Rotavirus genome consists of eleven segments of dsRNA, each encoding one single protein. Viral mRNAs contain an open reading frame (ORF) flanked by relatively short untranslated regions (UTRs), whose role in the viral cycle remains elusive. Here we investigated the role of 5’UTRs in T7 polymerase-driven cDNAs expression in uninfected cells. The 5’UTRs of eight genome segments  of the simian SA11 strain showed a strong inhibitory effect on the expression of viral proteins. Decreased protein expression was due to both compromised transcription and translation and was independent of the ORF and the 3’UTR sequences. Analysis of several mutants of the 21-nucleotide long 5’UTR of gs 11 defined an inhibitory motif (IM) represented by its primary sequence rather than its secondary structure. IM was mapped to the 5’ terminal 6-nucleotide long pyrimidine-rich tract 5’-GGY(U/A)UY-3’. The 5’ terminal position within the mRNA was shown to be essentially required, as inhibitory activity was lost when IM was moved to an internal position. We identified two mutations (insertion of a G upstream the 5’UTR and the U to A mutation of the fifth nucleotide of IM) that render IM non-functional and increase the transcription and translation rate to levels that could considerably improve the efficiency of virus helper-free reverse genetics strategies. Reoviridae family) are the major cause of gastroenteritis in infants and young children worldwide and infect many other animal species  were used. Expression downregulation was only present when IM was positioned at the 5’-terminal end in the 5’UTR, with GG as the initial dinucleotide, while insertion of the wild-type IM or the U5A control within the ORF or the 3’UTR did not show any difference (3), which showed strong expression levels  of gs11 was moved from its original 5’ end terminal position to a position either within the 5’UTR, downstream of a 15-nucleotide long arbitrary sequence that does not inhibit protein expression, or in the coding region or in the 3’UTR of a construct containing the EGFP ORF and the UTRs of gs11. The resulting constructs, indicated as 5’-dIM, IMfference . This con levels  thus indin vitro by T7 polymerase from a construct containing the EGFP ORF flanked by gs11 UTRs (EGFP-5’3’gs11) and from the corresponding one containing the U to A mutation in position 5 [EGFP-5’(A5)3’gs11]. Both mRNAs were produced at similar levels, with the last one transcribed only 1.5 times more (in vivo from the same two constructs in MA104 cells infected with vTF7.3 vaccinia virus (determined by qPCR) yielded, however, a five times higher level of the transcript from the U5A construct .We then determined whether compromised expression was the consequence of inhibition of transcription or translation. mRNAs were produced mes more . Transcronstruct . When te in vivo , while t in vivo . AltogetTo establish if IM has also an impact on transcripts stability, an assay of the decay rate was performed exploiting incorporation of the ribonucleotide homologue ethynyl uridine (EU), in presence of pactamycin and cycloheximide (two drugs that block translation). EU was fed to transfected cells for 1.5 hours and then incorporation into newly-made mRNAs was stopped by addition of the transcription inhibitor actinomycin D. RNA samples were collected at different time points and EU-labelled mRNAs quantified by qPCR. As shown in in vitro transcribed and then equal amounts of transcripts were in vitro translated in a HeLa cell lysate-based protein expression system. As shown in in vitro (coupled transcription/translation) expression system 3’gs11 mutant were  compare  with 6C.in vitro . Of notein vitro . Becausein vitro .In conclusion, the overall IM inhibitory effect on expression is due to an interference with both T7-mediated transcription and cellular translation.We tested the 5’UTRs of all remaining SA11 genome segments cloned upstream of EGFP. As expected, the 5’ UTRs of gs 3, 5, 6, 9, and 10 , which contain an IM identical to that of gs11, inhibited EGFP expression, whilst those of gs 1 and 4  containing an A in position 5 did not . InteresThus, a set of cDNA constructs encoding all genome segments under the control of T7 promoter and containing either of the described mutations (addition of an extra 5’ G or mutation of T5 into A) with exception of gs1, 2 and 4 should provide the basis for a more efficient helper virus-free reverse genetics system.To test whether rotavirus infection can overcome IM activity, we constructed a recombinant vaccinia virus (vT7-NE) with inducible expression of both the T7 RNA polymerase and an NSP5-EGFP fusion (NE) flanked by the gs11 UTRs. This construct was first validated by transfection experiments showing that the gs11 5’UTR inhibited NSP5-EGFP expression, independently of the 3’UTR . Co-infeKnowledge of the function of several rotavirus proteins and their precise roles in the viral replication cycle still presents unclear points. In part this is due to the lack of a broad reverse genetics system that could be universally applicable to all RV genome segments. Plasmid-based helper virus-free strategies were already successful with other viruses belonging to the same family of rotavirus (orbivirus and orthoreovirus) but failed when applied to rotavirus. One of the main reasons proposed to explain the difficulties encountered in having a helper virus-free system for rotavirus is the poor expression of transfected mRNAs or cDNAs. Indeed in the few cases in which recombinant RVs were obtained, robust protein production was guaranteed by the helper virus infection.The experience with bluetongue virus and the “double transfection strategy” highlighted the importance of a strong protein production in order to achieve the recovery of recombinant virus . It is ibona fide 5’ and 3’ terminal sequences of viral mRNAs.In this work we revealed that the 5’UTR of several  genome segments of rotavirus contain an inhibitory motif (IM) that downregulates expression of rotavirus cDNAs when expressed exploiting a T7 polymerase-encoding recombinant vaccinia virus. This is the preferred system used in all attempts to generate a recombinant rotavirus since the T7 promoter coupled with the HDV ribozyme provides Rotavirus mRNAs are known to form secondary structures involving interaction between 5’ and 3’ UTRs ,29, but in vivo there is a higher rate of transcription of the cDNAs in which IM was mutated in position 5. T7 polymerase is a single subunit enzyme that does not require protein co-factors. The T7 promoter is composed of two domains: the binding region and the catalytic domain. Since the binding region is located upstream position -5, IM is unlikely capable of interfering with the proper tight binding of the polymerase with its promoter [According to our studies, IM activity is the consequence of impairment of both transcription and translation. Quantification of the relative levels of mRNAs showed that promoter . During promoter . In thispromoter . The IM promoter .in vitro transcription kits presented a discrepancy. The mRNA levels in the coupled transcription-translation HeLa cell lysate-based system reflected what was observed in vivo , while in the in vitro transcription kits containing only T7 enzyme and an adequate transcription buffer, transcripts of cDNAs containing non-functional IM were only slightly more abundant than the counterpart. We questioned if this yield disparity could be explained by a major rate of degradation that could occur in vivo to IM-containing mRNAs. Our results showed no differences in mRNA stability. Since the difference in transcription levels was detectable only in presence of cellular cytoplasmic extracts, a possible explanation hypothesizes the presence of a cytoplasmic factor that can specifically bind to IM and therefore interfere with transcription.Interestingly, the quantification of mRNA levels using two different in vivo, transfecting in vitro synthesized transcripts into cells, and in vitro, using HeLa cell lysate-based in vitro protein expression systems fed with pre-formed mRNAs. This effect on translation upon deletion of the 5’UTR from gs11 was not due to a change of the nucleotide context of the start codon. Optimization of the nucleotides upstream the AUG codon did not modify expression of gs11 nor of the mutant lacking the 5’UTR. In both constructs, the nucleotide in position +4, which is not optimal in gs11 (U instead of G), was not changed. In a recent work with gs4 Gratia et al. reported that optimisation of both positions -3 and +4 is important for expression efficiency [AUGU), strongly suggests that the IM inhibitory activity on translation cannot be ascribed to a less effective start codon recognition. The UTRs of a number of viruses, such as JEV [The presence of IM affects also translation since mRNAs containing non-functional IM increase protein expression by 3 times compared with the ones containing functional IM. This difference was observed both ficiency . Howeverh as JEV , HIV-1 [h as JEV , HCV [37h as JEV , and polh as JEV  are bounh as JEV . These mh as JEV ). Althouh as JEV –45. A coh as JEV . In factRecently, it has been reported that certain adenosines within the 5’UTRs of cellular mRNAs are preferentially methylated under stress conditions and this post-transcriptional modification was found to promote translation initiation . AlthougWe found that rotavirus infection partially reverts IM-mediated expression inhibition suggesting that a viral component (or a cellular factor induced upon infection) interferes with the inhibitory mechanism in uninfected cells. It can be hypothesized that while in uninfected cells a trans-acting negative regulator has free access to the viral mRNAs containing an IM, in infected cells it is displaced from viral messengers possibly through binding competition with specific viral proteins. In this respect, we performed co-expression experiments with a panel of viral proteins . So far, we have not identified any individual viral protein able to revert IM inhibitory activity. Not even NSP3 could rescue protein expression. In the context of viral infection, specific changes in subcellular distribution and in quantity of the translation apparatus components, –50 resulIM and its non-functional versions are highly conserved among the same genome segments of different group A rotavirus strains . The preIn conclusion, our study is a deep analysis of the mechanisms underlying poor protein expression from rotavirus cDNAs and considers their possible involvement in the failure of helper virus-free reverse genetics strategies applied to rotavirus. We mapped two distinct single base mutations that increase both mRNA transcription and protein translation, which are likely compatible with genome segment packaging in infectious particles. The increased yields of mRNAs and proteins could be the key to obtaining infectious virus from cells transfected with all eleven genome segments.S1 FigAnti-NSP5 WB of cells transfected with polyadenylated versions of gs11 constructs, with or without 5’UTR. The polyadenylated mRNAs were T7-polymerase transcribed, derived from a synthetic gene fragment containing 30 adenines downstream of the 3’UTR. Tubulin used as loading control.(TIF)Click here for additional data file.S2 FigA-B) Anti-NSP5 WB of MA104 cells co-expressing different gs11 constructs with or without NSP3, either un-tagged (A) or SV5-tagged (B). In B anti-SV5 WB revealing expression of SV5-NSP3. Tubulin used as loading control.(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 TableNucleotides that render IM non-functional are indicated in red; genome segments containing a functional IM are marked with a tick. ALL indicates the six strains.(TIF)Click here for additional data file.


