CENs), the DNA sequences where kinetochores form, to attach chromosomes to microtubules. In contrast to most eukaryotes, which have broad centromeres, Saccharomyces cerevisiae possesses sequence-defined point CENs. Chromatin immunoprecipitation followed by sequencing (ChIP–Seq) reveals colocalization of four kinetochore proteins at novel, discrete, non-centromeric regions, especially when levels of the centromeric histone H3 variant, Cse4 (a.k.a. CENP-A or CenH3), are elevated. These regions of overlapping protein binding enhance the segregation of plasmids and chromosomes and have thus been termed Centromere-Like Regions (CLRs). CLRs form in close proximity to S. cerevisiae CENs and share characteristics typical of both point and regional CENs. CLR sequences are conserved among related budding yeasts. Many genomic features characteristic of CLRs are also associated with these conserved homologous sequences from closely related budding yeasts. These studies provide general and important insights into the origin and evolution of centromeres.Accurate chromosome segregation requires centromeres ( CENs) are chromosomal regions essential for proper chromosome segregation through their ability to establish evolutionarily conserved protein complexes called kinetochores. During mitosis, kinetochores attach to microtubules emanating from spindle poles, thus providing the mechanism for chromosome segregation. Eukaryotes have different types of CENs. Most eukaryotes have large multimeric centromeres lacking DNA sequence specificity. In contrast, the budding yeast, S. cerevisiae, has short punctate centromeres, comprised of specific DNA sequences. Combining chromatin immunoprecipitation and deep sequencing, we identified regions of the yeast genome that are bound by key kinetochore components; we refer to these regions as Centromere-Like Regions (CLRs). We found that CLRs can promote segregation on episomal plasmids and native chromosomes. Most CLRs are found in intergenic regions, close to native CENs. CLRs resemble point CENs by their short size and regional centromeres by their lack of determining DNA sequences. CLR sequences are conserved among related budding yeasts. Our findings indicate that, similar to other fungi and eukaryotes, S. cerevisiae possesses the ability to form sequence-independent centromeric structures. Establishment of centromeric elements outside regular CENs, or neocentromerization, can lead to chromosome missegregation and is a hallmark of cancer cells. CLR formation in budding yeast provides a simple model of neocentromerization.Centromeres ( Saccharomyces lineage has small, punctate CENs encompassing only 125 base pairs (bp) Saccharomyces cerevisiaeS. cerevisiae, Cse4 is commonly found outside centromeres using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq) The kinetochore is a conserved proteinaceous structure that assembles on centromeric DNA and is responsible for connecting chromosomes to the spindle, thus ensuring accurate chromosome segregation. The length of centromeric DNA differs among eukaryotes, from less than one kilobase pair (kb) to several megabase pairs (Mb) CENs, termed Centromere-Like Regions (CLRs). We showed that cloned CLRs can help the segregation of a CEN-less episomal plasmid and that endogenous CLRs can promote accurate segregation of a chromosome bearing an inactivated centromere. We found that most CLRs are found in larger than average intergenic regions and lie in close proximity to S. cerevisiae centromeres. Other genomic features associated with CLRs include a weak association to autonomously-replicating sequences  and an increased level of “AT” nucleotides over a short stretch of DNA. We observed sequence conservation of CLRs with members of the Saccharomyces sensu stricto and other budding yeasts carrying point CENs, but not with other yeasts and fungi bearing larger, regional CENs. Our results have implications for the origin and evolution of centromeres since CLRs might constitute evolutionary remnants from regional CENs.If Cse4 accumulation at non-centromeric sites is functional, i.e. imparts centromere-like activity, then additional kinetochore proteins should also be present. To investigate this possibility, we generated genome-wide binding profiles using ChIP-Seq to characterize four epitope-tagged kinetochore proteins, comparing a wild-type strain with normal levels of Cse4 (WT) to a strain overproducing Cse4 (Cse4 OP). Our ChIP-Seq data indicate recruitment of all tested kinetochore proteins to discrete sites outside −5ChIP-Seq data were generated for four epitope-tagged kinetochore proteins: Cse4 (CenH3), the outer kinetochore protein Ndc80 (Hec1), and the inner kinetochore components Mif2 (CENP-C) and Ndc10 (Cbf2). We compared a wild-type strain with normal levels of Cse4 (WT) to a strain overproducing Cse4 from the Gal1–10 promoter (Cse4 OP), with at least a 3-fold increase in Cse4 protein levels in Cse4 OP as measured by Western blots , especially among those displaying high levels of protein binding , and, compared to CEN-proximal CLRs, these centromere-distal CLRs are generally associated with reduced, yet significant, occupancy of the outer kinetochore protein Ndc80  for all four proteins  in mcd1-1, results remained unchanged; no individual CLR showed significant binding for all four proteins  and did not differ greatly from strains with functional Mcd1  were compared to ARS-only (ARS plasmid) and ARS-CEN plasmids (CEN plasmid) (CLR sequences tested (CLR1 and CLR15) produced colonies of intermediate size; these were larger than those obtained from ARS plasmids, but smaller than those from CEN plasmids (CLR plasmids (CLR7 and CLR10) behaved like ARS plasmids. Consistent with the requirement for Cse4 recruitment to extrachromosomal plasmids for their segregation CLRs, CLR1 and CLR15, had higher enrichment values for Cse4 than CLR7 and CLR10 . As another test of segregation proficiency, doubling times in selective medium  were measured. CLR1 and CLR15 decreased doubling time compared to an ARS plasmid, but to a lesser extent than CEN plasmids  for ∼4 generations CLR plasmids were maintained in a significantly greater fraction of cells  than ARS plasmids (20%) . The CEN plasmid was maintained in 91% of cells  with ARS plasmids bearing random inserts of similar sizes to CLR inserts, respectively 1 kb for ARS-R1 and 0.8 kb for ARS-R2 (ARS-CLR plasmids was re-assessed then in comparison to ARS-R1 or ARS-R2 and found to follow similar trends than those obtained with ARS-only plasmid as a control (CLR sequences can enhance plasmid segregation.To determine whether plasmid) . After tplasmids . Other Cof cells . Differeof cells . To ensur ARS-R2 . Statist control . Taken tCLR can function in its natural context, on a chromosome, to promote proper segregation. Galactose-driven transcription towards a native CEN inactivates the kinetochore, thus creating a conditional centromere that can be switched off when cells are grown in galactose CEN3 and CLR1, the only naturally-occurring CLR on chromosome 3. First, segregation was monitored by pedigree analysis; bud emergence in a daughter cell was assayed after CEN inactivation in an unbudded mother cell ). In a second assay, we followed segregation of a GFP-labeled chromosome 3 after a single nuclear division CEN3 is active . This improvement in faithful chromosome segregation is weaker than that provided by a natural centromere or by a physically-tethered synthetic kinetochore CEN. In C. albicans, Cse4 overproduction improves segregation in mutants defective in kinetochore proteins Dam1 and Dad2 CEN3 inactivation might be hindered at a higher degree in Cse4 OP due to excess Cse4 molecules per se.Second, we asked if a her cell  [19]. Bu strains . In conts active . Cse4 OPCEN3 is inactive, Cse4 OP significantly improves the segregation of chromosome 3. To test whether this improvement is due to CLR activity, we deleted CLR1 by gene replacement and then monitored chromosome segregation by pedigree analysis and GFP imaging. In both assays, deletion of CLR1 decreased the Cse4 overproduction-dependent rescue of chromosome segregation, by 46% in the case of the budding assay and by 39% for the GFP dots assay . To ensure that the observed decreased was caused by the loss of CLR1 and not by impacted kinetochore assembly at CEN3 arising from the experimental manipulation (gene replacement at CLR1), we verified binding of Cse4, Mif2, Ndc10 and Ndc80 at CEN3 in clr1 strains for WT and Cse4 OP. We found that binding levels of kinetochore components at CEN3 in clr1 strains were similar to those in CLR1+ strains, confirming that CEN3 integrity is intact in clr1 strains , the assembly of functional CLRs might share some similarities to that of native S. cerevisiae centromeres, such as the functional requirement on Ndc10. This is also supported by the significant binding of Ndc10 at CLRs determined by ChIP experiments.Ndc10 is a budding yeast-specific essential kinetochore component required for the centromeric localization of many proteins, including Cse4 appeared , as leveCLRs compare to native S. cerevisiae CENs with regard to DNA sequence? Searching across all 23 identified CLRs for sequences similar to the centromeric CDEI or CDEIII consensus motifs did not yield clear results, nor did we find any motifs enriched amongst CLR sequences. CLR sequences tend to encompass a significantly AT-enriched, 90-bp stretch of DNA , CLRs are often associated with ARSs, but not with tRNAs or retrotransposons  form mostly in large intergenic regions, and they are closely associated with replication origins CLRs did not reveal any enrichment for genes involved in particular cellular process (P<0.01).Which genomic characteristics best describe CLR and non-CLR regions), we compared Cse4 binding profiles to the genome-wide distribution of RNA polymerase II and Sono-Seq regions. Sono-Seq regions correspond to sites of highly-accessible chromatin −8). Cse4 has been shown to undergo proteolysis to ensure physiological levels and protect from extensive stable euchromatinization with deleterious effects K16R, subject to reduced degradation CLRs. From ChIP-qPCR analyses, Cse4K16R shows increased localization to the previously-tested CLRs , suggesting that non-centromeric localization of Cse4 does not require Scm3. This finding is supported by the fact that transient incorporation of Cse4 on non-centromeric sites occurs at regions of high histone turnover, linking this phenomenon with nucleosome incorporation and ejection dynamics in vitro reconstitution of Cse4-containing nucleosomes, two distinct populations of Cse4 nucleosomes have been reconstituted: one resembling canonical octameric nucleosomes and another found primarily at t AT-rich DNA characteristic of CENs, atypical in its inclusion of Scm3 Scm3 (HJURP) is a CLRs from a control group consisting of regions that did not pass statistical filters set during ChIP-Seq analysis . CLRs differed from the control group globally and across three individual variables: distance from CENs, overlap with an intergenic region, and nearby RNA polymerase II occupancy . Group membership of individual sites could be predicted quite accurately using discriminant analysis or k-means clustering  vs. group 2 (CLR13–23)), we observed that the strongest discriminant was the distance to CENs, with CLRs in the top tier usually closer to CENs. This is consistent with our previous observations from the ChIP-Seq data. The proximity of CLRs to centromeres suggests that pericentric chromatin creates a preferred environment for establishment of CLRs CLRs