Conditions were defined that facilitated analysis of GlnR-dependent induction of gene expression in response to rapid nitrogen starvation. Microarray analysis identified global transcriptional differences between glnR+ and glnR mutant strains under varying nitrogen conditions. To differentiate between direct and indirect regulatory effects of GlnR, chromatin immuno-precipitation (ChIP) using antibodies specific to a FLAG-tagged GlnR protein, coupled with microarray analysis (ChIP-chip), was used to identify GlnR binding sites throughout the S. venezuelae genome.GlnR is an atypical response regulator found in actinomycetes that modulates the transcription of genes in response to changes in nitrogen availability. We applied a global S. venezuelae. GlnR-binding regions were associated with genes involved in primary nitrogen metabolism, secondary metabolism, the synthesis of catabolic enzymes and a number of transport-related functions.GlnR bound to its target sites in both transcriptionally active and apparently inactive forms. Thirty-six GlnR binding sites were identified by ChIP-chip analysis allowing derivation of a consensus GlnR-binding site for S. venezuelae is extensive and impacts on many facets of the organism's biology. GlnR can apparently bind to its target sites in both transcriptionally active and inactive forms.The GlnR regulon of  Streptomyces coelicolor  encodes predicted homologues of GlnR and GlnRII, both of which occur in regions with a high degree of synteny with the respective chromosomal locations in S. coelicolor. S. venezuelae GlnR and GlnRII show 80.6% and 67% identity, respectively, to their S. coelicolor homologues.The S. venezuelae showed reproducible, vigorous growth in Evans medium containing 30 mM ammonium chloride as nitrogen source. A maximum growth rate of 0.27 h-1 was achieved during exponential phase and consistently dispersed mycelium throughout growth was verified microscopically (data not shown).To aid in the accurate interpretation of global transcriptional data, a minimal Evans medium  was usedamtB [amtB occurred in response to a switch from mid-log growth in Evans containing 30 mM ammonium chloride to Evans medium completely lacking a nitrogen source for a 30 min period. This induction was almost entirely reversed by the exogenous addition of 30 mM ammonium chloride to the starved culture for a 15 min period. Transcript levels of amtB, assessed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), were induced 53-fold by nitrogen starvation and this level was reduced 9-fold by ammonium addition  were used to produce cDNA for hybridisation to Affymetrix GeneChips. Three separate biological replicates of this experiment provided a global transcriptional profile of gene expression under conditions that had a marked effect on amtB expression. The microarray data confirmed the pattern of amtB expression measured using qRT-PCR, i.e. the gene was strongly induced (76-fold) upon N starvation and was repressed (15-fold) by exogenous addition of ammonium  to search for a sequence motif within the upstream regions of genes whose transcriptional profile was altered in the glnRII mutant. No common feature was detected.In contrast to the major transcriptional changes observed in the ty Table . 165 genram MEME   allows the expression of GlnR-Flag under the control of the native ent site . The cong Figure , and funglnII, a defined GlnR target, but not that of hrdB which encodes the major sigma factor of S. venezuelae and which did not respond to changes in nitrogen status in the microarray experiments . Samples were taken at each of the three time points used for transcriptional analysis, and hybridised along with Cy-5-labelled control samples of total DNA from the same time point that had not been subjected to immunoprecipitation. Plotting the Cy3/Cy5 ratio across the chromosome allowed identification of peaks corresponding to GlnR-associated sites. Peaks were considered significant when at least 2 consecutive probes had a Cy3/Cy5 ratio greater than 2.5 standard deviations above the mean of all signals. This analysis was carried out in duplicate on separate biological samples. As a further control, immunoprecipitation was carried out using the wild-type strain carrying the empty Flag vector (pIJ10500) and therefore not making the GlnR-Flag fusion protein. Peaks that were also present in this sample were considered to be artefacts. Figure glnII promoter region.Immunoprecipitation (IP) trials, followed by Western blotting, demonstrated enrichment of GlnR-Flag protein following IP Figure . DNA elus Figure . Having 0  many more GlnR