The ERK-regulated ternary complex factors (TCFs) act with the transcription factor serum response factor (SRF) to activate mitogen-induced transcription. However, the extent of their involvement in the immediate-early transcriptional response, and their wider functional significance, has remained unclear. We show that, in MEFs, TCF inactivation significantly inhibits over 60% of TPA-inducible gene transcription and impairs cell proliferation. Using integrated SRF ChIP-seq and Hi-C data, we identified over 700 TCF-dependent SRF direct target genes involved in signaling, transcription, and proliferation. These also include a significant number of cytoskeletal gene targets for the Rho-regulated myocardin-related transcription factor (MRTF) SRF cofactor family. The TCFs act as general antagonists of MRTF-dependent SRF target gene expression, competing directly with the MRTFs for access to SRF. As a result, TCF-deficient MEFs exhibit hypercontractile and pro-invasive behavior. Thus, competition between TCFs and MRTFs for SRF determines the balance between antagonistic proliferative and contractile programs of gene expression.  •Integrated ChIP-seq Hi-C analysis identifies over 700 TCF-dependent SRF target genes•Over 60% of TPA-inducible gene transcription is TCF-dependent•TCF-dependent transcription potentiates cell proliferation•TCF/MRTF competition for SRF determines contractility and pro-invasive behavior Two cofactor families, the TCFs and the MRTFs, compete for binding to SRF, a key regulator of the mitogen-responsive transcription. TCFs not only control proliferative gene expression programs but also regulate access of the MRTFs to SRF, thereby regulating cell contractility and pro-invasive behavior. Srf), which is required for Ras-induced cell-cycle re-entry but not for proliferation per se  are Ets domain proteins, regulated by Ras-ERK signaling  Elk-1, Net, and SAP-1  and RasGRP1 . TPA alsmulation A. We usemulation . The TPAmulation  associatmulation A and genmulation B. It wasmulation B, suggesTo investigate the role of TCFs in gene expression and cell behavior, we studied MEFs from embryos lacking all three TCFs . They were associated with the same transcription factor (TF) binding motifs as in NIH 3T3 fibroblasts . We first identified 2,577 high-confidence SRF binding sites . In TKO roblasts , althougroblasts F.Integration of Hi-C chromosome interaction maps with ChIP-seq data can reliably identify signal-induced genes controlled by remote regulatory sites . We therUsing principal component analysis (PCA) , we partKbtb2 and Myc, which interacts with five remote SRF sites, the closest at ∼200 kb  (Approximately 25% of the detectable SRF/Elk-1 binding sites (89 of 356) were associated with the Direct TCF-SRF target genes, and a further 30 were associated with TPA-inducible SRF target genes that were not scored as TCF-dependent . In contnd Hat1) . Thus, ENext we sought to understand why Elk-1 expression only partially restored TCF-dependent TPA induction. Although it cannot be ruled out that this reflects gene-specific targeting of Elk-1, we found that the “rescued” genes had TSSs that were in general significantly closer to SRF sites than genes that were refractory to Elk-1 expression G. PrevioThe expression of Indirect TCF-dependent target genes must either rely on basal levels of proteins encoded by direct TCF-SRF target genes, or reflect a secondary response to transcriptional regulators produced by immediate-early genes. The Direct TCF-SRF target gene set includes 54 different transcription factors, many of which are themselves sensitive to ERK activation , which inhibits MRTF . qPCR gene expression analysis was by standard methods. For primers, see ChIP and ChIP-seq have been described previously  with seqGSE75667.Samples were prepared using the GenElute mammalian total RNA miniprep kit (RTN350-1KT). DNA was removed by DNAase I and rRNA by the Ribo-zero rRNA removal kit (Epicenter). RNA-seq libraries were prepared from 1 μg RNA using the directional mRNA-seq Library Prep v1.0 protocol (Illumina). Libraries were subjected to 72-base single-end sequencing on the Hiseq analyzer, and trimmed to 50 bp. Raw and processed data are in the GEO: diff), assuming a quasi-normal distribution of gene read counts. Differential gene expression analysis was performed with Deseq  ≤ 0.01; minimum change of 10%). The effects of knockout and reconstituted background were estimated by comparing the degree of gene induction under the different conditions and identifying genes that are similarly affected  (default settings). Reads within annotated RefSeq genes  and reads containing intronic sequences (“intronic reads”) were annotated using the bam file obtained by BWA as input. Normalization was against a set of invariant genes across samples, defined as those with differences in read counts within 1σ from the mean difference  . Raw and processed data are online in the GEO: q (MACS) , retainihttp://homer.salk.edu/homer/interactions). Reads from NCBI SRA: SRX554530 (GSM1648486 and GSM1696042 (Z score ≥ 2). Chromosomal regions exhibiting preferential interactions were identified by using the automated PCA analysis on Hi-C data in HOMER (runHiCpca.pl), retaining those with a PCA score >100. Interactions mapping at all RefSeq-annotated TSSs  were retrieved using the annotateInteractions.pl pipeline.This analysis used the HOMER Hi-C analysis pipeline (RX554530 , and GEOM1696042  were aliM1696042 . A “univF.G. and C.E. designed, conducted, and interpreted experiments. F.G., S.H., and A.S. analyzed the genomic data and wrote scripts. N.M. performed the sequence analysis. R.T. conceived the project, suggested and interpreted experiments, and wrote the paper with F.G.


CDR1 gene in azole resistant isolate (AR) of Candida albicans. This study examines its genome-wide role by profiling Ncb2 occupancy between genetically matched pair of azole sensitive (AS) and AR clinical isolates. A comparison of Ncb2 recruitment between the two isolates displayed that 29 genes had higher promoter occupancy of Ncb2 in the AR isolate. Additionally, a host of genes exhibited exclusive occupancy of Ncb2 at promoters of either AR or AS isolate. The analysis also divulged new actors of multi-drug resistance, whose transcription was activated owing to the differential occupancy of Ncb2. The conditional, sequence-specific positional escape of Ncb2 from the core promoter in AS isolate and its preferential recruitment to the core promoter of certain genes in AR isolates was most noteworthy means of transcription regulation. Together, we show that positional rearrangement of Ncb2 resulting in either activation or repression of gene expression in response to drug-induced stress, represents a novel regulatory mechanism that opens new opportunities for therapeutic intervention to prevent development of drug tolerance in C. albicans cells.Ncb2, the β subunit of NC2 complex, a heterodimeric regulator of transcription was earlier shown to be involved in the activated transcription of  Candida albicans turns into a life threatening pathogen in immunocompromised individuals. For this lethality, C. albicans has an array of important traits, including yeast to hyphae transition, secretion of adhesion molecules and phenotypic switching12C. albicans is the fourth most common infections in hospitals which on several occasions are systemic and fatal45The commensal Candida cells employ several strategies to acquire resistance not only to the drug in use but also display collateral resistance to other unrelated drugs. Among various mechanisms which contribute in acquiring multi-drug resistance (MDR), enhanced drug export represents an important strategy adopted by the pathogen89C. albicans are shown to over-express CDR1, CDR2 or MDR1 drug efflux pump protein-encoding genes. Cdr1 and Cdr2 belong to the ATP binding cassette (ABC) super-family of transporter proteins and use energy derived from ATP hydrolysis to expel drugs to the cell exterior. Mdr1 on the other hand is an H+/drug antiporter which belongs to the super-family of major facilitators (MFS)1112Candida; the regulatory circuits which control their gene expression are different1314The CDR1 is a major drug efflux protein-encoding gene which is regulated at the transcriptional as well as at post transcriptional levels9161718192021CDR1 promoter harbours various cis-regulatory elements  as well as specific cis-elements such as Basal Regulatory Element (BRE), Negative Regulatory Element (NRE) and Drug/Steroid Response Element (DRE and SRE) in the 5′ flanking region172023CDR1 have also been identified. For example, Non dityrosine 80 (Ndt80), a homolog to a meiosis-specific transcription factor (TF) in Saccharomyces cerevisiae is an activator of CDR1CDR genes (Tac1) binds to the Drug Response Element (DRE) in both the CDR1 and CDR2 promotersCDR1 expression25ERG genes, also targets CDR1S. cerevisiae as an essential factor and the β and α-subunit-encoding genes are termed as NCB2 (YDR1) and BUR6, respectively384040In addition to the gene-specific transcription factors, there are a variety of accessory factors that act in global manner in regulating MDR genes. For example, chromatin-modifying enzymes and proteins positively or negatively affect the formation of an active transcription initiation complex2829313233343537CDR1 in azole sensitive (AS-Gu4) and azole resistant (AR-Gu5) isolates of C. albicans and also provided evidence that this cofactor may also be involved in the regulation of select genes, particularly those targeted by Tac1C. albicans. The study shows that Ncb2 occupies the promoters of 25–30% of C. albicans genes of almost every metabolic class in AR and AS isolates, thereby reinforcing its status as a global regulator of gene expression. Notably, there are hosts of novel MDR genes, which are regulated by Ncb2 in response to drug stress, leading to transcriptional activation.In an earlier report, we have shown the importance of the β-subunit of NC2 complex in the basal and activated transcription of CDR1 gene in azole resistant (AR) clinical isolate of C. albicans43C. albicans, we performed a genome-wide recruitment analysis of the transcriptional regulator by employing ChIP-on-chip assay and compared it between azole sensitive (AS) and AR isolates. Three biological replicates were processed for each of the isolate to identify DNA regions that were occupied by Ncb2. By using polyclonal anti-Ncb2 antibody, the cross-linked protein-DNA complexes were pulled downP-values and fold enrichments (in comparison with input DNA); I) genes with P-values ≤ 0.055 and fold enrichments ≥1.5 , II) genes with P-values ≤ 0.055 and fold enrichments ≥2.0 , III) genes with P-values ≤ 0.01 and fold enrichments ≥2.0 . Furthermore, to compare the genomic binding of Ncb2 between the isolates, the average binding profile for the promoter  was computed which showed that Ncb2 mainly recruited at the core promoter regions434547485051Ncb2, a part of the heterodimeric NC2 complex that controls transcription of genes was earlier shown to be involved in the activated transcription of P-values ≤ 0.01 and fold enrichments ≥2.0), and carried out a search at Candida Genome Database (CGD). A closer look at Ncb2 occupancy revealed its binding to the diverse functional classes of genes involved in metabolism, transport, cell wall, filamentous growth, virulence, mitochondrial function, signal transduction, biofilm formation and oxidative stress  of interest with different normalized log ratios (enrichments) and P-values. Their expression was checked by semi-quantitative end-point RT (reverse