may be disfavored due to a greater risk for instability, in the same way that increased distance between CENs in dicentric chromosomes increases instability CLR formation.Twelve different variables  were exaanalysis ; these col group . This din groups . There ianalysis . CEN-distal CLRs . In addiCLR-containing regions are conserved among the Saccharomyces lineage and other closely-related budding yeasts with point CENs CLR sequences are more conserved than a randomly-selected set of intergenic regions . Although the role of CLRs in wild populations of yeasts remains unknown, sequence conservation with other budding yeasts suggests that CLRs possess a conserved function.Saccharomyces sensu stricto database CENs, the association to tRNAs, the mean length of intergenic regions, and the mean AT content in the most AT-enriched 90-bp stretch of DNA, in closely-related budding yeast. Trends similar to those observed for S. cerevisiae CLRs were observed for sequences similar to CLRs present in S. mikatae, S. kudriavzevii and S. bayanus  and contain a short CDEII-like, AT-rich stretch of DNA. According to previous models of centromere evolution, an epigenetic regional centromere evolved to point centromeres in a few steps CEN repeats would diverge and/or disappear. Third, a segregation locus from a self-propagating genetic element (such as the STB locus in the yeast 2 µm plasmid) would integrate on the chromosome, with the potential to successfully hijack the segregation machinery. Once this is accomplished, it is likely that CEN repeats would diverge or disappear even more. Finally, acquisition of specific DNA modules and evolution of segregation proteins that bind this newly-integrated locus would create point centromeres with high specificity. If this model is correct, then CLRs may be remnants of regional CENs that were lost or diverged during evolution  or, as in the S. cerevisiae lineage, through the acquisition of specific DNA modules (point CENs and STB) to form stable, strong centromeres with high segregation fidelity.Overall, volution . CLRs miCLR formation affects chromosome segregation differently, depending on whether the chromosome has an active centromere or not. Recent data from an assay measuring the transmission fidelity of an artificial chromosome indicated a significant, although modest, increase in chromosome loss (about 2 fold) when Cse4 is overproduced and the normal centromere is functional CEN is active, the observed increase in chromosome instability is likely due to the formation of functional CLRs in a subset of the cell population. The modest effect is concordant with the lower levels of protein binding at CLRs vs. CENs, and with the higher incidence of CEN-proximal CLRs. Based on studies of dicentric chromosomes, CEN-proximal CLRs have potentially less deleterious effects than CEN-distal CLRs CEN is inactive, CLR formation promotes, at least partially, accurate chromosome segregation of the CEN-inactivated chromosome, which might be beneficial to yeast cells under this condition.CENs have been created artificially through two mechanisms: tethering of outer kinetochore components onto DNA CEN in C. albicansDrosophila and barley have revealed a predisposition for neoCENs to form in pericentric chromatin in vitro chromatin templates Drosophila cells CLR formation only at a subset of all non-centromeric Cse4 binding sites.New CENs also occur naturally. In humans, acentric chromosomes resulting from chromosome rearrangements can be stabilized through the establishment of neocentromeres at sites devoid of α-satellite repeats and containing little or no heterochromatin CENs. Many liposarcomas carry a supernumerary chromosome containing oncogenes and a neoCENCENs that have been recurrently used throughout primate evolution CLRs, evolutionarily-new centromeres, ectopic neocentromeres and bacterial centromere-like regions are likely to provide additional insights into the origin, evolution, establishment and maintenance of native centromeres.New ARS plasmid (pPL26) was generated by cloning a 0.9 kb fragment containing ARS1 into pRS305 CLR and CEN6 sequences were integrated into pPL26. ARS-CEN6 plasmid pRS315 was also used in this study.Yeast strains are isogenic with W303 . Cse4 is600 = 0.5–0.7). Proteins were crosslinked to DNA by treating cells with formaldehyde  for 15 minutes, then quenched with glycine. Cells were collected by filtration after two washes. After cell lysis using a FastPrep machine , chromatin was sheared by sonication using a Branson Digital 450 sonifier (Branson). Clarified, sonicated lysates were taken at this step for Sono-Seq, prior to immunopreciptation ChIP-Seq experiments were performed at least in independent biological duplicates, as described previously mcd1-1 background, cultures were grown overnight at the permissive temperature (25°C) to OD600 = 0.3–0.4 and then shifted to the restrictive temperature (37°C) for ∼2.5 h prior to crosslinking, similarly to other protocols for the abrogation of the pericentric intramolecular C loop To collect ChIP samples of K16R strains were grown in SC Raffinose/Galactose – URA overnight to OD600 = 0.3, overproducing normal and non-degradable Cse4 respectively. Cells were then transferred in rich media  and grown for ∼4 h to OD600 = 0.8–1.0, for easier comparison with our previous ChIP-qPCR data.To collect ChIP samples for the non-degradable Cse4 experiment S. cerevisiae S288c reference genome version 2 (SGD/UCSC sacCer2) by the ELAND algorithm (Illumina). The peak scoring algorithm PeakSeq Raw sequencing data were first processed by Illumina's analysis pipeline. Reads were then parsed according to the index. Remaining bases were aligned against CLRs, we took a conservative, stringent approach to minimize false positives lacking functional significance or failing qPCR validation. For each biological replicate, only putative regions with q-value <10−5 were considered CLR, 1) all four kinetochore proteins must be present given the q-value threshold; and 2) for proteins in direct contact with DNA, mean PeakSeq ratios between duplicates should be above 2.0 for open chromatin marker Cse4 and 1.5 for direct DNA binders Mif2 and Ndc10 CLRs and CENs, inspection of normalized signal tracks (high tagged/untagged ratio and low background in untagged controls desired), binding over a highly PolII-occupied ORF, and presence in HOT regions CLRs, met these criteria in Cse4 OP and none in WT (LCNCRs) that did not pass the aforementioned filters, used during computational analyses. For WT, only one low-confidence negative region, the rDNA array, was found after unmasking for repeated regions. GO analysis for this control group showed a significant enrichment for metabolic genes.To uncover ne in WT . Include−5 and PeakSeq ratio ≥2.00.Significant binding regions for RNA Polymerase II and Sono-Seq were determined using q<10CLRs. These six CLRs were randomly selected and spanned multiple confidence levels of our final ranked target list  http://frodo.wi.mit.edu/primer3/) and primer sequences are given in the p). Enrichments were calculated by the 2−ΔΔCp method Real-time quantitative PCR (qPCR) was performed to validate the presence of kinetochore components Cse4, Mif2, Ndc10 and Ndc80 at six Saccharomyces Genome Database.Signal track files were visualized in the Integrated Genome Browser, with y-axes scaled according to the number of uniquely-mapped reads and with annotations from the Target lists from different biological replicates were merged and annotated to find overlapping and/or nearest genomic features using various R and Bioconductor packages, mostly ChIPpeakAnno Target lists were first sorted by q-value and then by the difference between PeakSeq experimental and background reads. Pairwise comparisons of lists were done using ChIPpeakAnno with parameters maxgap = 0 and multiple = T. Spearman's rank correlation coefficients and associated p-values for overlapping peaks were computed.CLRs and LCNCRs. GO analysis for CLRs did not give any significant term. GO analysis for LCNCRs showed a significant enrichment for metabolic genes (data not shown).GO Biological Process Ontology analyses  were performed on SGD's website. We compared GO results from 600 = 0.8 were isolated, medium was removed and cells were frozen at −80°C. Cells were resuspended in water, an equal volume of 0.2 M NaOH was added and samples were incubated at room temperature for 5 min., after which NaOH was removed thoroughly. Samples were then boiled in 1× sample buffer containing 5% β-mercaptoethanol for 4 min. and the supernatant was kept. Samples were run for protein gel electrophoresis on a 4–12% Novex NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer. Proteins were then transferred a PDVF membrane on a semi-dry Trans-Blot SD apparatus (BioRad). Membranes were blocked with TBS+0.1% Tween with 5% dry milk. Primary antibodies were added for an overnight incubation: mouse anti-HA 12CA5 and mouse anti-β-actin (Abcam). After washes in 1× TBS+0.1% Tween, a HRP-conjugated anti-mouse IgG secondary antibody was added in 1× TBS+0.1% Tween with 5% dry milk for 1.5 h. Following washes in 1× TBS/T, the SuperSignal West Pico Chemiluminescent Substrate (Thermo) was added to the blot and detection was done on a STORM imager . Western blot images were processed and analyzed using the ImageQuant software . Cse4-3HA protein levels were normalized by the abundance of β-actin in each replicate.Whole-cell protein extracts were obtained using the post-alcaline yeast protein extraction method CLR sequences inserted into an ARS plasmid were conducted according to standard procedures Plasmid assays to test 6 cells in the same media. Optical densities were measured every 2–4 hours. Doubling times were calculated in R. Statistical significance was tested by a Bayesian analysis with Markov Chain Monte Carlo (MCMC), using R package rjags . MCMC simulations let the data and its variability generate sampling distributions of the maximum likelihood estimator without strong prior or test assumptions; p-values were calculated from 100,000 comparisons of this estimator between 2 groups.For doubling time analyses, cells were grown overnight in synthetic complete (SC) media lacking leucine (SC-Leu), with raffinose and galactose as carbon source. Cultures were diluted around 5×10For plasmid retention and colony formation analyses, cells were grown for ∼4 generations in rich medium, with raffinose and galactose as carbon sources. Cultures were diluted 10-fold or 100-fold and plated on SC-Ade-Leu and SC-Ade plates. Photos were taken after 4 days of growth. Pictures of transformation plates on SC-Ade-Leu with galactose/raffinose also represent 4 days of growth after transformation.cdc15-2 allele. Experiments including the additional temperature-sensitive allele ndc10-1 were performed similarly. About 98% of cells were large-budded. GFP dots were visualized in live cells and classified into two categories: 1) one GFP dot in each cell, or 2) two GFP dots in the same cell. A minimum of 200 cells were counted per replicate. Statistical significance was assessed using Fisher's exact test. In addition, for each sample, an aliquot was quick-fixed with ethanol and DAPI was added. >90% of cells had segregated DNA between their buds, as revealed by DAPI staining. Experiments plotted in CEN3 (OFF) or not (ON). Strains comprising conditional CEN3 were also resuspended in raffinose-only media (ON) and gave similar high percentages of cells with accurate segregation. Overnight growth of cells in raffinose-only media, with an active conditional CEN3 (ON), yielded >99% of cells with accurate segregation.Chromosome segregation analysis of GFP dots present on chromosome 3 was performed in biological triplicates as described CEN3 (OFF) or not (ON) were plated on galactose/raffinose plates. Strains comprising conditional CEN3 were also plated on YPAU+dextrose plates (ON) and gave end-point results comparable to those of Single-cell pedigree analysis was performed on strains containing a conditional centromere CLRs located within 5 kb of tRNAs, ARSes or retrotransposons. 