peaks were observed. The most highly enriched regions were those containing the glnA and glnII promoters. The amtB promoter was also highly represented in the IP material. This coincides with the maximal levels of expression of these genes during the time course. However, by T45 exposure of the starved cultures to exogenously added ammonium chloride did not abolish the GlnR binding detected at many promoter sites during N starvation, despite the fact that by T45 many of these genes, most notably glnII and amtB, showed a marked loss of transcriptional activity. This unexpected phenomenon was observed consistently for nearly all target genes identified, and in duplicate biological experiments. Therefore a simple model, in which nitrogen starvation leads to an increased affinity of GlnR for its target promoters, which is reversed by sufficient levels of nitrogen, does not adequately explain the observed binding activity. To investigate further, an extra ChIP experiment was carried out 30 min after addition of ammonium chloride (T60). However, even at this time point, GlnR was still associated with all target sites identified at T30. It would therefore appear that GlnR assumes different forms during the time course. At T0, GlnR is present , are known to be regulated by GlnR in S. coelicolor [S. venezuelae genes that are homologous to GlnR-regulated genes in S. coelicolor was enriched in this study.All of the reproducible GlnR-binding sites (observed in at least two experiments and absent within the control) were located within intergenic regions. A total of thirty six such peaks were identified and Table elicolor ,12. Howein vivo in S. venezuelae, 250 bp sequences of each promoter region were aligned and a consensus sequence  was derived using MEME [30 the level of transcript present in the wild-type strain is 14-fold greater than that in the glnR mutant) and GlnR is associated with the promoter region in vivo. EMSA experiments with purified GlnR (data not shown) revealed retardation of DNA sequences upstream of glnA, glnII, amtB and Sven_7046, all of which contain the conserved GlnR binding motif. Mobility of the promoter region of Sven_2720 was unaffected in the presence of GlnR.The identification of a consensus GlnR binding site has proven to be of great value in identifying GlnR-regulated genes . To idening MEME . MatchesS. venezuelae. We identified a large number of genes within the GlnR regulon but, like others [In this study we combined transcriptomic and ChIP-chip analyses to investigate the genetic control of nitrogen regulation in e others , we coulin vivo binding sites on the S. venezuelae chromosome for the nitrogen responsive transcriptional regulator GlnR, and these cover three major aspects of nitrogen metabolism.The genes identified here can be divided into four categories: i) those that responded to nitrogen availability in a GlnR-dependent manner and where GlnR was associated with the promoter region; ii) those that responded in a GlnR-dependent manner, but showed no GlnR interaction at the promoter and are potentially modulated through another regulatory protein iii) those that responded to nitrogen status independently of GlnR, and iv) those GlnR targets identified by ChIP-chip that showed no response to nitrogen status under the conditions studied. For categories i) and iv), we identified 36 As expected, genes that encode proteins known or likely to be directly involved in nitrogen metabolism are highly represented among the GlnR targets. Such functions include the assimilation of nitrogen, either from ammonium  or from alternative N sources such as urea or nitrate . GlnR also regulates production of N-acetyl-glutamate synthase, which, as part of the arginine biosynthesis pathway, synthesises N-acetyl-glutamate from glutamate and acetyl-CoA and whose activity is dependent on cellular nitrogen levels . All of Streptomyces species have evolved.Several genes downstream of GlnR-binding sites  are predicted to encode periplasmic binding protein (PBP) components of ABC transport systems with substrates such as amino acids and small peptides, suggesting roles in scavenging of alternative nitrogen sources during starvation. Other GlnR-targets encode predicted secreted proteins involved in the degradation of various macromolecules, such as the predicted glycosyl hydrolases, Sven_6731, encoding a xylanase, and Sven_6632, encoding a β-glucosidase, involved in the degradation of plant cell wall polysaccharides ,27, a prMany of the genes encoding secreted proteins and PBPs fall into category iv), i.e. GlnR targets in ChIP-chip experiments that were