transcription) PCR. CDR1 and CDR2 were used as positive controls. Inspite of the higher Ncb2 occupancy, the expression data showed a mixed response of gene expression. For instance, three genes  were up-regulated in AR isolate whereas PMT1 (Protein mannosyltransferase), RTA4  and ERG11 were down regulated in AR cells 2Cys6 family transcription factor involved in the transcriptional activation of CDR1 and CDR2919CDR1 and CDR2, Ncb2 could regulate other Tac1 target genes i.e. RTA3, IFU5, LCB4, GPX1 and orf19.1887Previous ChIP-on-chip experiments in CDR1 in the AR isolate whereas its shift in binding to upstream of the TATA-box region in AS isolate resulted in its repressionCDR1-activated transcription, Ncb2 together with TBP moves on the DNA, we explored if positional shift of Ncb2 is also associated with the regulation of other genes42ZCF8, NDH51, orf19.7459, SUN41 PDX1, CRZ2, ECE1), filamentous growth , response to stress , response to drug , translation , transport , cell wall organization , and pathogenesis , as identified by GO slim analysis  while 5 genes were repressed  in the AR isolate  assay. For this, genes that displayed shift in occupancy of Ncb2 and showed regulated gene expression in AS and AR isolates were chosen. CRIP assay was performed with genes such as A hypothetical model depicting differential positional occupancy at the promoter regions is shown in CDR1 by the transcription regulator, Tac1. We observed that Ncb2 occupied the promoter regions of a few of the Tac1-regulon genesΔtac1 strain was determined. We observed that Ncb2 was associated at the promoter regions of selected MDR genes from all categories  which included RTA4, ERG11, CDR2, HEM15, orf19.6898.1, NOP14, CDR1, DFI1, PDX1, RTA3 and MDR1, however, orf19.1698, CHK1 and orf19.7063 showed no occupancy of Ncb2. Notably, all the three genes which appeared to be Tac1 dependent belongs to the AR exclusive Ncb2 enriched category. Among control genes included in the analysis, Ncb2 was seen to be bound to the ADH1 promoter in Δtac1 strain, while it was not seen on the ACT1 promoter  complex to the DNA bound TBP results in the mobilization of TBP away from the TATA box or allows it to escape and return to the TATA binding mode probably by inducing a conformational change in the TBP-DNA complexC. albicans.There were also a set of genes, which showed exclusive occupancy of Ncb2 on their promoters in AR isolate. Interestingly, most of such genes showed positive regulation of transcription. These results imply that an exclusive occupancy of Ncb2 in the AR isolate may exert a positive impact on gene expression by recruiting active pre-initiation complex at the promoters. In comparison, the genes whose promoter regions showed comparatively higher enrichment of Ncb2 occupancy in the AR isolate resulting in an activation or repression, points that the higher occupancy may not always lead to positive regulation of the transcription since it also depends on the promoter occupancies of other transcriptional activators46The analysis of Ncb2 bound DNA regions showed that the frequency of Ncb2 motif is significantly higher as compared to the random sequences. Importantly, Ncb2 motif was found to be distributed around the detected peaks and constitute A-rich sequence . This isC. albicans in response to drug stress by alternating its binding to the core promoters/TATA regions to either activate or repress transcription. Since, negative or positive regulation by Ncb2 does not correspond to the strength of the promoter occupancy; it may probably depend on the downstream promoter occupancies of other co-activators. Considering that MDR is a multifactorial manifestation of many regulatory mechanisms, this study provides an example that a global regulator may actively be involved in MDR acquisition in C. albicans.In conclusion, we demonstrate a new mode of action of Ncb2 in C. albicans azole resistant (AR) and azole sensitive (AS) isolates were grown in YEPD medium  in 30 °C incubator at 200 rpm for routine experimental purposes. Strains details are given in All the primers used in this study are listed in 2, 1 M sorbitol, and 30 mM DTT) for the formation of spheroplast. Spheroplasts were washed twice with cold TBS containing 1 M sorbitol, resuspended in ChIP lysis buffer . SDS was added at a final concentration of 0.5%. Cells were sonicated for 25 sec at amplitude of 6 for seven cycles. After sonication cell suspensions were centrifuged and termed soluble total chromatin (STC). Fifteen μl samples were run on 1% agarose gel to check the size of the fragmented chromatin (approximate size ~250 bp to ~500 bp). STC was diluted 5 times with ChIP lysis buffer and pre-cleared. After pre-clearing the samples pre-immune serum (negative control) and anti-Ncb2 antibody was added at a final concentration of 2–5 μg and incubated O/NChIP experiments were carried out as described earlierC. albicans (SC5314) was used. It covered 6204 orfs, representing 99.7% of the total genome comprising 180000 unique 60 mer probes in situ synthesized using “SurePrint” technology (Agilent) that was optimized for DNA-protein binding studies. To study the Ncb2 genome-wide association, ChIPed DNA purified by Qiagen PCR purification kit send to Genotypic technology Bengaluru for linear amplification by LMPCR (ligation mediated polymerase chain reaction) and size distribution of amplified fragment (~250–500 bp) as well as yields were determined. LMPCR was performed by using 200 ng grams of input and immunoprecipitated DNA was blunted at 12 °C for 20 min using T4 DNA polymerase as per the Mammalian ChIP-on-chip Protocol Version 10.1 of Agilent Technologies. The blunted DNA was cleaned by phenol:chloroform extraction and the precipitated DNA were re-suspended in 25 μl of water and the entire DNA was ligated to linkers overnight. The linker-ligated DNA was subjected to two rounds of LMPCR as per protocol and the product was precipitated and re-suspended in 25 μl of water.A high-density oligonucleotide microarray with 5000 bp upstream (promoters) and 500 bp downstream regions (genes) of http://www.ncbi.nlm.nih.gov/geo/] with accession number GSE768558 for the tiling array data.Two μg of input, ChIPed or immunoprecipitated DNA were labeled using Agilent Genomic DNA labeling kit PLUS and Cy3 (for ChIPed or immunoprecipitated DNA) or Cy5 (for input DNA) dUTP from Agilent. DNA yield and incorporation of label (specific activity) was measured using Nano Drop spectrophotometer. Labeled DNA were hybridized at 65 °C for 24 hr to Agilent 60 mer oligonucleotides. Hybridized arrays were scanned using high throughput Agilent scanner with “SureScan” technology, and process through automated feature extraction using Agilent feature extraction software. After quantification and normalization of the signals (ChIPed-DNA or immunoprecipitated DNA/input DNA), the binding profiles were represented as normalized log ratios. Normalization of data and statistical analysis were performed using Agilent’s DNA analytics software . Normalization methods used were median blanks subtraction and intra-array (intensity-dependent) LOWESS normalization. In blanks subtraction normalization for each channel (IP or input) of each replicate of each array, the median of the intensities of the blank spots is subtracted from each intensity on that replicate. As in all normalization steps, any negative signal intensities that result will cause the probe to be flagged as ‘excluded.’ Intra-array normalization attempts to correct for artifacts caused by nonlinear rates of dye incorporation, as well as inconsistencies in the relative fluorescence intensity between some red and green dyes. The LOWESS  algorithm normalizes the channels within each array using a nonlinear polynomial fit to the data, and effectively normalizes by probes and by arrays. Whitehead per-array neighborhood model was used to detect the protein-DNA binding event in DNA Analytics software . This method uses the distribution of all probes on each array to compute robust regions of increased probe signal (termed ‘peaks’). The algorithm does this by examining groups of probe triplets which are significantly enriched, yielding robust binding event detection. The Whitehead model samples every probe and its immediate upstream or downstream neighboring probe to identify a robust estimate of the location of bound protein. Three ChIPed DNA samples were processed for each strain. The data sets that support the results of this article are available in the GEO repository [ ACT1 was used as an endogenous control. For every RT-PCR analysis, c-DNA was synthesized from RNA isolated from three different preparations. The PCR-amplified products were analyzed by agarose gel electrophoresis and quantified by densitometry.Total RNA from AS and AR isolates were extracted by using Trizol reagent (Sigma). Briefly, cells from 15 ml of log phase cultures were centrifuged and washed twice with DEPC treated water. Three hundred mg of acid washed sterilized beads were added with 1 ml of Trizol reagent. Bead beating was done for 1 min, 3 times at 1 min interval on ice. After bead beating, 200 μl of chloroform was added, cell suspensions were mixed and centrifuged at 13 K for 10 min. Clear supernatants were taken out and 600 μl of isopropanol was added in each sample and stored at −20 °C O/N. Next day, samples were centrifuged at 13 K for 10 min at 4 °C and RNA pellet was washed with 75% ice cold ethanol. Samples were air dried and dissolved in 30 μl of DEPC treated water. Reverse transcription-PCR (RT-PCR) was performed as mentioned in the RevertAidTMH Minus kit . Briefly, a total of 10 μg RNA was treated with DNase for 30 min at 37 °C. Reactions were terminated by adding 1 μl of 25 mM EDTA, pH 8.0, and incubated further at 65 °C for 10 min. One μl of DNA free total RNA (1 μg) was primed with oligo(dT)18 and random hexamers for first strand c-DNA synthesis at 42 °C for 60 min. Reverse transcription reaction was terminated by heating at 70 °C for 5 min. Half a microlitre of c-DNA was used in PCR (25 μl reaction volume) primed with specific forward and reverse primers. Twenty-five to thirty cycles of amplifications were carried out to synthesize second strand unless otherwise stated. http://rsat.sb-roscoff.fr) and Multiple EM for Motif Elucidation (MEME) program (http://meme-suite.org/tools/meme). For the analysis of Ncb2 consensus motif, 500 promoter sequences of the genes that showed highest Ncb2 binding normalized log ratio were selected. Three hundred bp sequence of the promoter regions were chosen around the detected peaks. The program was allowed to discover motif with oligo and position analysis with oligomer length above 6–7 bp and five motifs per algorithm searched on both strands. To find out the Ncb2 consensus sequence for the genes that showed core promoters/TATA-box upstream binding in AS isolate, 250 bp sequences were used around the detected binding peak. Furthermore, for the analysis of conserved sequences in the AR isolate in which Ncb2 binds at core promoters/TATA-box regions, 125 bp upstream and downstream sequences were taken from the ATG codon.To find out the consensus binding site of Ncb2, the promoter sequences were analyzed with Regulatory sequence analysis tool . Before digestion, chromatin was precipitated at room temperature by using 1 volume of isopropanol, and the pellet was recovered after centrifugation at 8,000 rpm for 10 min at room temperature and was washed twice with 70% alcohol at room temperature. The air dried pellet was solubilized in 1X universal Fast digest buffer, and 10 μl of respective restriction