23 sites were randomly chosen on chromosomes containing a putative CLR. The number of chosen sites on a chromosome paralleled the chromosomal distribution of CLRs. The number of random sites falling within 5 kb of a feature was determined for 100,000 iterations. The p-value is given by the fraction of iterations with greater or equal feature association than found across CLRs. For these discrete genomic features, we adjusted p-values using a Bonferroni correction.We determined the number of For centromere proximity, a similar procedure was followed. A site is centromere-proximal if located within 25 kb of the centromere.S. cerevisiae, the number of random sites chosen followed the number of CLR sequences deemed conserved by blastn in each species  was performed on genomes deposited at NCBI  with the following parameters: expect value (E) <1, and other values as default For each CLRs with at least one significant hit  and the average hit score across all CLRs.A phylogenetic tree indicates, for each species, the fraction of CLR values with 160 randomly-selected intergenic regions of same length to determine whether sequence conservation is greater in CLRs. Blastn was carried out as described above for this random set. Statistical significance of average blastn scores was tested by MCMC simulations.We compared CLRs and 38 LCNCRs, data from 12 variables were obtained  and second (y-axis) principal components. A 95% confidence ellipsis was added to the score plots.Principal component analysis was performed for the 23 high-confidence CLRs and LCNCRs were performed on standardized, scaled data in R, to identify variables that would distinguish these two well-defined groups. When all 12 variables were present, the percentage of a binding site overlapping an ORF was a very strong discriminator but could be perceived as arbitrary, depending on the length of the binding site. Therefore discriminant analysis was also performed with all variables except that one. Discriminant score plot was generated using the discriminant function with all variables (x-axis) and the discriminant function with all variables except percent overlap with an ORF (y-axis). We used stepwise discriminant analysis to determine the variables that discriminate best between groups, in SAS, and also obtained similar results. Overall discriminative power was tested using cross-validation (leave-one-out classification). As a comparison, k-nearest neighbors classification was performed on the same scaled, standardized dataset, using k = 3.Linear discriminant analyses between Figure S1Western blot analysis of Cse4 levels in WT and Cse4 OP strains. (A) Western blot image showing the levels of Cse4-3HA  in Cse4 OP and WT strains, as well as β-actin as a loading control. (B) Quantitation of Cse4 protein levels in WT and Cse4 OP strains. Cse4 abundance was normalized by the β-actin protein levels. Normalized Cse4 levels (means in arbitrary units (a.u.)+/−standard errors of the mean (SEM)) are plotted on a linear scale. Individual enrichments were obtained from four biological replicates.(TIF)Click here for additional data file.Figure S2CENs are occupied by four kinetochore proteins, in WT and Cse4 OP strains. ChIP-Seq signal tracks for Cse4 (red), Mif2 (blue), Ndc10 (green) and Ndc80 (orange) are scaled according to the number of uniquely-mapping reads, in WT (A) and Cse4 OP strains (B). Significant binding sites are represented by a liked-colored box under its corresponding signal track. Control samples (immunoprecipitates from untagged strains) are shown in grey. Open reading frames (ORFs) are represented by purple boxes. The black circles indicate centromeres. Horizontal scale bars represent 1 kb.All (TIF)Click here for additional data file.Figure S3CLRs. (A) All tested CLRs were not bound by all four proteins in WT. Only Cse4 in CLR1 and Ndc10 in CLR7 displayed significant binding . (B) In Cse4 OP, significantly enriched protein binding was confirmed at the six CLRs tested, for all four proteins examined . Normalized enrichment ratios (means in arbitrary units (a.u.)+/−standard errors of the mean (SEM)) are plotted on a linear scale. Individual enrichments were obtained from qPCR reactions run in triplicates and performed in at least two biological replicates. Note the different scales between (A) and (B). A normalized enrichment of 1 indicates no enrichment over a negative control region not enriched for kinetochore proteins.ChIP-qPCR validation of 6 (TIF)Click here for additional data file.Figure S4CLRs when the pericentric intramolecular C loop is abrogated in a cohesin-deficient mcd1-1 background. (A) All tested CLRs were not bound by all four proteins in strains with normal Cse4 levels (WT) in a mcd1-1 background, similarly to CLR1 and Ndc10 in CLR7 displayed significant binding . (B) In strains with elevated Cse4 levels (Cse4 OP) in a mcd1-1 background, significantly enriched protein binding was confirmed at the six CLRs tested, for all four proteins examined , similarly to Kinetochore proteins are present at (TIF)Click here for additional data file.Figure S5CLR plasmids generate colonies of intermediate size, which are on average larger than those carrying ARS plasmids and smaller than those containing CEN plasmids. After a few generations of growth in non-selective rich medium, strains carrying various plasmids were plated on selective medium (-Ade -Leu) for four days. Strains plated on permissive medium (-Ade) do not differ in colony size.Colony formation on plates from plating assays. Strains bearing (TIF)Click here for additional data file.Figure S6ARS-CLR plasmids and ARS plasmids bearing random inserts of similar sizes in plasmid segregation assays. (A) Doubling times in selective medium of strains carrying different plasmids (Means+/−SEM). (B) Fraction of plasmid-containing cells after growth in non-selective media for strains bearing various plasmids (Means+/−SEM). P-values were computed using MCMC simulations  (A–B). ARS-R1 and ARS-R2 refer to ARS plasmids bearing random inserts of 1 kb and 0.8 kb, respectively.Comparison of (TIF)Click here for additional data file.Figure S7CEN3.Time course analysis of daughter cell budding after transfer of mother cells to a galactose plate. Values (Means+/−SEM) are given for various genotypes at 3-h intervals refer to . ON and (TIF)Click here for additional data file.Figure S8CEN3 are similar in clr1 and CLR1+ strains. ChIP-qPCR confirms that the presence of kinetochore proteins at CEN3 is not affected when CLR1 is deleted. Individual protein enrichments at CEN3 were normalized and compared in strains containing CLR1 (CLR1+) and missing CLR1 (clr1), for WT (A) and Cse4 OP (B). Normalized enrichment ratios (means in arbitrary units (a.u.)+/−SEM) were plotted on a log 10 scale. A normalized enrichment of 1 indicates no enrichment over a negative control region not enriched for kinetochore proteins.Binding levels of kinetochore proteins at (TIF)Click here for additional data file.Figure S9−8), in concordance with the presence of Cse4 around transcribed regions Cse4 marks a subset of open chromatin. Cse4 is associated with promoters, accessible chromatin and RNA polymerase II-bound regions. Cse4 shows a broader euchromatin distribution upon its overproduction, consistent with previous reports (TIF)Click here for additional data file.Figure S10CLRs compared to non-CLR Cse4 binding sites at gene promoters. (A) ChIP-qPCR comparative analyses of CLRs and non-CLR promoters bound by Cse4 indicate that non-degradable Cse4K16R is relatively more abundant than normal Cse4 at both CLRs and non-CLR Cse4 binding sites, in similar proportions. Non-degradable Cse4 enrichments for 6 CLRs +/−SEM). (B) ChIP-qPCR data depicting normalized enrichments for non-degradable Cse4 are presented for 6 previously-tested CLRs Click here for additional data file.Figure S11SCM3 was deleted. Despite this discrepancy, binding sites are highly correlated . Control samples (immunoprecipitates from untagged strains) are shown in grey. Open reading frames (ORFs) are represented by purple boxes. Horizontal scale bars represent 1 kb. Significant regions of protein binding are represented by a like-colored box under the corresponding signal tracks. (D) Overlap of binding regions between Cse4 OP SCM3 (red) and Cse4 OP scm3Δ (blue). Note that the Venn diagram is not drawn to scale.Localization of Cse4 to non-centromeric regions does not require the centromere chaperone Scm3. Cse4 binding at extra-centromeric sites is not greatly affected by the presence or absence of the essential chaperone Scm3 in Cse4 OP strains. (A–C) Cse4 ChIP-Seq binding profiles are compared in the presence (red) or absence (blue) of Scm3, upon overproduction of Cse4. Examples on chromosomes 1 (A), 6 (B) and 3 (C) are depicted. A 66% increase in the number of Cse4 non-centromeric binding sites was observed when (TIF)Click here for additional data file.Figure S12CLRs are conserved in sequences homologous to CLRs in the Saccharomyces sensu stricto. Comparison of all sequences homologous to CLRs in S. mikatae, S. kudriavzevii and S. bayanus that were deemed conserved by blastn scores (CENs (within 25 kb) (A), association with tRNAs (within 5 kb) (B), mean AT content of the most AT-rich 90-bp stretch of DNA (C), and average length of intergenic regions (D). Annotations and sequences were obtained from the Saccharomyces sensu stricto database CLRs and CENs in S. cerevisiae (Genomic features associated with n scores  for assorevisiae  and deta(TIF)Click here for additional data file.Figure S13CEN2. Shown is ChIP-Seq signal for kinetochore proteins in Cse4 OP strains (blue) compared to WT (red). Plots depict the log ratio of read enrichment for Cse4 (A), Mif2 (B), Ndc10 (C) and Ndc80 (D), centered at CEN2, on log 2 scales.Aggregated signal plots for individual kinetochore components at (TIF)Click here for additional data file.Figure S14CEN5. Shown is ChIP-Seq signal for kinetochore proteins in Cse4 OP strains (blue) compared to WT (red). Plots depict the log ratio of read enrichment for Cse4 (A), Mif2 (B), Ndc10 (C) and Ndc80 (D), centered at CEN5, on log 2 scales.Aggregated signal plots for individual kinetochore components at (TIF)Click here for additional data file.Figure S15CLRs and negative control regions (LCNCRs) using a χ2 distribution. (A–C) χ2 normal quantile plots, including data points (red), an ideal fit line (black) and 95% confidence intervals (blue), are presented for all 61 sites (CLRs and negative control regions) (A), for 23 CLRs (B), and for 38 negative control regions (C). On the horizontal axis are the theoretical quantiles, and on the vertical axis are the data quantiles.Data used in this study fit multivariate normal distributions. Transformed data for 12 variables  have bee(TIF)Click here for additional data file.Table S1CLRs and 38 control regions (LCNCRs).Raw data from 12 variables for 23 (DOC)Click here for additional data file.Table S2Yeast strains used in this study.(DOC)Click here for additional data file.Table S3Primer sequences used for qPCR.(DOC)Click here for additional data file.


