG1 is required for the proper formation of photoreceptors in the developing rodent retina [Dlx5-Dlx6 intergenic regions and controlled Dlx5, Dlx6 and Gad1 expression through cis and trans acting mechanisms. Evf2 mouse mutants appeared to have reduced numbers of GABAergic interneurons in early postnatal hippocampus and the dentate gyrus. This situation is restored to normality in Evf2 mutant adult hippocampus although reduced synaptic inhibition still occurred [lncRNAs are implicated in cell fate determination events in multiple cells lineages, including the nervous system. Taurine upregulated gene 1  locus. FSHD is the third most common myopathy and is predominantly caused by a contraction of specific repeats mapping on chromosome 4q35 [MALAT-1, which is another lncRNA associated with various cancers and metastasis  was founome 4q35 . It is some 4q35 . Furtherome 4q35 .6.Recent evidence demonstrate that lncRNAs contribute to the complex biological system organization and gene regulatory networks of the central nervous system (CNS), affecting brain patterning, neural stem cell maintenance, neurogenesis and gliogenesis, stress responses, and synaptic and neural plasticity.Mercer and colleagues identified 849 lncRNAs (among the 1328 examined), that are expressed in the adult mouse brain and found that the majority were associated with specific neuroanatomical regions, cell types, or subcellular compartments . A complet al. discovered more than 1000 evolutionarily conserved intergenic lncRNAs in mouse by analyzing chromatin signatures from four mouse cell types [Guttman ll types . A functll types .Sox2 gene within one of its introns and is subsequently transcribed in the same direction [in vitro enhances their differentiation along the OL lineage, in part, by inducing an increase in Nkx2.2 mRNA levels [Detailed analyses of specific lncRNAs, dynamically expressed in the CNS, reveal potential roles in mediating neural cell fate decisions. The Sox2OT lncRNA, which contains the irection , is exprirection . Moreoveirection . The lncA levels .BC1 and primate-specific BC200 lncRNAs, are selectively targeted to postsynaptic dendritic compartments, where they regulate local protein synthesis by repressing the initiation of translation through an eIF4A-dependent mechanism [LncRNAs also modulate synaptic plasticity and promote long-term changes in synaptic strength. The rodent-specific echanism –69. Simiechanism . Anotherechanism .6.1.6.1.1.-as is a lncRNA transcribed antisense to the maternally expressed Ube3a gene, mutated or deleted in AS, suggesting that Ube3a-as may repress paternal Ube3a expression. Other studies have shown that repression of Ube3a is dependent on Ube3a-as [Ube3a can occur in the absence of Ube3a-as and implies a more complex regulatory relationship underlying the imprinting of Ube3a [Angelman syndrome (AS) is a neurodevelopmental disorder associated with genomic imprinting and characterized by severe neurologic abnormalities . Ube3a-aUbe3a-as ,74. Howeof Ube3a .FMR1, the gene encoding the neuronal development protein, FMRP. The lncRNAs ASFMR1 and FMR4 are generated from the FMR1 gene locus. ASFMR1 has multiple alternative splicing patterns and overlaps the 5′ untranslated region (UTR) CGG repeat region of FMR1 [FMR1 start site. Alternative splicing of ASFMR1 seems to exhibit pre-mutation-specific profiles and is also silenced in FXS patients and upregulated in pre-mutation carriers, suggesting that a common process is responsible for regulating the expression of these transcripts. FMR4 is also silenced in FXS patients because of a CGG expansion repeat in the 5′UTR of the FMR1 gene and upregulated in pre-mutation carriers [LncRNAs may influence the pathogenesis of fragile X syndrome (FXS), which is characterized by a triplet nucleotide repeat expansion in the 5′UTR of  of FMR1 ; FMR4 iscarriers ; thus, tAnother microsatellite expansion disease in which lncRNAs are involved is the spinocerebellar ataxia type 8 (SCA8), an autosomal dominant disorder, characterized by bidirectional transcription of this expansion repeat from opposite strands, forming both a protein-coding transcript encoding a polyglutamine expansion, ATXN8, and a lncRNA transcript containing a CUG expansion, ATXN8OS . This suHtt gene, which encodes a ubiquitously expressed 3144 amino acid protein of unknown function, leading to a toxic gain of function in the mutant protein [HAR1 locus is under control of REST [HAR1 region contains lncRNAs, HAR1F and HAR1R. HAR1F is specifically expressed in the neurons of the marginal zone during development of the cortex and in the frontal cortex, hippocampus, cerebellum, thalamus and hypothalamus in the adult brain [Huntington’s disease (HD) is caused by an expansion repeat mutation in the  protein , which p of REST . The HARlt brain . The levlt brain .6.1.2.APP) and the generation of amyloid peptides which can aggregate and form plaques. Faghihi and colleagues characterized a conserved non-coding antisense transcript for BACE1, called BACE1-AS, which functions as a regulator of BACE1 gene expression. BACE1-AS upregulates BACE1 levels in response to a variety of stresses, including Aβ 1–42 exposure, and is elevated in several brain regions of patients with AD. These findings imply that BACE1-AS is deregulated in AD, which induces feed-forward regulation of BACE1, increases Aβ levels, and thus may promote the pathogenesis of the disease [β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a crucial enzyme in AD pathophysiology, involved in the cleavage of the amyloid precursor protein  and other customized oligonucleotide approaches with the capacity to reprogram disease-associated mRNAs, are now being developed . These a