enzyme was added. Digestion was performed O/N at 37 °C. After completion of restriction digestion, chromatin was precipitated by absolute ethanol and dissolved in ChIP lysis buffer. ChIP experiment then was performed as described earlier.The chromatin restriction digestion-coupled immunoprecipitation (CRIP) assay was performed as described earlier for the ChIP assay, with minor changes as describedAccession codes: GSE768558.How to cite this article: Shariq, M. et al. The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans. Sci. Rep.7, 46084; doi: 10.1038/srep46084 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Caenorhabditis elegans (C. elegans), the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) through hydrolysis of an endogenous XRN inhibitor 3’-phosphoadenosine-5'-phosphate (PAP). Here, we find through unbiased screening that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript. De-repression appears to be triggered such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Indeed, we find that two distinct XRN2 repression mechanisms are alleviated at different thresholds of XRN2 inactivation. Like more than 15% of C. elegans genes, xrn-2 occurs in an operon, and we identify additional operons under its control, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but a part of the mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons.XRN2 is a conserved 5’→3’ exoribonuclease that complexes with proteins that contain XRN2-binding domains (XTBDs). In  C. elegans. In response to reduction of XRN2 activity, this self-repression is alleviated, increasing xrn-2 mRNA and thus protein production, which restores robust XRN2 activity. Although XRN2 and its upstream gene are transcribed from a single promoter as a gene expression unit called “operon”, XRN2 regulates only itself. It does so by inactivating a cryptic promoter that exists between the two genes and by destabilizing its own nascent transcript. Many other C. elegans genes (>15%) occur in operons, and we identify additional operons that XRN2 regulates through an analogous mechanism. Thus we find a novel function of XRN2 in modulating expression of genes in operons including itself. As one of the mechanisms could operate on genes outside operons, XRN2 may also regulate gene expression in organisms lacking operonic gene organization.XRN2 is a conserved eukaryotic protein that controls gene expression by degrading or processing various types of RNA. Here we find that XRN2 negatively regulates its own levels in the nematode  Caenorhabditis elegans (C. elegans) [Drosophila melanogaster [C. elegans genes are predicted to be in operons [Polycistronic gene expression is common in prokaryotes: multiple genes are arranged tandemly and transcribed from a single promoter, as one RNA precursor. This organization of genes into an operon permits regulation of functionally related genes in one unit. By contrast, protein-coding genes in eukaryotes are usually organized monocistronically, i.e., one promoter drives the expression of one gene. However, operons do occur in some eukaryotes, such as the nematode elegans) , and thenogaster , 3, 4. I operons , 6.C. elegans are separated prior to translation, in the nucleus, by a process termed trans-splicing [cis-splicing, which uses the spliceosome to excise the introns and fuse the exons of eukaryotic pre-mRNAs. However, rather than joining two fragments of the same precursor, it links a 5’-capped, 22-nucleotide RNA sequence, the splice-leader (SL), which is transcribed separately, to a splice acceptor site on the nascent transcript. Trans-splicing occurs on both monocistronic genes and genes in operons, but with splice-leaders of different sequences: whereas monocistronic genes and those most promoter-proximal in operons are spliced to SL1, the other operon-contained genes exhibit preferential albeit not exclusive splicing to SL2 [trans-splicing occurs subsequent to cleavage of the immediately upstream pre-mRNA at the 3’ end [Although the polycistronic transcript is the template for protein synthesis in prokaryotes, the individual cistrons in splicing , 8. Thise 3’ end , 8.C. elegans operons do not appear to be enriched for functionally related genes [C. elegans operons are estimated to have internal promoters [Unexpectedly, ed genes  and, coned genes . One mecromoters  in interromoters . Varyingromoters , 11. AddXRN2 is a 5’→3’ exoribonuclease that is conserved in eukaryotes. XRN2 is predominantly localized in the nucleus, and several nuclear RNA species have been reported as its targets , 13. XRNC. elegans, XRN2 is essential for embryogenesis, larval development and fertility. The xrn-2 gene is encoded in an operon as the second gene and shows ubiquitous expression throughout development [C. elegans [paxt-1 null (paxt-1(0)) animals cannot survive at temperatures ≥ 26°C due to degradation of XRN2. This phenotype is suppressed by an increased xrn-2 gene dosage [In elopment . We have elegans , 24. paxe dosage  and recaZK430.2/bpnt-1 gene as a suppressor of paxt-1(0) lethality. Depletion of BPNT1 protein induces accumulation of xrn-2 mRNA and thus XRN2 protein through inhibition of XRN2 activity. This autoregulation requires the ICR upstream of xrn-2 and does not affect the expression of its polycistronic partner rpl-43. A genome-wide RNA sequencing analysis identifies a subset of operons that are controlled by XRN2 in an analogous manner, revealing a novel role of XRN2 in polycistronic gene expression.Here we identify a loss-of-function mutation in the C. elegans animals at temperatures ≥ 26°C [ZK430.2 gene. As the gene encodes a protein that shows structural and functional conservation with the human BPNT1 , completely suppressed the larval arrest phenotype of paxt-1(0) (paxt-1(0) animals seems exclusively caused by destabilization of XRN2 [bpnt-1(xe22) allele could suppress xrn-2 phenotypes. We utilized xrn-2(xe34), a temperature-sensitive xrn-2 allele that we obtained from a genetic screen that will be described elsewhere. The xrn-2(xe34) allele has a missense mutation that changes a glutamic acid at position 699 to lysine, and the mutant animals show developmental defects and are not maintainable above 25°C (xrn-2(xe34) animals were arrested as larvae when cultured at 26°C, while bpnt-1(xe22); xrn-2(xe34) animals developed to adult (bpnt-1(xe22) allele can suppress both the paxt-1 and xrn-2 phenotypes.We have recently reported that XRN2 is stabilized by forming a complex with PAXT-1 and that PAXT-1 is required for larval development of s ≥ 26°C , 24. In n BPNT1 ( and see axt-1(0) . Since t of XRN2 , 24, we ove 25°C . xrn-2 allele changes tryptophan at position 294 to stop (W294*). A strain with the same mutation, VC40114, had been isolated in the Million Mutation Project [bpnt-1(xe22) allele was responsible for suppression of the paxt-1(0) phenotype, we removed unrelated mutations from the VC40114 strain by outcrossing three times followed by crossing the bpnt-1(gk469190) allele into the paxt-1(0) background. The bpnt-1(gk469190) allele, like the bpnt-1(xe22) allele, completely suppressed the larval arrest phenotype of paxt-1(0) (The  Project . In ordeaxt-1(0) .xe22 encoded a gain-of-function allele that enhanced BPNT1 activity on PAP. However, when modeling C. elegans BPNT1 on the published crystal structure of rat BPNT1 [flag::bpnt-1 and the flag::bpnt-1(xe22) transgenes, respectively, which we integrated in the same genomic locus using Mos1-mediated single copy insertion (MosSCI) [xe22 results in a loss- rather than gain-of-function allele. We confirmed this by crossing the single copy-integrated flag::bpnt-1 transgene into paxt-1(0); bpnt-1(xe22) animals, which reinstated temperature-sensitive lethality  mutant lethality.Given that BPNT1 negatively regulates a negative regulator of XRN2 PAP in yeast , it seemat BPNT1 , we noti(MosSCI) . As predethality . Thus, xpaxt-1(0); bpnt-1(xe22) animals. XRN2 protein signal intensity was increased more than two-fold in both bpnt-1(xe22) and paxt-1(0); bpnt-1(xe22) animals as compared to wild-type animals at both normal and elevated temperatures, while it was reduced in paxt-1(0) animals as previously reported [bpnt-1 mutation in paxt-1(+), i.e., wild-type, animals at 20°C.A major substrate of BPNT1 proteins is PAP, which inhibits activity of XRN2 . Thereforeported   animals relative to wild-type animals (bpnt-1(xe22) animals results, at least in part, from an increase in its mRNA levels.In order to establish how BPNT1 depletion boosted accumulation of XRN2 proteins, we quantified  animals , indicatxrn-2 gene is the downstream gene in a two-gene operon (WormBase ID: CEOP2697), where rpl-43 is the upstream  gene. However, transcriptional upregulation of the operon does not appear to account for the increase of XRN2 in the bpnt-1 mutant animals. This is because both mature mRNA and pre-mRNA levels of rpl-43 were unaltered by bpnt-1(xe22).  In striking contrast, however, we observed a consistent upregulation of pre-xrn-2 mRNA levels with two sets of specific primers  animals as compared to wild-type animals  was dispensable for XRN2 regulation, we created a reporter construct that contained the promoter of rpl-43~xrn-2 operon (rpl-43Prom) followed by the rpl-43 gene body (rpl-43Body) and the rpl-43ICR. A sequence encoding green fluorescent protein (GFP) and the nuclear protein histone H2B with the 3’ UTR of the unrelated unc-54 gene was fused to the construct, generating rpl-43Prom::rpl-43Body::rpl-43ICR::GFP::H2B::unc-54 3’ UTR. This and all subsequent reporter transgenes in this study have GFP::H2B::unc-54 3’ UTR (which we will thus omit when referring to transgenes in the following) and were inserted at an intergenic genomic locus on chromosome V by MosSCI. rpl-43Prom::rpl-43Body::rpl-43ICR promoted ubiquitous GFP expression as we reported previously [Pxrn-2. However, the bpnt-1(xe22) allele increased the GFP signal in hypodermal cells and in vulval cells  upregulated the rpl-43Prom::rpl-43Body::rpl-43ICR reporter as evidenced by an enhanced GFP signal in hypodermal cells  in the apparent absence of increased rpl-43Prom operon promoter activity, we wondered if rpl-43ICR might exhibit promoter activity. To test this, we utilized a reporter construct, rpl-43Body::rpl-43ICR, which lacked rpl-43Prom. As expected, this reporter did not show detectable GFP signal in untreated animals. However, XRN2 depletion induced GFP expression in the hypodermis and the intestine , which appears unaffected by XRN2 depletion. Specifically, mRNA levels for the first two genes of this eight-gene operon are comparable for xrn-2(RNAi) and mock RNAi animals (ran-4Prom), the ran-4 gene body (ran-4Body) and the ICR between the first and the second genes (ran-4ICR) induced GFP expression in many cell types including those of hypodermis, vulva and intestine, and, as expected, neither XRN2 depletion nor the bpnt-1(xe22) allele had obvious effects on the expression  animals show no obvious phenotype, we speculated that the cryptic promoter is de-repressed when XRN2 activity is severely reduced. To test this, we used the xrn-2 temperature-sensitive allele, xrn-2(xe34) (xrn-2(xe34) allele increased GFP signal of the rpl-43Prom::rpl-43Body::rpl-43ICR reporter in hypodermal, intestinal and vulval cells as compared to wild-type xrn-2 (xrn-2(+)) both at 23°C  allele promotes cryptic promoter-independent accumulation of xrn-2 both at 23°C and 26°C, while it activates the cryptic promoter only at 26°C. To confirm that the cryptic promoter is inactive at 23°C, we examined gfp mRNA transcribed from the rpl-43ICR by RT-qPCR. Since we failed