MIRs) is evolving rapidly. Here we investigate the effect of 3D architecture of chromatin on the transcriptional regulation of MIRs. We demonstrate that MIRs have transcriptional features that are similar to protein-coding genes. RNA polymerase II–associated ChIA-PET data reveal that many groups of MIRs and protein-coding genes are organized into functionally compartmentalized chromatin communities and undergo coordinated expression when their genomic loci are spatially colocated. We observe that MIRs display widespread communication in those transcriptionally active communities. Moreover, miRNA–target interactions are significantly enriched among communities with functional homogeneity while depleted from the same community from which they originated, suggesting MIRs coordinating function-related pathways at posttranscriptional level. Further investigation demonstrates the existence of spatial MIR–MIR chromatin interacting networks. We show that groups of spatially coordinated MIRs are frequently from the same family and involved in the same disease category. The spatial interaction network possesses both common and cell-specific subnetwork modules that result from the spatial organization of chromatin within different cell types. Together, our study unveils an entirely unexplored layer of MIR regulation throughout the human genome that links the spatial coordination of MIRs to their co-expression and function.Our knowledge of the role of higher-order chromatin structures in transcription of microRNA genes ( MIRs) have been identified in the human genome  are a large family of small noncoding RNAs ∼21 nt) that have emerged as key posttranscriptional regulators of gene expression in eukaryotic organisms. More than 1500 miRNA genes   are actually other types of RNAs, such as tRNAs, Y RNAs and Vault RNAs, which are transcribed by RNAPIII  displayed no occupancy by RNAPIII  . There h RNAPIII . However RNAPIII , while o RNAPIII . These mMIRs originate from precursor molecules (pri-miRNAs). These transcripts can be encoded as independent transcription units (TUs), in polycistronic clusters or within the introns of protein-coding genes (MIRs are organized into introns of protein-coding genes (intragenic MIRs) and are likely transcribed in parallel with their host transcripts (MIRs located within intergenic regions (intergenic MIRs) are believed to be derived from independent TUs  from the Encyclopedia of DNA Elements (ENCODE) project .In this study, we have integrated comprehensive 3D chromatin interaction data  and geno project . We estaMIR regulation . We began our analysis with the identification of putative MIR promoters using integrative data sources and prediction methods (see below). We then used ChIA-PET with RNAPII peak data and ChIP-seq of histone modifications and DNA methylation data to characterize the chromatin features of MIRs. We also correlated the chromatin status with gene expression data, and examined the expression patterns between MIRs and their nearby protein-coding genes. Next, we developed a statistical model to assign MIRs into different chromatin interaction models based on ChIA-PET interaction data, and thus obtained a global MIR–MIR interaction network. Finally, we integrated RNA-seq data and disease information to systemically uncover the relationship between chromatin organization, cell-specific MIR regulation and disease biology.The huge genome-wide data sets from the ENCODE project  provide http://encodeproject.org/ENCODE/cellTypes.html for detailed information). The data sources used in this analysis are available in Supplementary Table S1 and summarized as below.In this study, we focus our analysis on K562 (chronic myelogenous leukemia) and MCF7  cell lines  protein-coding genes with HGNC (symbol from the HUGO Gene Nomenclature Committee) symbols were downloaded from Ensembl  and RefSeq database (http://www.ncbi.nlm.nih.gov/RefSeq/). The gene ontology (GO) annotation and ID mapping data were retrieved from Ensembl using the BioMart tool. The best-curated list of known disorder–gene associations was obtained from Online Mendelian Inheritance in Man database . We only considered entries with the ‘  and NCBI/GEO (GSE33664). The ChIA-PET data can be used to simultaneously identify protein binding sites and chromatin interactions in a whole-genome, de novo and unbiased manner . The mapped files (in bam format) were directly downloaded from the ENCODE Web site and were subjected to expression estimation. The expression levels in FPKM (fragments per kilobase of exon per million fragments mapped) estimated by Cufflinks  using small RNA-seq data.ufflinks  were aveEpigenetic modification data. DNA methylation data as well as ChIP-seq data for histone modifications were also retrieved from the ENCODE data repository site (http://genome.ucsc.edu/ENCODE/). To characterize the chromatin features, such as histone modifications, transcription factor bindings, DNase I hypersensitive sites and DNA methylation, for the transcriptional regulation of MIRs, we used the epigenetic mark data to measure their profile around MIR promoters. For each location , the number of peaks or tags within ±5 kb from the center of the locations were counted.Hi-C data. The processed topological domains for each chromosome were obtained from a recent Hi-C study (http://chromosome.sdsc.edu/mouse/hi-c).-C study , which pmiRNA-target interaction data. Predicted targets of miRNAs were retrieved from six databases: TargetScan  by integrating three different data sources . Firstly, the predicted TSSs were retrieved from the miRStart database (http://mirstart.mbc.nctu.edu.tw/)  . Previous studies have shown that H3K4me3 can be considered as a useful chromatin mark for identifying active MIR promoters  showed shared promoters with nearby protein-coding genes. Several examples can be found in Supplementary Figure S3. The full list of MIR TSSs is used for further analysis and provided in Supplementary Data Set S1. The RNAPII binding peak intensity within ±2 kb from the MIR TSS site is available as Supplementary Data Set S2.We annotated the edu.tw/) , which sAGE tags  from theFANTOM4) . This lePET data , which ita . Some detected ChIA-PET interactions may result from cancer-related genome translocation. We, therefore, removed genomic regions that mapped proximal to genomic structural variations in their respective genomes (MIRs) within ±2 kb from their predicted TSS sites to the two interacting anchor boundaries are considered to be chromatin linked to each other. These linked genes form complex interaction networks based on the connectivity of overlapping anchors from one duplex interaction to the others. We did this analysis for each replicate of the samples separately and for each type of cell lines (K562 cell line with three replicates and MCF7 with four replicates). To avoid gene interactions detected by chance, each pair of interacting genes should be recovered by at least two replicates. The whole chromatin interaction network consists of 2292 modules, which were referred to as chromatin communities . The full list of chromatin communities is provided in Supplementary Data Set S3.Because  genomes  before cMIR-related chromatin interaction models, we focus on only MIRs in the whole network. Each interacting MIR pair should be supported by at least two independent replicates, unless they are neighbors on the genome (in which case they should be recovered by at least one replicate), as neighboring interactions are believed to be more reliable to detect in ChIA-PET technology (MIRs (in clusters) prefer to be transcribed together  of which were intrachromosomal and 83.3% of which were supported by at least two replicates. Although most (62.4%) interchromosomal interactions were linked indirectly, they were relatively reliable, as they were supported by at least two independent data sets. We also incorporated RNAPII-associated ChIA-PET peak data to investigate transcriptional regulation of MIRs that are not assigned to the defined interaction models. We assigned all the annotated MIRs to three interaction models based on how the MIRs were involved in the interaction networks: (i) ‘basal transcription’ model in which MIR overlapped with standalone RNAPII peaks in its promoter regions, but did not overlap with any interaction anchors; (ii) ‘MIR-related chromatin interaction’ model that involved a MIR(s) that interacted with other MIRs or protein-coding genes; while (iii) other ‘not assigned’ model in which the promoters of the MIRs were not supported by any RNAPII peaks or interactions . We found 293 (19%) MIRs involved in the basal transcription model and nearly half of the MIRs  in the ‘MIR-related chromatin interaction’ models . The list of MIR-related chromatin interaction models can be found in Supplementary Data Set S4.To define chnology  and adjatogether . It is nSupplementary Data Set S5). However, only 0.2% (160) of the mapped pairs was to arise from within the same communities. We observed that 104 (65%) of these self-interacting pairs were from the giant community, indicating targets show significant underrepresentation within the same community that ‘their’ MIRs come from, compared with that of by chance . To associate significantly overrepresented chromatin community interactions with the mapping of miRNA–target pairs, two communities were considered to be linked if there were at least 10 mapped miRNA–target interactions between them. The statistical significance of their interactions was tested by a hypergeometric test corrected for multiple comparison by a false discovery rate (FDR) at 0.001 level. The criterion of at least 10 mapped miRNA-target pairs between two communities is based on the observation that each target in the chromatin interaction network was predicted to be targeted by 10 MIRs on averages. With an extension analysis, we relax this criterion , and performed the analysis analogous to that shown in We outlined the strategy in 3 independent randomizations for each analysis. The Mann–Whitney U test was used to test the significance of distributions between the random control and observed data sets, and to calculate the P-value. Specifically, (i) to obtain the random control of the distributions of miRNA–target pairs between and within communities in MIRs). We then calculated the percentage of genes targeted by MIRs between or within communities. (ii) To assess the significance of function similarity between communities linked by miRNA–target pairs in MIR–MIR interactions from the same family or disease category in MIR interactome and then calculated the distribution. FDR values were calculated from the 103 random data sets as the probability of random values significantly greater than observed values (with the Mann–Whitney U test P < 0.001).We performed several random control analyses during this study. The basis was to extract the same number of genes/interactions chosen randomly from the same background sample, and then to calculate similar properties. To obtain significant statistics, we performed 10http://www.r-project.org/; release 2.14.1). GO term enrichment analysis was performed using hypergeometric test corrected for multiple comparison by the Benjamini–Hochberg method (FDR < 0.05), implemented in GO::TermFinder software   was usedMIRs using RNAPII-associated ChIA-PET data . Based on searching RNAPII peaks within 50 kb upstream of pre-miRNAs, we identified 180 novel putative MIR promoters , which were not supported by other public databases (MIRs with predicted promoters (Supplementary Data Set S1). From the combined data sets from K562 and MCF7 cell lines, we found that nearly two-thirds  of the annotated MIRs showed high-confidence RNAPII binding sites around their predicted promoter regions . Most of these MIRs associated binding peaks are enriched around the predicted transcription start sites (TSSs) of the MIRs , indicating that most, if not all, intragenic MIRs are co-transcribed with their host genes . For some intergenic MIRs, they have their own promoters for transcription . Some intergenic MIRs share common promoters with their nearby protein-coding genes, suggesting co-transcription . Notably, MIRs and nearby genes with opposite directions of transcription showed higher correlation of RNAPII binding distributions (r = 0.62) as compared with those with identical directions , suggesting the importance of the bidirectional arrangement of promoters as a regulatory mechanism within the human genome . The higher the RNAPII occupancy, the higher the MIR expression level tends to be . Besides, we observed that MIRs and their host (for intragenic MIRs) and nearby protein-coding genes (for intergenic MIRs) had coordinated expression output .Next, we determined whether m ENCODE  and obseMIRs could also be subject to epigenetic control at the chromatin level, similar to that seen for protein-coding genes. We observed high enrichment of active chromatin marks, such as histone modifications including H3K4me3, H3K4me2, H3K4me1 as well as histone acetylation and histone variant H2A.Z, within MIR promoter regions, that correlated well with RNAPII binding and expression output . By contrast, when these marks were weakened or totally lost, the binding peaks and expression levels changed accordingly.In addition to transcriptional regulation directed by RNAPII, MIRs is just tracking the expression of their host protein-coding genes being regulated by the epigenetic factors, we performed association analysis for intergenic and intragenic MIRs separately. We observed that the overall patterns are consistent ; Robert Bosch Stiftung ; Henry Lester Trust ; Fundamental Research Funds for the Central Universities [M.C. and L.Y.F.].Funding for open access charge: Conflict of interest statement. None declared.