to quantify its levels due to little or no expression in the presence of wild-type xrn-2, we examined its expression by RT-PCR  and xrn-2(xe34). At 26°C, on the other hand, gfp mRNA showed elevated expression in the xrn-2(xe34) background. Thus, the two XRN2 repression mechanisms are alleviated at different thresholds of XRN2 activity, where activation of the cryptic promoter requires more severe reduction of XRN2 activity. Failed activation of the cryptic promoter in the intestine by xrn-2(xe34), in contrast to xrn-2 RNAi (xrn-2(xe34) and/or very efficient intestinal XRN2 depletion through RNAi by feeding.The cryptic promoter in the epletion . Given tw growth  and a mow growth  while bp-2(xe34) . The xrn at 23°C  and 26°C at 23°C . On the  at 26°C  but not  at 26°C . These ry RT-PCR . At 23°Cn-2 RNAi , might btrans-splicing to the spliced leader RNA SL2 [trans-spliced to SL1, and the proportion increases particularly for those that have an internal promoter in their upstream ICRs [xrn-2 mRNA accumulates upon the cryptic promoter-independent XRN2 de-repression, we examined xrn-2(xe34) animals cultured at 23°C. While rpl-43 mRNA showed no change, xrn-2 mRNA levels increased approximately 40% . To identify XRN2-sensitive operons, we calculated fold changes for each gene upon xrn-2 versus mock RNAi, and plotted these for the second against the first gene for the 1388 annotated operons (Wormbase release: WS249) -RNA sequencing  . Althougrpl-43~xrn-2 operon (cri-3~clpf-1 operon (WormBase ID: CEOP3108) whose first and second genes showed relatively strong expression for further examination (xrn-2 RNAi (xrn-2(xe34)-mediated XRN2 inactivation  ICRs like the 2 operon . Since omination . Quantifn-2 RNAi  or xrn-2tivation . In contcri-3 and clpf-1 (cri-3ICR) is responsible for the susceptibility to XRN2 depletion, we replaced ran-4ICR of the ran-4Prom::ran-4Body::ran-4ICR reporter by the ICR between cri-3 and clpf-1 (cri-3ICR). Like rpl-43ICR, cri-3ICR reduced the GFP signal of the reporter in a wild-type situation  animals. Formally, we cannot exclude that C. elegans BPNT1 differs in function from its orthologues in other eukaryotes. However, we prefer an alternative interpretation, namely that these data reveal an unanticipated, non-linear behavior of the XRN2 pathway. Such behavior was not readily inferable from previous biochemical and other knowledge on the pathway’s components, thus highlighting the value of unbiased, phenotype-based genetic screens. The interpretation is consistent with, first, conservation of BPNT1’s function from yeast to mammals [C. elegans protein, and, third, most importantly, our elucidation of XRN2 autoregulatory pathways that can account for this non-linear behavior. Thus, XRN2 functions to reduce xrn-2 mRNA levels and, conversely, loss of XRN2 function increases xrn-2 expression. This negative feedback autoregulation functions as a buffer to keep XRN2 accumulation within a certain range. As XRN2 has multiple and essential functions in RNA metabolism and development, robust maintenance of XRN2 levels may be important to protect the organism from sudden changes in environment. XRN2 autoregulation then works in concert with other mechanisms that ensure robustness of XRN2 activity, such as its stabilization by PAXT-1 [Loss of BPNT homologues enhances or recapitulates phenotypes of XRN mutant yeast  and plan mammals , 30, secy PAXT-1 , 24. Finxrn-2 being in an operon, as shown by the fact that we identify two mechanisms, both of which rely on the ICR between xrn-2 and its upstream gene but on different thresholds of XRN2 activity for induction. The two mechanisms also differ with respect to their reliance on the upstream operon promoter. One mechanism can function in the absence of this promoter, suggesting that the ICR that separates xrn-2 from rpl-43, although unusually short [et al. [rpl-43~xrn-2 operon  agar seeded with Escherichia coli OP50 according to the standard methods described previously [eviously .Cloning and site-directed mutagenesis were performed by PfuUltra II Fusion HS DNA Polymerase  according to the supplier’s protocol using specific primers . PCR-ampMos1-mediated Single-Copy transgene Insertion (MosSCI) was performed according to the previous report using the EG8082 strain [DNA fragments were inserted into the pCFJ150 vector by Multisite Gateway Technology (Life Technologies) according to the supplier’s protocol. 2 strain .paxt-1(xe5) worms were harvested, washed and incubated with 50 mM EMS in 6 ml of M9 buffer for 4 hours at room temperature. The worms were washed three times with M9 buffer and cultured at 25°C. The L3- or L4-stage larvae of the F1 generation were cultured at 26°C, and their progeny were screened for normal development. One mutant line that was maintainable at 26°C was isolated and backcrossed 6 times with the parental paxt-1(xe5) strain to remove unrelated mutations. Genomic DNA was extracted and purified using Gentra Puregene Tissue Kit . DNA libraries were created from 50 ng of genomic DNA using Nextera DNA Library Prep Kit . The sequencing data were generated using a HiSeq 2500 (Illumina).About 6,000 L4-stage C. elegans assembly (obtained from http://hgdownload.soe.ucsc.edu/goldenPath/ce6/chromosomes/) using ‘‘bwa” [Escherichia coli (E. coli) genomes , which typically resulted in less than 1% aligned reads. Potential PCR duplicates were identified and removed using Picard , reducing the number of reads to 93% to 95%. Sequence variants were identified using GATK [bpnt-1 gene.Sequence data were processed following a similar workflow as described previously . Sequencg ‘‘bwa”  . DIC and fluorescent images were obtained using an Axio Observer Z1 microscope and AxioVision SE64 (release 4.8) software . For GFP reporter assays, presence or absence of differences in signal intensity between conditions were evaluated by visual inspection of at least twenty worms. Where this revealed consistent patterns of difference, fluorescence and DIC Images of at least five randomly selected worms per condition were acquired for hypodermis, intestine, and vulva, and examined. In the experiments for Figs About 6,000 worms were harvested, washed three times with M9, resuspended in 100 μl of SDS lysis buffer (63 mM Tris-HCl (pH 6.8), 5 mM DTT, 2% SDS, 5% sucrose) and heated for 5 min at 95°C, followed by sonication. After centrifugation at 10,000 x g for 10 min at 4°C the supernatant was collected. 100 μg of the extract was subjected to SDS-PAGE and Western blot. A rat anti-XRN2 antibody , a mousexrn-2 was obtained from the Ahringer library [xrn-2 RNAi causes slow growth, worms were treated with control or xrn-2 RNAi from L1 to L4 stage for 40~42 or 48~52 hours, respectively, at 20°C. Vulval morphology was observed to confirm mid-L4 stage.The RNAi clone against  library . RNAi wa library : bacteri15 primers (for mature mRNAs) or random primers (for pre-mRNAs) according to the supplier’s protocol. RT-qPCR was performed with specific primers  and frozen in liquid nitrogen. Worms were broken open by five repeats of freeze and thaw using liquid nitrogen and a 42°C heating block, before RNA was extracted and purified according to the supplier’s protocol with the modification that RNA was incubated with 50% 2-propanol at -80°C overnight for efficient precipitation. The purified RNA was treated with DNA-free Kit  to remove DNA. For mRNA quantification by RT-qPCR, cDNA was generated from total RNA by ImProm-II Reverse Transcription System  using oligo(dT) primers , the SYB primers . For polC. elegans assembly from UCSC [https://www.bioconductor.org/) and setting the parameter “splicedAlignment = TRUE”, which calls the SpliceMap aligner with default parameters [ftp://ftp.wormbase.org/pub/wormbase/releases/WS220/species/c_elegans/) by counting reads overlapping all annotated exons for each gene. Samples were normalized by the mean number of counts mapping to all exons, and gene-level counts were log2-transformed after adding a pseudocount of 8. Operon annotations were downloaded from WormBase (version WS249) (ftp://ftp.wormbase.org/pub/wormbase/releases/WS249/species/c_elegans/PRJNA13758/), comprising 1,388 operons in total. For each operon, the log2 fold-changes in expression for xrn-2 RNAi vs mock were calculated for both the first and second gene in the operon  in the RStudio environment (version 0.99.484).In order to make use of the most recent operon annotations from WormBase, RNA-sequencing reads were aligned to the October 2010 (ce10) rom UCSC . Alignmerom UCSC , with thrameters . The resrameters  , with each showing the highest correlation to the 32h timepoint ; the second pair (replicate 2) were less well-matched and showed the highest correlations to the 35h (xrn-2 RNAi: r = 0.94) and 36h (mock: r = 0.96) timepoints. We therefore focused our analysis, as described above, on the replicate 1 pair. We validated our initial results using the less well-matched pair by plotting the difference between the log fold-change of the second gene in operons and the log fold-change of the first gene in operons for each replicate against one another (cri-3~clpf-1 operon (CEOP3108), fell into the upper right quadrant of the plot, indicating that most of the expression changes were captured in both replicates.Extensive rhythmic gene expression during tal time . To dete another . The corS1 Figxrn-2(xe34) or xrn-2(xe34); xrn-2::gfp animals were cultured from L1 at 20°C, 23°C or 26°C for 72 hours and observed by stereomicroscopy at the same magnification. xrn-2(xe34) animals developed normally to adults at 20°C while were arrested and died as larvae at 26°C. The temperature-sensitive phenotype of xrn-2(xe34) animals were rescued by expression of GFP-fused wild-type xrn-2 (xrn-2::gfp).wt, (PDF)Click here for additional data file.S2 FigS. cerevisiae , R. norvegicus (Rn Bpnt1) and C. elegans (Ce BPNT-1) are aligned by Clustal Omega  and displayed by Jalview .The most frequent amino acids are in blue, similar amino acids in light blue, based on BLOSUM62. Amino-acids numbers relative to the first methionine are shown. The bpnt-1(xe22) allele has a mutation that changes the 294th tryptophan to stop. (B) Structure of R. norvegicus Bpnt1. The corresponding region missing in the C. elegans BPNT-1(xe22/W294*) is shown in orange.