Asp synthetase (GluQ-RS) is a paralog of the catalytic domain of glutamyl-tRNA synthetase and catalyzes the formation of glutamyl-queuosine on the wobble position of tRNAAsp. Here we analyze the transcription of its gene in Shigella flexneri, where it is found downstream of dksA, which encodes a transcriptional regulator involved in stress responses.Glutamyl queuosine-tRNAdksA-gluQ-rs, is conserved in more than 40 bacterial species. RT-PCR assays show co-transcription of both genes without a significant change in transcript levels during growth of S. flexneri. However, mRNA levels of the intergenic region changed during growth, increasing at stationary phase, indicating an additional level of control over the expression of gluQ-rs gene. Transcriptional fusions with lacZ as a reporter gene only produced β-galactosidase activity when the constructs included the dksA promoter, indicating that gluQ-rs do not have a separate promoter. Using bioinformatics, we identified a putative transcriptional terminator between dksA and gluQ-rs. Deletion or alteration of the predicted terminator resulted in increased expression of the lacZ reporter compared with cells containing the wild type terminator sequence. Analysis of the phenotype of a gluQ-rs mutant suggested that it may play a role in some stress responses, since growth of the mutant was impaired in the presence of osmolytes.The genomic organization, gluQ-rs depends on the dksA promoter, and strongly suggest the presence and the functionality of a transcriptional terminator regulating its expression. Also, the results indicate a link between glutamyl-queuosine synthesis and stress response in Shigella flexneri.The results presented here, show that the expression of  Acidithiobacillus ferrooxidans and Helicobacter pylori (genes gltX1 and gltX2) [Lactococcus lactis, which has similarity with the catalytic domain of histidyl-tRNA synthetase, is involved in histidine biosynthesis [Salmonella enterica has shown that PoxA, encoded by poxA/genX, has similarity to the carboxy-terminal catalytic domain of lysine-tRNA synthetase and is required for posttranslational aminoacylation of bacterial elongation factor P. A poxA mutant has reduced colonization and virulence, possibly due to misregulated expression of proteins encoded by the SPI-1 pathogenicity island [The fidelity of the translation process depends on the aminoacyl–tRNA synthetase enzymes (aaRS). These essential enzymes are responsible for the correct attachment of the corresponding amino acid onto the cognate tRNA, therefore organisms have at least 20 synthetases . The enzd gltX2) ,5. aaRS d gltX2) . Some ofynthesis . A receny island ,9.Escherichia coli glutamyl-tRNA synthetase paralog, glutamyl queuosine-tRNAAsp synthetase (GluQ-RS) has approximately 35% amino acid similarity with the catalytic domain of GluRS. This includes the amino acids involved in recognition and activation of glutamate. Although GluQ-RS is missing the carboxyl-terminus domain responsible for the tRNA recognition, in E. coli this enzyme is able to activate the amino acid in the absence of the tRNA. Further, once the aminoacyl-adenylate has been formed, the enzyme attaches the glutamate to the nucleoside queuosine present onto the tRNAAsp. Therefore, this enzyme is involved in the synthesis of a new modified nucleoside glutamyl-queuosine (GluQ) present in tRNAAsp[E. coli in rich or minimal media [Asp, tRNAAsn, tRNAHis and tRNATyr[E. coli only tRNAAsp is a substrate for the GluQ-RS enzyme.An n tRNAAsp,11. Thisal media . Queuosid tRNATyr; howeverAspQ34 might improve recognition of both GAC and GAU codons [Shigella flexneri it has been shown that mutations in genes required for tRNA modifications, miaA and tgt decreased virulence. miaA is required for 2-methylthio-N6-isopentenyladenosine modification at position 37 of the anticodon loop and tgt is involved in queuosine modification at position 34 within the anticodon loop [gluQ-rs gene in the major human pathogen, S. flexneri.The presence of modifications within the anticodon loop of the tRNA, could enhance the accuracy of the codon binding . Then thU codons  and stimU codons . In Shigdon loop -18. In tAsp[http://www.uniprot.org/), we were able to identify GluQ-RS in more than a hundred bacterial species, primarily proteobacteria . Further genomic analysis indicated that the gluQ-rs gene is found primarily in two genomic arrangements, either alone or located immediately downstream of dksA. Searching within the String database [dksAgluQ-rs gene organization was conserved in more than 40 different species, all of which were within the gammaproteobacteria group. These included species of Aeromonadales, Alteromonadales, Enterobacteriaceae, including E. coli and S. flexneri, Pseudomanadales, and Vibrionaceae  at pH 7.4. The analysis of the dksA and gluQ-rs transcripts shows that for both mRNAs, the level is stable during the growth curve, with an increase of 1.3-fold at stationary phase compared to the early mid log phase . In order to confirm those results, a transcriptional fusion strategy was used. Different segments of the operon were cloned and fused to the lacZ reporter gene in pQF50, and promoter activity was assayed by β-galactosidase activity [dksA. By mean of bioinformatics tools, including BPROM from the Softberry software package (http://linux1.softberry.com/berry.phtml), we identified those promoters in S. flexneri and included all three promoters in the constructs indicated in Figure dksA promoters to the beginning of the gluQ-rs gene, with the first five amino acids of GluQ-RS, named pVCPDT, represents the full length dksA gene with its native promoters  than the plasmid containing the wild type sequence. This result suggested that the intergenic region upstream of gluQ-rs contains a transcriptional terminator.In order to explain the difference observed in expression of ng mFold  was perfr Figure . In order Figure . As showEscherichia fergusonii that also have the terminator structure, a methionine is not present at that location. In the S. flexneri sequence, there is another AUG codon in the same reading frame 27 nucleotides downstream from the one in the terminator. In order to determine which methionine is the start site for translation of the S. flexneri GluQ-RS, we constructed a vector that included the intergenic region from the stop codon of the dksA gene to the end of gluQ-rs cloned into the expression vector pET15c. This allowed expression of C-terminal His-tagged GluQ-RS under T7 promoter control. The protein was partially purified by affinity chromatography as described elsewhere [2-T-D-T-Q-Y-I-G-R-F-A-P, which corresponds to the amino acid sequence after the second methionine. Therefore, the initiator methionine is not the one indicated in the database, and the protein is 298 amino acids. Surprisingly, there is no obvious Shine-Dalgarno sequence adjacent to the initiator methionine we identified . The major difference observed for the mutant was impaired metabolism when grown under osmotic stress conditions , suggesting that GluQ-RS is not essential for invasion or intracellular growth of S. flexneri.Because expression of irulence , and groAsp in bacteria. The protein is present in Firmicutes, Actinobacteria, Cyanobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria  by isoleucine, leucine or valine at that position (Ile47 of S. flexneri GluQ-RS). This substitution is also conserved in all enzymes analyzed here, including those from the Firmicutes group.GluQ-RS, a paralog of GluRS synthetase, is involved in the formation of GluQ, the nucleoside located at the wobble position of tRNAa Figure . From thgluQ-rs gene is widely distributed in the bacterial domain; however, its genome organization is variable. We observed that only in members of the gammaproteobacteria, namely Aeromonadales, Alteromonadales, Pseudomonadaceae, Enterobacteriaceae and Vibrionaceae, the gluQ-rs gene is located immediately downstream of the dksA gene , indicating that the expression of the gluQ-rs gene may be under control of its own promoter, a question that remains to be addressed.The e Figure . A more gluQ-rs-specific promoter. This is consistent with our results, in which we did not detect any activity from promoters other than those upstream of the dksA gene , we cannot discount the possibility that the gene is specifically regulated under some other conditions. The regulon database (http://regulondb.ccg.unam.mx/index.jsp) indicates that the E. coli gluQ-rs gene has a recognition site for the σ24 subunit of RNA polymerase. From our analysis, this sequence is identical to S. flexneri, but there is no experimental evidence of this recognition. Interestingly, when the gluQ-rs gene was deleted in S. flexneri, the mutant showed impaired growth in the presence of osmolytes , we didAspQ34 might improve recognition of both GAC and GAU codons, consequently the interaction of the codon GAU with the anticodon of tRNAAspG34 could be less efficient. In fact, in S. flexneri there are a few genes such as sitA, virF and proX (an inducible gene under osmotic stress) that have a bias toward those codons that favor the modified tRNA. Thus, while there is no obvious loss of plaque formation in the gluQ-rs mutant (data not shown), the absence of GluQ-RS may influence the expression of proteins such as SitA that are required for fitness of Shigella in the host [tRNA modifications present at the anticodon loop might be important for the accuracy of codon reading during the translation processes . Morris the host .gluQ-rs, a gene codifying an enzyme involved in the formation of GluQ present on the tRNAAsp, is under the control of the dksA promoter. Also, we show the presence of a functional terminator that controls the expression of gluQ-rs. Finally, we present data that suggest that the presence of modification of the tRNAAsp is important for survival of the human pathogen Shigella flexneri under osmotic stress conditions.In this work we have shown that the expression of E. coli strains were maintained on LB-agar , Shigella strains were maintained on Trypticase Soy Agar plus 0.01% congo red. All strains were stored at −80°C in LB broth plus 20% glycerol. The bacteria were grown in LB broth adjusted to pH 7.4 with 40 mM MOPS (Merck) or M9 minimal media [600).The bacterial strains and plasmids used in this study are described in Table al media . When nehttp://www.uniprot.org/) and