(A) Protein sequences of BPNT homologues in (PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Figrpl-43ICR reporter animals in xrn-2(+) or xrn-2(xe34) genetic background were cultured from L1 to L3 at 23°C followed by 26°C to L4. Levels of indicated mRNA from the cri-3_clpf-1 were quantified by RT-qPCR and normalized to act-1 mRNA levels with values of xrn-2(+) animals defined as 1 . Values are shown in (PDF)Click here for additional data file.S5 FigSee (PDF)Click here for additional data file.S6 FigSee (PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableSee (XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Pyrococcus furiosus. The protein is located in an operon structure together with the hypothetical gene pf1089 and western blot as well as end-point RT-PCR experiments revealed an extremely low expression rate of both proteins. Furthermore, conditions to induce the expression of the operon are not known. By introducing an additional copy of tfb-RF1 using a Pyrococcus shuttle vector we could circumvent the lacking expression of both proteins under standard growth conditions as indicated by western blot as well as end-point RT-PCR experiments. A ChIP-seq experiment revealed an additional binding site of TFB-RF1 in the upstream region of the pf1011/1012 operon, beside the expected target of the pf1089/tfb-RF1 region. This operon codes for a putative ABC transporter which is most-related to a multidrug export system and in vitro analysis using gel shift assays, DNase I footprinting and in vitro transcription confirmed the activator function of TFB-RF1 on the corresponding promoter. These findings are also in agreement with in vivo data, as RT-qPCR experiments also indicate transcriptional activation of both operons. Taken together, the overexpression strategy of tfb-RF1 enabled the identification of an additional operon of the TFB-RF1 regulon which indicates a transport-related function and provides a promising starting position to decipher the physiological function of the TFB-RF1 gene regulatory network in P. furiosus.Transcription factor B recruiting factor 1  is a transcription regulator which activates transcription on archaeal promoters containing weak TFB recognition elements (BRE) by recruiting TFB to the promoter. The mechanism of activation is described in detail, but nothing is known about the biological function of this protein in  The core transcription machinery of Archaea resembles a simplified version of the eukaryotic RNA polymerase (RNAP) II system, which consists of only one multi-subunit RNA polymerase (RNAP). For initiation of transcription three general transcription factors (TFs), the TATA-binding protein (TBP), TFB, and TFE are necessary for recognition of the two core promoter elements, the TATA box and the TFB recognition element BRE; . The firIt is interesting to note that the regulation of this eukaryotic-like transcription machinery is controlled by a system very similar to that found in Bacteria . Correspin vitro analysis of archaeal activation mechanisms revealed so far two basic principles: the activator promotes either binding of TBP or TFB  using phase-contrast microscopy.eviously . GelriteAll used strains, plasmids, and primers are shown in the supplement . Cells lysis was induced by sonication , cell debris was removed by centrifugation and the protein concentrations of the supernatants were determined by Bradford assay. Western blot experiments were done as previously described  using recombinantly expressed and purified TFB-RF1 . The IgGescribed . The sigP. furiosus cells harboring the pYS5 plasmid were cultured in a fermenter in the presence of 40 mM sodium pyruvate for formaldehyde-crosslinking. For immunoprecipitation 10 μg of purified polyclonal antibodies raised against TFB-RF1 was coupled to 50 μl Dynabeads Protein G  according to the manufacture’s instructions. After elution from the beads DNA was purified and subsequently used for library preparation. Input sample was prepared as described previously . For multiplex sample preparation the NEBNext® Multiplex oligos  were used and libraries were PCR amplified by the NEBNext®High Fidelity Master Mix . Libraries were pooled in equimolar ratios and sequenced using the Illumina HiSeq 2000 platform (read length = 50 b) . For further analysis only the demultiplexed and quality filtered reads were used.Library preparations were done according to the NEBNextP. furiosus DSM3638 genome using Bowtie2 with default settings (1pfk promoter region); and ‘mask’ 1613140 1629427 (PF1937-PF1952). Called peaks were verified by repeating the analysis using additional genomic regions or no specified genomic region as ‘norm_region.’ Downstream analyses were performed as recommended for the software. Statistical analysis was carried out using SPSS Statistics 21 (IBM). In addition, only binding sites were considered for further analysis, which were present in the three replicates of IP, which do not correspond to the ribosomal gene locus in the P. furiosus genome and which show higher enrichment scores than the regions corresponding to genes and promoter or terminator regions present as an additional copy on the plasmid pYS5 . 10 to 20 ml cell culture was pelleted and cells were lysed by addition of 1 ml TriFast followed by rigorous shaking for 15 min. After adding 0.2 ml 2 M sodium acetate pH 4.0 RNA was isolated according to the manufacture’s instructions. Contaminating DNA was removed via the TURBO DNA-freeTM Kit . The integrity of the total RNA was assessed by formaldehyde agarose gel electrophoresis and purified RNA was stored at -80. Three independent RNA samples were prepared from both culture conditions and strains.Reverse transcription was done using a QuantiTect Reverse Transcription Kit  in combination with a Random Primer Mix . For each assay 415 ng total RNA was used. RT-PCR and RT-qPCR primer pairs were designed using the Primer3 software package (Supplementary Table RT-PCR reactions were assembled using +RT cDNA (1:10 diluted), -RT cDNA (1:10 diluted) or no cDNA  and corresponding primer pairs with a final concentration of 1 μM. Amplification was done with the Phusion DNA polymerase  and RT-PCR products were analyzed by agarose gel electrophoresis. One representative result is shown in the manuscript.TM SYBR® No-ROX Kit . Primers were added to a final concentration of 0.3 μM and the total volume of the DNA samples in each reaction was 4 μl. The efficiency of the primer pairs, determined by serial dilutions, was in the range between 0.9 and 0.95  were assembled in a 15 μl reaction volume according to FAM-labeled template DNA and TFB-RF1 were incubated under the conditions used for the gel shift assay. DNase I was added for 1 to 5 min at 37°C and the reaction was stopped by the addition of 95% formamide. DNA was ethanol precipitated and resuspended in 3 μl of formamide buffer. A DNA sequencing ladder using the same primer was generated as a molecular mass standard. Samples were loaded onto a 4.5% denaturing polyacrylamide gel and analyzed using an ABI 377 DNA sequencer. DNase I footprints were repeated two times and a representative experiment is shown.32P]-UTP (111 TBq/mmol) as described previously  and TFB-RF1 activates transcription of this operon , but no signals were detected in crude extracts from P. furiosus cells grown under both conditions , except control signals with an antibody raised against subunit F of the RNAP (lower part). To check the expression of the pf1089/tfb-rf1 operon on the RNA level, end-point RT-PCR was performed. Using a PCR amplicon spanning both genes , only a weak signal could be detected in cells grown on starch or pyruvate  in comparison to the housekeeping gene pf0256 which encodes the RNAP subunit E′ (lanes 4 and 10). These data indicate that the overall expression of the operon is extremely low and most likely special conditions are required for induction.The transcriptional regulator TFB-RF1 (PF1088) activates transcription on archaeal promoters containing weak BRE elements by recruiting TFB to the promoter . We haves operon . To get pf1089/tfb-rf1 operon we used a Pyrococcus/Escherichia coli shuttle vector for overexpression of TFB-RF1 . A growth behavior analysis of Pyrococcus pYS5 under glycolytic as well as gluconeogenic conditions showed only minor differences compared to cells harboring the empty shuttle vector pYS3 as control . Using crude extracts from Pyrococcus pYS3, TFB-RF1 is also not detectable similar to the wild type . In contrast, using Pyrococcus pYS5 a weak signal could be detected in crude extract from cells grown with starch and two increased signals in crude extract from pyruvate cells . The two signals correspond to the monomer and the dimer of TFB-RF1 and all attempts to achieve complete denaturation of the protein with increased SDS concentrations were not successful (data not shown). It is interesting to note that the ratio between the monomer and the dimer is the other way round in comparison to the recombinant protein produced in E. coli .To circumvent the low expression rate of the  TFB-RF1 . The cons Figure A. A growin trans on the activation of the genome-located pf1089/tfb-rf1 operon we performed RT-PCR reactions as described before. Using RNA from Pyrococcus pYS5 grown on pyruvate, an enhanced pf1089/tfb-rf1 signal could be detected even stronger than the signal of the house-keeping pf0256 . As the promoter is only active under gluconeogenic conditions we obtained wild type results under glycolytic conditions . This is also the case for RNA from the control strain Pyrococcus pYS3 . Taken together, these results demonstrate that the strategy to bypass the missing natural expression of TFB-RF1 by introducing an additional plasmid-encoded copy of tfb-rf1 is working and TFB-RF1 is able to stimulate transcription on corresponding promoters located in the genome.To check if the plasmid-expressed TFB-RF1 is able to act Pyrococcus genome we performed ChIP-seq experiments as described previously . This leads to an enrichment of these regions in the input samples. However, two chromosomal positions were identified, which are also located in close proximity to promoter elements. The first one corresponds to the upstream region of pf1089 and proofs that transcriptional activation of the operon  is correlated with in vivo binding of TFB-RF1 upstream of pf1089 . The additional signal for tfb-rf1 in both samples, IP and input DNA, resulted from overexpression on the plasmid. The second peak covers the promoter region upstream of the operon pf1011/ pf1012, which encode most likely an ABC transporter system . The ChIP-seq approach indicates that the regulatory function of TFB-RF1 is not only restricted to auto-activation of its own operon, but also controls in addition operon pf1011/ pf1012.To investigate if TFB-RF1 recognizes additional binding sites within the eviously . We useds genome . Furtherpf1011/ pf1012 a gel shift assay was performed . Using the upstream region of pf1011 as template with increasing concentrations of TFB-RF1 a specific DNA protein complex was formed , but there is no interaction with the strong gdh promoter (lanes 7 to 10). To determine the binding site of TFB-RF1 in more detail, we also applied DNase I footprinting experiments . TFB-RF1 protected a region on the non-template strand from -47 to -70 upstream of the translation start site . This region shows striking sequence similarities to the one found in the pf1089 promoter region in our previous study . Additionally, the binding sites predicted with peak calling using PIQUE are located within both footprint regions . A comparison of the BRE of pf1011 with the Pyrococcus consensus sequence 5′-VRAAA-3′  did not lead to a strong shift . In contrast, in the presence of TFB-RF1 a TBP/TFB/TFB-RF1/DNA complex was efficiently build even under TFB-limiting conditions . Furthermore, TFB-RF1 stimulates transcription from the pf1011 promoter in a cell-free transcription assay , whereas transcription from a control promoter is not affected . For the pf1011 promoter we observed sixfold activation in comparison to sevenfold activation on the pf1089 promoter . Taken together, the in vitro experiments clearly demonstrate that the newly identified binding site upstream of pf1011 belongs to the TFB-RF1 regulon and also requires this protein for transcriptional activation.To test this hypothesis we applied a gel shift assay with the upstream region of promoter . We assuin vitro data with the in vivo situation, we isolated RNA from Pyrococcus pYS5 and pYS3 cells grown on starch or pyruvate and analyzed RNA levels by RT-qPCR. Both transcripts, pf1089/tfb-rf1 as well as pf1011/1012, are highly upregulated in the pYS5 strain after growth on pyruvate , whereas in the pYS3 strain these transcripts are almost not affected by switching from starch to pyruvate . A direct comparison of the RNA levels between pYS5 and pYS3 strains revealed a 54-fold activation for the