then were searched in the GenomeNet (http://www.genome.jp/) to confirm the genomic organization. A selected number of GluQ-RS enzymes were aligned using the MUSCLE algorithm [Methanocaldococcus jannaschii and Archaeoglobus fulgidus as an outgroup. All positions containing gaps and missing data were eliminated. There were a total of 199 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 [The protein sequences were obtained from the Uniprot database . The purified nucleic acid was treated with RNase- free DNase (Fermentas) and its concentration was estimated by measuring the optical density at 260 nm (OD260). Approximately 1 μg of total RNA was subjected to reverse transcription using M-MuLV polymerase (Fermentas) and random primers following the provider’s protocol. The cDNA was amplified using specific PCR primers for each gene of interest . The 1,110 bp amplified fragment was cloned in the vector pTZ57R/T and sequenced to verify the mutation. This plasmid was digested with BamHI and HindIII and the fragment subcloned in to the vector pQF50.A possible transcription terminator between am Mfold . Site diam Mfold . Using tgluQ-rs, the recombinant plasmid pATGGQRS was constructed. A PCR reaction was performed using the primers ATGGQRSF and ATGGQRSR  strain and the His-tagged protein was partially purified by affinity chromatography as described previously [In order to establish which is the first AUG codon of SR Table  and genoeviously . The eluBglII and NcoI enzymes and replaced by the TRC promoter obtained from pTRC99a plasmid by amplification and digestion with BamHI and NcoI to obtain the pTRCGQ plasmid. The empty plasmid (pCM) was constructed by incorporating the TRC promoter into the pET15c plasmid.This plasmid was constructed from the pATGGQRS plasmid in which the T7 promoter was removed by digestion with gluQ-rs was carried out using the λ red recombinase method [S. flexneri 2457T carrying pKD46 and prepared as described elsewhere [E. coli ΔgluQ-rs::kan mutant strain using primers dksAF and pcnBR . Strains were grown at 30°C overnight and 5 ml of LB was inoculated with a 1:100 dilution and grown at 37°C to reach an OD650nm of 0.5. The cells were then washed and resuspended to 2.5 x 107 cfu/ml and diluted 200 fold in to a solution of IF-10a medium (Biolog). Each well was inoculated with 1.2 x 104 cfu (0.1 ml per well) into the corresponding plates and incubated for 24 hrs at 37°C. The metabolism was recorded and analyzed by the Omnilog software (V 1.20.02) .The effect of the deletion of the Asp synthetase and its codifying gene; tRNAAsp: Transfer ribonucleic acid codifying aspartic acid; GluQ: Glutamyl-queuosine; aaRS: Aminoacyl–tRNA synthetase; GluRS: Glutamyl-tRNA synthetase; Q: Queuosine; SPI-1: Pathogenicity islands of Salmonella; miaA: tRNA Δ(2)-isopentenylpyrophosphate transferase; tgt: tRNA guanine transglycosylase; RT-PCR: Reverse transcription-polymerase chain reaction; LB: Luria Bertani; MOPS: 3-(N-morpholino)propanesulfonic acid; M-MuLV: Moloney Murine Leukemia Virus Reverse Transcriptase; PVDF: Polyvinyl difluoride.GluQ-RS: Glutamyl queuosine-tRNAThe authors declare that they have no competing interests.ex vivo assays of the wild type and mutant strains of Shigella and the enzymatic activity under different growth conditions. CM: performed the RT-PCR of the mRNA isolated from Shigella flexneri. CRF: performed the Biolog assay in collaboration with JCS. JCS: Analysis of the data, experimental design, N terminal determination of GluQ-RS. SMP and JCS: analyzed the results and wrote the paper. All authors contributed to the editing and approved the final paper.VCC: performed cloning, the enzymatic assay, data analysis. VPT: performed


Though plant Ca2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.Plasma membrane Ca2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The −1478 to −886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for −1210 and −886 bp flanking region. The −1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The −1210 and −886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the −886 bp and −519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.The 1478 bp promoter sequence of rice plasma membrane Ca2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology.The rice plasma membrane Ca Second messengers are involved in phosphorylation cascade which ultimately leads to activation of transcription factors controlling sets of stress regulated genes.Abiotic stress  is the most harmful factor for growth and productivity in crops worldwide. These problems increase consistently due to climate change. Many researchers have obtained transgenic plants with improved stress tolerance by overexpressing genes with stress-protecting functions 2+ is required in almost all the stages of plant growth and development, playing a fundamental role in regulating polar growth and participating in plant adaptation to various stress factors 2+ plays crucial roles in plant membrane stability, cell wall stabilization, and cell integrity 2+2+/CaM). It has been well evidenced that various environmental conditions and hormone signal molecules as light stress, low temperatures, salt, alkali, gibberellins, or abscisic acid (ABA) can trigger alteration of cytosolic Ca2+ concentrations, leading to different plant adaptation responses 2+ATPases are members of p-type ATPase superfamily involved in the restoration and maintenance of ion homeostasis by pumping calcium ions out of the cytosol in all eukaryotic cells. Plant p-type Ca2+ATPases have been divided into two groups, type IIA and IIB. The latter contains an N-terminal autoinhibitory domain that binds to calmodulin and activates the Ca2+pump As a second messenger of paramount significance, Ca2+ATPases and the expression levels of different plant Ca2+ATPase genes  were shown to be up-regulated under salinity stress, Arabidopsis ACAs, type IIB Ca2+ATPases, may be involved in cytosolic Ca2+ signal shaping in response to several stresses. For example, AtACA8 was found to be up-regulated, whereas AtACA10 was down-regulated in response to cold stress 2+ATPase plays key functions in the development of cold resistance in winter wheat 2+ATPase under low temperature stress, indicating that the Ca2+ATPase activity was mainly localized at the plasma membrane in wheat seedling cells growing at normal temperatures 2+ATPases present Ca2+/CaM binding regions, contributing to ABA-induced drought signal transferring under PEG stress, since ABA synthesis was related with cytoplasmic Ca2+ concentrations 2+ in guard cells, including Ca2+ influx across the plasma membrane Arabidopsis, the expression of ACA8 and ACA9 genes might be stimulated by ABA, suggesting for an indirect role of plant Ca2+ATPases in stress signalling 2+/CaM messenger system was involved in controlling stress resistance in rice seedling, blocking messenger transduction, drought resistance, salt resistance and decreasing chilling resistance 2+ treatment increased protection against membrane lipid peroxidation and membrane stability and therefore resulted in the increase of drought resistance in rice seedlings 2+ appeared to reduce the devastating effects of stress by elevating the content of proline and glycine betaine, thus improving the water status and growth and minimizing the injury to membranes 2+ plays important roles in plant responses to drought resistance. Previously, Romani 2+ATPase inhibitor) prevented both the increase in Ca2+ efflux and the transient reactive oxygen species (ROS) accumulation in Egeria densa in response to ABA treatment. This result was explained by assuming an important role of PMCa2+ATPase in switching off the signal triggering ROS production. Another report from the same group implicated PMCa2+ATPase activation in plant adaptation to osmotic stress 2+ATPase not only regulates plant development and abiotic stresses, but also protects plant from pathogens by activating salicylic acid (SA)-mediated programmed cell death (PCD) pathways 2+ATPases in shaping cytosolic Ca2+ signatures under abiotic and biotic stresses.The central role in calcium signalling seems to be played by Ca2+ATPases in the plant ability to tolerate abiotic stress, it is desirable and feasible to exploit stress-inducible promoters to drive the expression of relevant transgenes. In this study, we isolated and analyzed a stress inducible promoter OsPMCa2+ATPase from rice and investigated it with regards to tissue specific expression pattern and relative expression activities, using transgenic analysis in tobacco under different stresses. We also identified the shortest promoter region by making random deletion and found promoter regions sufficient for tissue specific expression and stress induced expression activity. This promoter drives high levels of transgene expression under abiotic stress conditions and will be useful for the development of stress tolerant transgenic plants.Considering the key role played by Cahttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/, 29) and PLACE  were used to determine the cis-acting regulatory elements and to analyze the OsPMCa2+ATPase promoter sequences (http://rice.plantbiology.msu.edu).DNA sequences were analysed by using DNAMAN software, while PLANT CARE (Oryza sativa (Var. IR 64) by CTAB method and used as template for PCR amplification of OsPMCa2+ATPase promoter. Sequences of DNA adaptors and primers used for promoter amplification are provided in 2+ATPase promoter sequence. To construct the various promoter deletions of OsPMCa2+ATPase promoter with GUS fusion products, a PCR series was carried out with four primer pairs, F/RS, D1/RS, D2/RS and D3/RS , D1, D2, and D3. In addition, the CaMV35S promoter was used as positive control and wild-type tobacco as negative control, in order to determine OsPMCa2+ATPase promoter activity.Genomic DNA was extracted from leaves of nd D3/RS , respectNicotiana tabacum cv. Xanthi) leaf discs were transformed using a standard procedure as described earlier Agrobacterium tumefaciens (LBA4404) containing promoter-GUS (β-glucuronidase) fusion constructs in pCAMBIA-1391Z. Primary transgenic explants were grown in tissue culture chamber at 26°C under a 16 h light/8 h dark cycle. The transgenic plants were screened for integration of the intact promoter-GUS chimeric gene into the genome by PCR. PCR products were analysed on 1% (w/v) agarose gel. Total genome DNA was isolated from leaves of hygromycin resistant tobacco plants using the CTAB method. PCR analysis was carried out using F/RS, D1/RS, D2/RS and D3/RS primers, hygromycin specific primers and GUS specific primer pair  and methyl viologen (MV) treatments were chosen. Flowers, stems, leaves and roots of transgenic tobacco were cut into pieces and subjected to soaking in Petri dishes filled with 200 mM NaCl, 20% PEG, 100 µM ABA, 2 mM SA, 200 µM MeJA and 100 µM MV solution or sterilized water, for 24 h at room temperature. For cold stress, tissues were incubated at 4°C for 48 h. Tissues in absence of stress were used as control. All the above treatments were carried out under a growth regime of 16 h light/8 h dark at 20±1°C unless otherwise mentioned.Leaves, shoots, roots and flowers were vacuum infiltrated for 1 h in the GUS reaction mixture containing 2 mM 5-bromo-4-chloro-3-indolyl- β-D-glucuronide (X-Gluc) and 100 mM sodium phosphate buffer as described by Jefferson 2+ATPase promoter induced by various stresses was studied using transgenic tobacco seedlings. Fluorometric analysis of GUS activity was performed using 4-methylumbelliferyl-b-glucuronide (4-MUG). The extracted proteins were mixed with GUS assay buffer . The addition of the stop buffer (0.2 M Na2CO3) halted the reaction. Next, 4-MUG was hydrolyzed by GUS to produce 4-methylumbelliferone fluorochrome (4-MU). GUS activity was determined in triplicate with a microplate spectrofluorometer . The excitation wavelength was 365 nm and the emission wavelength 455 nm. Protein concentration was determined using Bradford assay, according to the manufacturer’s instructions.The behavior of OsPMCa2+ATPase promoter was isolated from Oryza sativa genomic DNA using the OsPMCa2+ATPase specific primer sets  was cloned at HindIII and BamHI sites of promoter less vector, pCAMBIA1391Z  (2+ATPase promoter deletions −1210 bp and −886 bp are sufficient to drive gene expression.To identify the expression profiles of transgenic plants driven by the four OsPMCa−519 bp) . The res2+ATPase gene expression, its promoter activity was studied in tobacco transgenic plants submitted to drought, salt and cold treatments. The presence of abiotic-stress-responsive elements inside the promoter sequence encouraged the research. In consequence, GUS activity was measured through fluorometric assay. Distinct GUS expression was evidenced for each of the four  OsPMCa2+ATPase promoter segments (GUS activity) deletions in shoots  coiling, flowering, seed and fruit maturation. An increase in MeJA levels affects flowering time, flower morphology and the number of open flowers. MeJA induces ethylene-forming enzyme activity, which increases the amount of ethylene necessary for fruit maturation. Increased amounts of methyl jasmonate in plant roots have shown to inhibit their growth. Treatments with MeJA for 24 h proved sufficient to trigger nstructs . The MeJf leaves . Deletio to MeJA . Fluorom by MeJA . The varGUS staining experiment by razorblade section was used for tissue-specific expression of OsPMCa2+ATPase promoter, two month-old transgenic containing full-length and its deletions tobacco plants were used. Transgenic plants driven by the CaMV35S promoter, and wild-type tobacco, were the respective positive and negative controls. Blue points were detected in vascular bundles, mesophyll and cortex of leaves; vascular bundles, pith and cortex of stems; and vascular bundles, tips and caps of roots of transgenic tobacco driven by the CaMV35Spromoter  construct still showed the strongest GUS expression activity of the four OsPMCa2+ATPase promoter constructs examined.A promoter . No GUS promoter  was obse2+ATPase full promoter and deletions were used. In some deleted segment low expression was found for corolla, calyx and stalks  is required for binding to nuclear factor(s) in response to pathogen or salt-induced stress 2+ATPase promoter which imply that a GT-1-related transcription factor positively regulates OsPMCa2+ATPase gene expression under the conditions of pathogen attack or NaCl stress. In our result we also identified some GT-1 element at −519 to −1 bp but under salt stress these segments did not respond, indicating that this GT-1 element may not be sufficient for OsPMCa2+ATPase promoter activation by salt treatment. Only −1210 bp promoter segments responded in leaves and shoots of tobacco transgenic plants submitted to MV treatments. Oxidative damage in plants caused by MV may be due to the excess generation of superoxide radicals, which are normally detoxified to oxygen and hydrogen peroxide (H2O2) by superoxide dismutase OsPMCa2+ATPase promoter region. The −519 bp did not respond to ABA treatment in case of leaves and shoots as we found no cis-regulatory element in this region. The −593 bp deletion construct did not respond to ABA treatment 2+ATPase promoter region. The −1210 to −886 bp is sufficient for MeJA-induced GUS activity in OsPMCa2+ATPase promoter. However, the −519 bp deletion drastically reduced jasmonic acid-responsive promoter activity in leaves but not in shoots and roots. This may be due to the fact that some inhibitory elements might be present in this region. Previous studies have implicated a number of different types of regulatory elements in conferring MeJA-responsiveness in plant promoters. Some shares sequences containing the TGACG (or its inverse CGTCA) motif, as in the case of the AS-1-type element in the glutathione S-transferase gene Lox l) gene 2+ATPase promoter region located at −1261 bp. It had been suggested to bind with SA-dependent and pathogen-induced transcription factors WRKY and TGA The plant growth regulators appear to play a predominant role in the conversion of environmental signals into changes in plant gene expression 2+ATPase promoter was a phloem-specific promoter. It is interesting that in some deleted fragment GUS activity did not occur in xylem but only in phloem, so long as the cambium was formed. Moreover, root sections showed that staining was not only in the vascular bundle but also in the root tip meristematic zone. It was also interesting that GUS staining was weekly detected in root cap, which differed to results for the CaMV35S promoter. The mechanism behind this is still unclear. The members of the DOF TF family have been found to control vascular tissue-specific gene expression by binding to the core recognition sequence CTTT 2+ATPase promoter. Consequently, this confirmed that the motif ‘CTTT’ and its homologous sequences determined the promoters’ phloem-specific expression pattern. Other conserved motif like ATAAGAACGAATC also involved in the phloem strength and specificity was identified by Hehn 2+ATPase promoter has role in survival adaptability by responding to stresses and hormones.Transverse section of the stems and petioles indicated that the OsPMCa2+ATPase promoter showed very high level of GUS expression in anther, stigma as well as other floral organs upon treatment with NaCl, drought, ABA and MeJA, while relatively low expression was evidenced in case of cold-induced stress. No anther and stigma specific activity was observed under MV treatment. Surprisingly, none of the deleted fragments showed any expression for anther and stigma, while varied expression was found in corolla, calyx and stalks. This might be due to the presence of repressor elements located outside the deleted fragment, which could block the activity in anther and stigma. This is the first report where OsPMCa2+ATPase promoter conferred anther-specific transgene expression under various abiotic and hormonal stresses. The results presented here suggest that the OsPMCa2+ATPase promoter may play an important signalling and/or defensive role during the flowering stages. Previously it was reported that MeJA induced flower specific expression in Tomato Prosystemin Promoter ArabidopsisTo attain anther-specific transgene expression, an anther specific promoter is necessary. Full-length OsPMCa2+ATPase promoter expression in tobacco leaves. Induction of disease resistance-related plasma membrane proteins by plant hormones has not been reported. Cold-regulated plasma membrane protein genes are induced in wheat and rice by ABA treatment 2+ATPase promoter may be efficient at mediating and enhancing plant defense responses against abiotic stresses.Plasma membrane proteins are involved in the recognition and transduction of endogenous hormonal signals 2+ATPase promoter from Oryza sativa is a reproductive organ-specific as well as vascular-specific promoter. The OsPMCa2+ATPase promoter contains drought, salt, cold, MV, ABA and MeJA related cis-elements, which regulated gene expression in roots. It was also revealed that in the leaves these cis-elements might exist outside the region contained in the −519 bp deletion construct. Overall, the tissue-specificity and inducible nature of OsPMCa2+ATPase promoter could grant wide applicability in plant biotechnology. Abiotic stress tolerance in rice is a complex trait, and single transgene introduction may not be sufficient to impart stress tolerance under field conditions. Further strategies involving rice genetic transformation with multiple transgenes expressed in an inducible manner will help to improve its stress tolerance. In consequence, the promoter analyzed in the present study could be of great use to drive transgenes based on expression pattern and extent of required inducibility.In the present study we demonstrated by histochemical analysis that the full-length OsPMCaFigure S1PCR analysis to detect the presence of different promoter deletions in transgenic tobacco. A) PCR of full-length promoter B) PCR of D1 promoter deletion C) PCR of D2 promoter deletion and D) PCR of D3 promoter deletion.(PDF)Click here for additional data file.Figure S2GUS localization and intensity in floral parts of transgenic transformed with different deleted promoter segments.A) GUS localization and intensity in tobacco flowers/florets for D1. B) GUS localization and intensity in tobacco flowers/florets for D2. C) Reproductive organs GUS localization and intensity in tobacco flowers/florets for D3. Flower parts were stained with GUS staining solution to study the localization and intensity under different stress condition. Details of stress treatments are described in (PDF)Click here for additional data file.Table S1List of primers used in this study.(PDF)Click here for additional data file.