pf1089/tfb-rf1 amplicon and 32-fold for the pf1011/1012 amplicon under pyruvate conditions. In the presence of starch the activation is reduced to 4.5-fold for pf1089/tfb-rf1 and 2.6-fold for pf1011/1012.To correlate our P. furiosus. Although the activation mechanism is studied in detail . These data indicate a strong phylogenetic conservation of this gene regulatory network and for all of these promoters TFB-RF1 most likely acts as activator of transcription by supporting the recruitment of TFB to an imperfect BRE.We identified the sequence  and furtpf1011/pf1012 operon as an additional target of the TFB-RF1 regulon may give new implications for unraveling the physiological function. Sequence analysis revealed that the corresponding protein PF1011 contains two Pfam domains, an ATP-binding domain of ABC transporters (ABC_tran/PF00005) in the N-terminal region and an additional domain of unknown function (DUF4162/PF13732) in the C-terminal region (ABC2_membrane_3/PF12698). This is a separate group of ABC transporters where the six-TMS topology resulted from intragenic duplication of a primordial three-TMS-encoding genetic element (The identification of the l region . The ATPl region . The dom element .8) using BLAST revealed that the homologs for both proteins with the lowest E-values belong to the drug exporter-1 family (3.A.1.105; Streptomyces peucetius and LmrA is a multidrug transporter from Lactococcus lactis (P. furiosus remains speculative. It is interesting to note that a computational analysis of PF1089 also revealed four TMS in its sequence, which denotes it as integral component of the membrane with unknown function. In summary, both so far identified TFB-RF1-regulated operons point to a transport-related function and provide a promising starting position to decipher the physiological function of the TFB-RF1 gene regulatory network.A more detailed sequence similarity search against the Transporter Classification Database (TCDBs lactis . The euks lactis , but this lactis . Due to Pyrococcus strains. KR did the RNA work and the western blots and SD the in vitro transcription experiments. RR performed the ChIP-seq experiments and the characterization of the DNA protein interactions. WH and RR wrote the manuscript. WH, RR, and MT coordinated and supervised the work. All authors agreed to the final version of the manuscript.MK constructed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stimulator of interferon genes (STING) plays an important role in host defense, autoimmune disease, osteoclast differentiation and anti-tumor response. Although many downstream targets have been studied in depth, the regulation of STING gene expression remains largely unknown. Here we demonstrate that transcription factors CREB and c-Myc maintain the transcriptional activity of STING. By 5′-rapid amplification of cDNA ends analysis, we identified the transcriptional start site (TSS) of STING. We illustrated that the region -124/+1 relative to TSS was sufficient for full promoter activity by a series of 5′ deletion promoter constructs. Transcriptional activity of the STING minimal promoter was dependent on CREB and c-Myc binding motifs and was abolished after mutation of these two DNA elements. Chromatin immunoprecipitation assays demonstrated that transcription factors CREB and c-Myc bind to STING promoter in vivo. Overexpression of CREB and c-Myc increased the STING promoter activity. Meanwhile, knocking-down of CREB and c-Myc by a small interfering RNA (siRNA) strategy markedly reduced endogenous STING expression. In summary, these results demonstrated that transcription factors CREB and c-Myc are involved in the regulation of STING transcription. Choe in vivo .Although functions and downstream targets have been studied in detail, cis elements or trans factors responsible for regulating STING gene expression remained largely unknown. In this study, we identified the minimal promoter of STING is located within the region -124/+1 bp relative to the transcription start site (TSS) and demonstrated that transcription factors CREB and c-Myc maintain the transcriptional activity of STING.The transcription start site of the human STING gene was identified by 5′ RACE analysis as described in the materials and methods. Three DNA fragments resulting from the 5’RACE nested PCR were obtained and products of three separate PCR reactions were cloned and sequenced. Direct sequencing of the nested PCR products indicated that the major transcription start site locates at 334 bp upstream from the ATG codon.http://www.cbs.dtu.dk/services/Promoter/) to search potential sequences. It showed a putative promoter locating within sequences near nucleotides -400 of the STING 5’ flanking region. Sequence analysis by using the TFSEARCH program (www.rwcp.or.jp/papia/) revealed that human STING promoter region contains several putative transcription factor-binding sites, such as CREB, Sp1, E2F, HOX and c-Myc  into the promoterless pGL3-Basic luciferase reporter plasmid and designated the plasmid pGL-1887. Then the plasmid pGL-1887 was transiently transfected into HEK 293 and HeLa cells. As shown in Figure To locate the minimal sequence responsible for the transcription of the human STING gene, a series of deletion constructs were performed by inserting different fragments of the human STING promoter with a fixed 3′ end at the +267 bp position (relative to TSS) but a different 5′ end into the pGL3-Basic vector and then were transiently transfected into HEK 293 and HeLa cells. Luciferase assays revealed pGL3+1 have little promoter activity. Much stronger promoter activities were observed in pGL3-124, 436, 677, 953 and 1887  revealed the presence of several putative binding sites, including CREB, Sp1, E2F, HOX and c-Myc, in the promoter region of STING gene. In order to evaluate the contribution of the putative binding sites to the regulation of the STING promoter, we made point mutations of them as described above in the context of the reporter construct, and these constructs were subjected to reporter gene assays in comparison to the wild-type construct. Mutation of the CREB and c-Myc sites reduced STING promoter activity to 31% and 43% of the wild-type promoter activity, respectively. Double CREB and c-Myc mutation reduced the promoter activity to 21% of the wild-type promoter activity Figure . HoweverIn order to confirm the critical regulatory function of CREB and c-Myc sites, transcription factors CREB and c-Myc were overexpressed. The results Figure  showed tTo further confirm the role of CREB and c-Myc in the regulation of STING promoter activity, we used RNA interference to knock down CREB or c-Myc expression in HEK 293 cells and HeLa cells. We cotransfected STING reporter plasmid together with CREB siRNA, c-Myc siRNA or negative control siRNA into HEK 293 cells and HeLa cells, respectively. The results showed a 50~70% reduction of the luciferase activity in the presence of CREB- or c-Myc-Specific siRNA compared to that with negative control siRNA Figure . In addiBased on the above experimental results, we conclude that CREB and c-Myc positively regulate the basal transcriptional activity of STING.To determine whether CREB and c-Myc bind to the STING promoter region, we performed chromatin immunoprecipitation (ChIP) experiments on HeLa cells. CREB- and c-Myc-associated DNA fragments were immunoprecipated using an antibody that recognizes the CREB or c-Myc subunit, respectively. Normal rabbit IgG was used as a negative control. DNA fragments were then amplified by real-time quantitative PCR using primers surrounding the potential binding sites for CREB and c-Myc. Figure To examine whether CREB and c-Myc affected the expression of STING, the mRNA and protein levels of STING were determined in HeLa cells after transient transfection with CREB or c-Myc expression vector or siRNA against CREB or c-Myc by real-time quantitative RT-PCR and western blot analysis. The results Figure  showed tIt was reported that transcription factor CREB can cause repression of HCMV replication and may contribute to the development of new anti-HCMV strategies . The fea2.Human embryonic kidney (HEK) 293 cells and Human cervical cancer (HeLa) cells were obtained firstly from the American Type Culture Collection (ATCC). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100unit/ml penicillin and 100 mg/ml streptomycin at 37°C in an atmosphere of 5% COCellular mRNA was prepared from HeLa cells using Trizol reagent followed by chloroform-isopropanol extraction and ethanol precipitation. 5′RACE was performed using a 5′/3′RACE Kit (Roche), according to the manufacturer's instructions. Three antisense STING gene-specific primers were needed for the first-strand cDNA synthesis and nested PCRs, including 5′-GGTCAGCCATACTCAGGTTATCAGG-3′, 5′-CCACAGTCCAATGGGAGGAGAAT-3′, 5′-ACCCCGTAGCAGGTTGTTGTAA TG-3′. Products from the nested PCRs were electrophoresed in a 1.2% agarose gel, purified, and then sequenced.Kpn I and Bgl II sites of pGL3-Basic (Promega). The forward primer was: 5′-CGGGGTACCAACCCAGCCTCG CCCGTCTGTCTA-3′ (a restriction site for KpnI underlined). The reverse primer was: 5′-GGAAGATCTAGCCTCCATTCCATTGCCCTTTG-3′ (a restriction site for BglII underlined). Similarly, the 5′ deletion clones were constructed with PCR by the above reverse primer, which was paired with the following forward primers:GGTACCTGGGACTATAGGCGTGTTCCGTC-3′);(-953 to/+267: 5′-CGGGGTACCGGGCTAGAATTATACCTACCTGA-3′);(-677 to+267: 5′-CGGGGTACCTGGAGTGCAGTGGCCTAATCTCT-3′);(-436 to+267: 5′-CGGGGTACCCAGGCTCTCTTGGAGAAGTCACA-3′);(-124 to +267: 5′-CGGGGTACCGTTCATTTTTCACTCCTCCCTCC-3′); DNA sequence analysis confirmed these sequences.(+1 to +267: 5′-CGGA 2154 bp 5′-flanking region of human STING gene was amplified by polymerase chain reaction (PCR) and then was cloned into the Kpn I and Bgl II sites of pGL3-Basic (Promega). The expression plasmids CREB, pcDNA empty vector were stored by our group. The expression plasmids psp-myc, psp empty vector were kindly provided by Dr. Therése Wahlströom. CREB siRNA, c-Myc siRNA and the negative control were designed and synthesized in Genepharma company . Sequences targeted in the CREB and c-Myc mRNA, as well as the negative control sequence were listed below:CREBi: 5′-AGUAAAGGUCCUUAAGUGCTT-3′c-Myci: 5′-GGUGAUCCAGACUCUGACCUU-3′control: 5′-UUCUCCGAACGUGUCACGUTT-3′The amplified promoter fragments were cloned into the Mutations of the CREB and c-Myc binding sites found in the STING promoter were performed using the QuikChange Site-directed Mutagenesis kit (Takara) according to the manufacturer's instructions. Oligonucleotides with site-specific mutations at the critical nucleotides necessary for transcription factor were listed in Table Transfections were carried out in HEK 293 cells and HeLa cells by using Lipofectamine-2000 transfection reagent (Invitrogen) according to the manufacturer's suggestion. Cells having been seeded into 48-well plates 24 h before transfection were cotransfected with 400ng of each of the luciferase containing plasmids together with 4ng of a control pRL-TK plasmid as an internal control. After 48 h of transfection, cells were harvested and Luciferase assay was performed by using the Dual Reporter assay system (Promega) and TD-20/20 Turner Designs luminometer according to the manufacturer's instruction. Results were representative of at least three independent experiments performed in triplicate. For overexpression or RNAi, the expression plasmid (300ng) or siRNA (50nM) was individually cotransfected into HEK 293 cells and HeLa cells, together with STING promoter reporter plasmids (100ng) by using