Pax6-expressing progenitors isolated from Pax6+/+ and Pax6−/− cortices and identified many negatively regulated cell-cycle genes, including Cyclins and Cdks. Biochemical assays indicated that Pax6 directly represses Cdk6 expression. Cyclin/Cdk repression inhibits retinoblastoma protein (pRb) phosphorylation, thereby limiting the transcription of genes that directly promote the mechanics of the cell cycle, and we found that Pax6 inhibits pRb phosphorylation and represses genes involved in DNA replication. Our results indicate that Pax6’s modulation of cortical progenitor cell cycles is regional and direct.The mechanisms by which early spatiotemporal expression patterns of transcription factors such as Pax6 regulate cortical progenitors in a region-specific manner are poorly understood. Pax6 is expressed in a gradient across the developing cortex and is essential for normal corticogenesis. We found that constitutive or conditional loss of Pax6 increases cortical progenitor proliferation by amounts that vary regionally with normal Pax6 levels. We compared the gene expression profiles of equivalent  •Pax6 loss increases cortical progenitor proliferation by region-specific amounts•The size of this effect correlates directly with normal Pax6 expression levels•Expression of many key cell-cycle regulators is increased in the absence of Pax6•Pax6 directly represses Cdk6 expression and controls pRb phosphorylation The transcription factor Pax6 regulates multiple aspects of forebrain development. Mi et al. show that it exerts regional control of cortical cell proliferation. It inhibits cell-cycle gene expression, directly repressing Cdk6, and limits the activity of the pRb-E2F pathway. Understanding how transcription factors that regulate specific spatiotemporal patterns of gene expression control cellular development remains a major challenge. Although many transcription factors essential for tissue specification have been discovered and cellular processes that they influence have been identified, how they operate is largely unknown. Current evidence indicates that these transcription factors control large gene regulatory networks comprising many, possibly thousands, of target genes, some of which encode other transcription factors . The numPAX6 result in eye defects and neurological abnormalities linked to structural alterations of the brain  progenitor cells, including those of the mammalian cerebral cortex . In humahe brain . PAX6 hahe brain . Mice wi defects . Several defects . ThroughOur first aim was to examine the nature and significance of Pax6’s regulation of cortical progenitor proliferation by examining mouse models with either complete or conditional loss of Pax6 function. Previous studies have suggested that gradients of Pax6 expression present across the cortex at early stages of corticogenesis are important for its regionalization in terms of later differentiation , but whePax6-expressing cortical progenitors depend on whether these cells express Pax6 protein or not, we isolated Pax6-expressing progenitors using a line of reporter mice carrying a YAC transgene (DTy54) that expresses GFP under the control of PAX6’s regulatory elements , Pax6 is expressed in a gradient across the embryonic cortex with high levels rostrolaterally and low levels caudomedially . We used of Pax6 G–1I. In Pax6. Their genotypes were as follows: (1) Pax6loxP/loxP; Emx1-CreERT2; R26R-YFP (inducible knockout [iKO] embryos); and (2) Pax6loxP/+; Emx1-CreERT2; R26R-YFP embryos  by E12.5 . In rostral and central-lateral areas , the mean Tc was longer than in central-medial and caudal areas in controls  and was significantly reduced in iKOs  loss of Pax6, since the rapid onset of a defect would strengthen the possibility that Pax6 influences the cell cycle directly. We analyzed mice carrying a tamoxifen-induced, cortex-specific deletion of by E12.5 J and 1K. in iKOs L–1O. TheR26R-YFP allele occurred within 48 hr  of control values within 48 hr of E10.5 tamoxifen administration, and to 9.8%  and 1.6%  by 72 hr and 96 hr, respectively. Immunohistochemistry showed no obvious loss of Pax6 protein from the cortex 48 hr after tamoxifen administration  or E13.5 (iKOE13.5tamox) are shown in E10.5tamox embryos, i.e., shortly after loss of almost all Pax6 protein, increases in the numbers of cells in S phase occurred specifically in the rostral cortex . This proved to be true . Interes embryos . It may cortices . We founl cortex L, but 2 e cortex M. In iKOl cortex P and 2Q.The results of these experiments indicate that during corticogenesis, Pax6 exerts a repressive action on the proliferation of progenitors, and the dynamics of the Pax6 expression gradient and rates of progenitor proliferation are correlated. At E12.5, when the Pax6 gradient is steepest, areas of highest expression correlate with regions where Tc is longest. Loss of Pax6 causes shortening of Tc only in these areas. In normal embryos at older ages, the Pax6 gradient becomes progressively more uniform across the cortex, as does Tc, and loss of Pax6 causes shortening of Tc in all areas.Pax6−/− mutants. To do this, we generated litters of mice containing Pax6−/− and Pax6+/+ E12.5 embryos that also carried the DTy54 transgene. This GFP reporter can be used to distinguish cells in which the endogenous Pax6 locus is transcriptionally active irrespective of whether it contains a WT or mutant allele  scores ≥ 9.3/10 for Agilent microarray comparisons. In a preliminary statistical analysis of the normalized data, we assigned a p value to the change in expression of each gene using an empirical Bayes moderated t test (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38703). Further quality control assessments are included in We carried out three independent experiments, each of which used samples of mRNA from a different pool of d t test . These pd t test . We idenWe used Gene Ontology (GO) databases to identify the biological processes most frequently associated with the sets of up- and downregulated transcripts. The ten most significantly overrepresented processes for each set are given in Pax6+/+ and Pax6−/− embryos by qRT-PCR , Cdca7, and Smad2 in control and iKO  cortex 72 hr after tamoxifen administration at E13.5, and, as in the Pax6−/− cortex, we found significant increases in the levels of all four in the iKO cortex , we carried out an additional experiment to test whether the levels of expression of X77 line . Pax6 prX77 line . The levCdk6 coding sequence and focused further work on testing for direct regulation of this gene by Pax6.As a next step toward defining a biochemical pathway through which Pax6 might regulate cortical progenitor cell cycles, we used bioinformatics to identify potential Pax6 binding sites in genomic regions surrounding some of the cell-cycle genes regulated by Pax6. Using a 21 bp consensus binding motif that is known from previous work to be recognized by the Pax6 paired domain P6CON; , 1994b, Cdk6 coding region. These candidate sites were filtered by conservation analysis across five vertebrate species  using the Mulan program. Five putative binding sites  databases, TRANSFAC and JASPAR, and identified 14 putative Pax6 binding sites within the 10 kb genomic regions immediately upstream and downstream of the BS1–BS5; A with atoter has . By aligtes BS1; C.We tested whether BS1–BS5 A could sGab1 and Syt8 that were previously shown to be Pax6 bound and Pax6 nonbound, respectively, were used to generate positive and negative control data  were significantly enriched by amounts similar to or greater than that of the Gab1 positive control (Cdk6 sites (BS1–BS5), but binds to only four of them in E12.5 cortex in vivo.We extracted chromatin from E12.5 cortex to test for binding of Pax6 to the predicted Pax6 binding sites BS1–BS5 in vivo by quantitative chromatin immunoprecipitation qChIP; A and 5B.rol data . Followita  using luciferase assays in cells that do not express endogenous PAX6 (HEK293 cells). We generated a set of eight luciferase reporter constructs to test each site individually C. We firor alone Di. CotraCMV-Pax6 D led to CMV-Pax6 Di. To teCMV-Pax6 A to geneCMV-Pax6 C. The muCMV-Pax6 Di, indicCdk6 transcription start site  individually. Short DNA fragments spanning each of the binding sites were cloned into plasmid pBS1mut-luc (which drives reporter expression and is not itself repressed by Pax6). PCR fragments including BS2 or BS3 were placed immediately upstream of the 2.3 kb promoter in pBS1mut-luc, whereas fragments including BS4 or BS5 were placed downstream of the luciferase coding sequence, mimicking their normal position relative to the art site A. Constrart site B nor BS5Cdk6 construct inhibited E12.5 cortical progenitor proliferation  protein levels (Pax6−/− cortex (We first tested the effects on pRb phosphorylation of transfecting Pax6 nonexpressing cells (HEK293) with increasing amounts of the Pax6 expression construct pn levels A, consisn levels B. Loss on levels A. Becausn levels , we usedn levels A. We the− cortex C. In allPax6−/− embryos by immunohistochemistry  cortex  cortex K. In PaxCdk6 (through binding to sites close to the Cdk6 coding sequence) and Cyclin D1/2 (either directly or indirectly), Pax6 can limit the levels of cyclin/Cdk complexes and hence the phosphorylation of pRb, one of the primary substrates of Cdks in G1 phase progression , which is known to be directly and positively regulated by E2Fs  respond to Pax6 by repressing gene expression. The failure to detect Pax6 binding to BS3 by ChIP suggests low or no occupancy of this relatively distant site by Pax6 in cortical progenitors in vivo, in line with previous work indicating that many potential transcription factor binding sites are unoccupied in vivo . To separate Pax6-expressing cells for gene profiling, we used the DTy54 transgene (Cdk6 (DN-Cdk6) and GFP-expressing construct described previously  Act 1986. For constitutive inactivation of nic line . For conPax6loxP , BAC tra-CreERT2 , and R26R26R-YFP  alleles.ransgene . Electroransgene  using eieviously .IdU and BrdU were used to label proliferating cortical cells as described in −1; Biochemical Dissociation Kit; Worthington) and GFP-expressing cells were sorted by FACS. RNA was extracted from dissociated cells or from telencephalic tissue using QIAshredder Spin Columns and QIAGEN RNeasy Kits; any traces of genomic DNA were removed by on-column DNase digestion. For microarray experiments, the quality of each sample was assessed using the Agilent Bioanalyzer to obtain an RIN of 1–10 . The Agilent Low RNA Input Linear Amplification Kit was then used to produce complementary RNA (cRNA) labeled with either Cyanine 3 (Cy3) or Cyanine 5 (Cy5) fluorescent labels. Labeled cRNA samples were hybridized to Agilent Dual-dye Whole Mouse Genome Arrays (G4122A). After hybridization, arrays were scanned in an Agilent Scanner for Cy3 and Cy5, and images were analyzed using Agilent’s Feature Extraction Software (version 7.1). Normalization and statistical analysis of the microarray data were performed using R-based software (http://www.R-project.org). For qRT-PCR, reverse transcription and real-time amplification were carried out using standard protocols with the primers listed in Telencephalic cells were dissociated with papain  and FunNet Transcriptional Networks Analysis (http://www.funnet.info), using the GO (http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes  databases. To predict Pax6-binding sites, we used TRANSFAC  and JASPAR (http://jaspar.cgb.ki.se) combined with conservation analysis using the Mulan (http://mulan.dcode.org) program. To reduce false positives, the threshold scores of the core motif match and the matrix match were set to 0.8 in the TRANSFAC program. The scanning cutoff was chosen such that the probability of getting a false-positive prediction in sequences of 500 bp length was <5% (fixed type I error). The probability of a given DNA sequence functioning as a cis-regulatory element was set to 80.0% for the JASPAR database.GO analysis was carried out with WebGestalt . Double-stranded oligonucleotide probes containing WT or mutant Pax6 binding sites (Pax6 proteins were obtained from full-length ng sites A were raChromatin was extracted from 20 E12.5 mouse cortices. DNA-protein complexes were precipitated with anti-Pax6 antibody (Covance) or with anti-igG antibody (Abcam). ChIP was performed as described by Cdk6 were isolated from a BAC clone (RP23-53B17) or genomic DNA using the primers listed in firefly luciferase reporter vector (Promega). Site-directed mutagenesis used the QuikChange Site-Directed Mutagenesis Kit (Stratagene) with the PCR primers listed in CMV-Pax6 construct, generated by inserting full-length Pax6 cDNA into pCMV-Script plasmid (Stratagene). The Renilla luciferase vector was pRLSV40 (Promega). HEK293 cells were harvested 48 hr after transfections and analyzed with the Dual Luciferase Reporter Assay System (Promega).Fragments around The primary antibodies were mouse anti-Pax6 , rabbit anti-Pax6 , rabbit anti-Cdk6 , rabbit anti-cyclin D2 , mouse anti-pRb , rabbit anti-phospho-pRb , rabbit anti-phospho-pRb , and rabbit anti-beta actin . Alexa-coupled secondary antibodies were used and blots were quantified using the Li-Cor scanning system.