Lipofectamine-2000. Luciferase assay was performed 48 h after transfection.7 subconfluent HeLa cells were fixed with 1% formaldehyde for 10 min at room temperature. After fixation, cells were washed twice in ice-cold PBS and harvested by centrifugation at 4°C. The cell pellets were resuspended in 500μl nuclear lysis buffer and sonicated using six 15 sec pulses with 50 second rest in between pulses at 5% of maximum output strength on a Sonicator Ultrasonic Processor . The antibody used in the ChIP assays was an anti-CREB antibody (Abcam), an anti-c-Myc antibody (Abcam) and rabbit IgG control antibody (Millipore) was used as a negative control. ChIP purified DNA was amplified by quantitative PCR (Q-PCR). The ChIP primers were as follows: forward 5′-GCTCCTACCTAATATCATCCCC-3′ and reverse 5′-AGTTATTTCCGGTAACAAGAGC-3′. Q-PCR was performed in an ABI 7300 real time PCR system (Applied Biosystems Inc.) and SYBR Green was used as fluorescent dye with each sample loaded in triplicate. A comparative Ct method (ΔΔCt) was used to calculate the relative fold enrichment of the immunoprecipitated DNA in all samples.The chromatin immunoprecipitation assay was performed by using the Magna ChIP™ kit (Millipore) following the manufacturer's instructions. Briefly, 1×10CREB: forward 5′-CATTAACCATGACCAATGCAG-3′,reverse 5′-CTGTGCGAATCTGGTATGTTT-3′;c-Myc: forward 5′-CACCAGCAGCGACTCTGA-3′,reverse 5′-GATCCAGACTCTGACCTTTTGC-3′;STING: forward 5′-GGGCTGGCATGGTCATATTA-3′,reverse 5′-TACTCAGGTTATCAGGCACC-3′;GAPDH: forward 5′-GTCAACGGATTTGGTCTGTATT-3′,reverse 5′-AGTCTTCTGGGTGGCAGTGAT-3′.Total RNA extraction was performed by using Trizol reagent followed by chloroform-isopropanol extraction and ethanol precipitation. Subsequently, duplicate samples of 2μl of each cDNA were used as a template. The quantification of gene transcripts was performed by real-time PCR using SYBR green I dye (Takara) and the ABI 7300 real time PCR system (Applied Biosystems Inc.). The specificity of amplification was assessed for each sample by melting curve analysis. Expression values were normalized with control GAPDH. The primers used are as follows:All samples were lysed in Laemmli buffer, boiled, and electrophoresed on SDS-polyacrylamide gel, then separated proteins were transferred onto polyvinylidene difluoride membranes. To block non-special sites, the membranes were incubated in 5% dry milk in TBS-T saline  for 1 h and then protein blots were incubated for overnight at 4°C with primary antibodies against GAPDH (Santa Cruz Biotechnology), CREB, c-Myc, STING . Membranes were washed twice with TBS-T and treated with either a horseradish peroxidase-linked goat anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology). Reactive proteins were visualized by enhanced chemiluminescence .p value ≤0.05 was considered statistically significant. Data are presented as the means±SD and all experiments were done in triplicate and repeated at least three times.The results in the present were analyzed using paired two tailed student's-t test. A


Cyclin D1 (Ccnd1) during the early developmental stages of the mouse cerebral cortex. We found evidence that SP8 binds to the Ccnd1 locus on exon regions. In vitro experiments show SP8 binding activity on Ccnd1 gene 3′-end, and point to a putative role for SP8 in modulating PAX6-mediated repression of Ccnd1 along the dorso-ventral axis of the developing pallium, creating a medialLow-lateralHigh gradient of neuronal differentiation. Activation of Ccnd1 through the promoter/5′-end of the gene does not depend on SP8, but on βcatenin (CTNNB1). Importantly, alteration of the Sp8 level of expression in vivo affects Ccnd1 expression during early corticogenesis. Our results indicate that Ccnd1 regulation is the result of multiple signals and that SP8 is a player in this regulation, revealing an unexpected and potentially novel mechanism of transcriptional activation.Multiple signals control the balance between proliferation and differentiation of neural progenitor cells during corticogenesis. A key point of this regulation is the control of G1 phase length, which is regulated by the Cyclin/Cdks complexes. Using genome-wide chromatin immunoprecipitation assay and mouse genetics, we have explored the transcriptional regulation of  The cerebral cortex is the most complex structure of the mammalian brain. It is the site of numerous higher-order sensory, motor, and cognitive functions. Cortical function relies on the proper formation of specialized cortical areas as well as on their sophisticated interconnections  of the dorsal telencephalon and, following cell-cycle exit, migrate radially to the cortical plate. Previous work has shown that regional differences in the proliferative programs in the GZ have far reaching consequences for histogenesis of cortical areas  dynamic expression levels have been shown to be at the heart of a regulatory network that control the balance between cortical progenitor proliferation and differentiation  in phosphate buffered saline (PBS) at 4°C. The tissue was cryoprotected by immersion in 30% sucrose/PBS, embedded in OCT (Tissue-Tek), and cryostat sectioned at 20 μm.In situ RNA hybridization on cryostat sections was performed as previously described , Ccnd1 , Axin2 , and Pax6 .Gene expression patterns were compared between brains of different genotypes by matching the plane of section according to multiple anatomical features. Whenever possible, this was performed for multiple planes of sections for each gene, and from at least three brains for each genotype.Foxg1tTA/+ and the tetO-Sp8-IE mice were used as control; differences in phenotype were not observed between these two lines or between Foxg1tTA/+ and the tetO-Sp8-IE mice and the wild type embryos.7 nuclei/mL using a diagenode sonicator (Bioruptor Plus). The DNA fragments bound by SP8 were isolated using a goat polyclonal anti-SP8 antibody , a rabbit polyclonal anti-SP8 antibody , or rabbit polyclonal H3K27ac  coupled to magnetic beads . The beads were washed 5 times with LiCl Wash Buffer  and finally with TE buffer .Dorsal telenchephalon  was dissected from E12.5 CD1 mouse embryos. The cells were crosslinked with 1% formaldehyde for 10 min. The formaldehyde reaction was quenched by adding glycine to a final concentration of 0.125 M for 10 min. Cells were then pelleted, rinsed once in cold phosphate-buffered saline (PBS) with 1 mM PMSF and once in cold lysis buffer  to obtain nuclear pellets. Nuclei were sonicated in RIPA buffer  at a concentration of 5 × 103) to reverse the formaldehyde crosslink and was purified using a QIAquick PCR Purification Kit (Qiagen), following the manufacturer protocol. To check for fragment size distribution after sonication, a small fraction of the sample was reverse cross-linked for 2 h at 65°C, purified using DNA purification columns from Qiagen, then loaded onto a 2% agarose gel.The DNA was incubated o/n at 65°C in elution buffer . Pearson's correlation on the two replicates calculated with a call to wigCorrelate (http://hgdownload.soe.ucsc.edu/admin/exe/macOSX.x86_64/) or Wiggletools  using the BWA algorithm , or Sp8 , along with the different Ccnd1 fragments identified by ChipSeq . The fragment named Ccnd1 exon 2.3 contains Ccnd1 exons 2 and 3. The vector pG4.74[hRLuc/TK] (Promega), containing the Renilla luciferase gene, was co-transfected for normalization. The TK promoter of the pG4.74[hRLuc/TK] vector was substituted with the human βglobin minimal promoter (from vector BGZ40)  were maintained in growth medium: Alpha Minimum Essential Medium with ribonucleosides and deoxyribonucleosides (ThermoFisher) completed with 7.5% bovine calf serum and 2.5% fetal calf serum (ThermoFisher)  in OPTIMEM medium (ThermoFisher) following the manufacturer's instructions, and cultured after 6 h in growth medium. Twenty-four hours after the transfection the cells were harvested in lysis buffer (Promega), and the luciferase and renilla luciferase activities were measured using the Dual Luciferase Assay protocol (Promega). Each transfection experiment was performed in triplicate and repeated at least two times. Reporter gene activities shown in Cyclin D1 is a key regulator of G1 phase progression in neural progenitor cells. We analyzed the mRNA expression of Cyclin D1 in the embryonic forebrain at E12.5. We found that while Ccnd1 is strongly expressed in the ventricular zone (VZ) of the basal ganglia  of the lateral ganglionic eminence (LGE) . We found that SP8 binds the Ccnd1 locus in vivo decorating Ccnd1 exons  on exons 1 and 2 indicated that these regions correspond to active chromatin domains Figures . The Ccn Figures . H3K27acCcnd1 5′UTR showed H3K27ac signal and it contains a CpG island suggests a role for this region in the transcriptional regulation of Ccnd1 in E12.5 cortical progenitor cells    Eto, . However+96 Eto,   transgenic mouse system, Sp8 is over-expressed during forebrain development  and a reduction of 0.8 folds in the Sp8 LOF mutants  (Table These data were further confirmed by RNASeq experiments (data not shown) performed on E12.5 mouse pallial cells showing an increase of 9) Table .Ccnd1 expression during mouse corticogenesis in vivo.These findings indicate that SP8 is a critical player in the regulation of Cyclin D1 is a major cell-cycle regulator  needs further investigation.In contrast to PAX6, βcatenin was able to activate transcription from the Ccnd1 Ex5 fragment. Consistently with our luciferase results, we found a cluster of putative SP8 binding sites at the end of the ORF in Ex5 fragment; this cluster overlapped with the SP8 summit identified in our ChipSeq experiments (Table Ccnd1 gene in addition to the classical enhancer/promoter regulative domains located upstream of the target genes.Our results show that SP8 is able to specifically activate gene expression from the ts Table . These fCCND1 3′UTR region has been shown to act as a critical regulatory element. Different miRNAs are predicted to bind human and mouse Ccnd1 3′UTR and regulate the level of Cyclin D1 expression (Deshpande et al., CCND1 3′UTR alter the stability of the CCND1 transcript activating its oncogenic potential (Lebwohl et al., CCND1: Snp rs7178, localized on CCND1 3′UTR, is involved in neuroblastoma (Wang et al., CCND1 3′UTR, is involved in cognitive behavior (Rietveld et al., Ccnd1 3′UTR (as revealed using the ECR Browser Ovcharenko et al., Ccnd1 3′UTR.Human Ccnd1 transcription from a region located at the end of the ORF in exon 5 and close to the 3′UTR, suggest that the 3′-end of the Ccnd1 gene may be a target of gene regulation at multiple levels, including the transcriptional one. The in vitro validation of the activity of SP8, as well as the interaction with PAX6 and βcatenin on the Ccnd1 locus, is based on an assay commonly used to screen the activity of genomic regulative regions. In addition, we provide further evidence based on manipulation of SP8 levels of expression in vivo in GOF and LOF transgenic mice as well as on RNAseq data that both clearly show a role for SP8 for Ccnd1 expression regulation at early stages of pallium development.Our data, showing that SP8 binds and specifically regulates Ccnd1 transcription in mouse pallium during corticogenesis, resulting in a complex pattern of Ccnd1 expression. SP8 appears as a major player in this regulation, uncovering a potential novel role of the Sp-transcription factor family in transcription regulation, which awaits further analysis.In summary, multiple signals regulate UB: Conceived the work; UB, BB, and ED: Collected and analyzed the data; UB, BB, DP, and CD: Wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.