not identified as nitrogen responsive in the transcriptional studies. However, the absence of a nitrogen-dependent transcriptional response may reflect the starvation conditions used in this study. The proteins encoded by these genes are not involved directly in nitrogen metabolism, but may, in some circumstances, perform useful roles under nitrogen-limited conditions. Therefore, for optimal induction, these systems may require additional input signals in addition to nitrogen limitation, such as substrate-dependent induction, that were absent in our experimental conditions.jadR1 and jadR2 genes, which encode transcriptional regulators that activate and repress, respectively, expression of the jadomycin biosynthetic genes [jadR1 is activated by GlnR. Expression of jadR1 is induced 3.3-fold in response to nitrogen starvation in the wild-type strain and this level is repressed 1.2 fold upon ammonium addition. Consistent with this, levels of jadR1 transcription in the glnR mutant during nitrogen starvation are 1.7-fold lower than in the wild-type strain -like gene clusters. All three were down-regulated in response to nitrogen limitation, but their expression, and that of adjacent genes, was not significantly altered in the S. venezuelae are also regulated by GlnR in S. coelicolor. Of the 15 genes identified as GlnR targets in S. coelicolor [S. venezuelae . Four of these 13 genes, namely the amtB-glnK-glnD operon and glnII, were among the twenty genes showing the largest induction upon N-starvation (Table ureA encoding the γ-subunit of urease and Sven_1860 (SCO2195) encoding a small protein of unknown function, also responded significantly .Many of the genes responsive to changes in nitrogen availability in elicolor , 13 have_1860 SCO195 encodelicolor , inductiS. venezuelae, N-starvation was achieved by complete removal of any N-source, whereas in S. coelicolor N-starvation was achieved by a switch from growth on ammonium to growth on nitrate. S. venezuelae nirB, encoding a nitrite reductase subunit, did not respond to N-starvation but the gene was induced in a GlnR-dependent manner in S. coelicolor. Hence the presence of nitrate/nitrite may be required for nirB induction. Similarly, whereas the complete absence of nitrogen induced ureA in S. venezuelae, S. coelicolor ureA was repressed upon N-starvation, perhaps indicating a preference for nitrate over urea as a nitrogen source in the latter species. We have not investigated the in vivo promoter binding activity of S. coelicolor GlnR and there is no published data on this. So it is currently unknown whether S. coelicolor GlnR also binds to target promoters in vivo under conditions in which transcriptional activation does not occur.The lack of a marked response to N-starvation by some candidate genes could reflect species differences in the GlnR regulon, or differences in experimental conditions. In this study with S. coelicolor but with some significant differences. The binding site (GlnR box) proposed by Tiffert et al. contained an "a-site" (gTnAc) located 6 bps upstream of a "b-site" (GaAAc), giving a consensus sequence of gTnAc-n6-GaAAc [S. coelicolor nasA (encoding nitrate reductase). GlnR recognition of the nasA promoter is mediated by two GTAAC "a-sites" separated by 18 bps, leading the authors to suggest that a "b-site"might not be obligatory. The motif we identified in S. venezuelae, GTnAC-n6-GTnAC , and may be biased towards a higher affinity "b-site". The promoters of amtB and glnA in S. venezuelae both contain a "b-site" with the sequence GAAAC, and both have two tandem copies of the minimal binding sequence , an arrangement suggested to be the predominant GlnR-binding site in S. coelicolor [in vivo in S. venezuelae, where a single occurrence of GTnAC-n6-GTnAC is the most common motif.Tiffert et al.  suggesteelicolor . This taet al. [Escherichia coli [Caulobacter crescentus [Bacillus subtilis SpoA [in vivo binding sites that were not bound in an in vitro assay. Local changes in DNA topology, or the co-operative interactions of multiple transcriptional factors in vivo, may reduce the requirement for the consensus sequence [Nine of the thirty-six regions identified in ChIP-chip experiments do not contain a GlnR binding motif. Transcription factors binding to non-consensus sequences are a common observation in ChIP-chip studies . Examplhia coli  and CtrAescentus . Likewislis SpoA  revealedsequence  and facinasA of S. coelicolor, GlnR is capable of binding to two distantly separated copies of the GTnAC motif. Inspection of promoter regions that do not contain the full consensus sequence reveals several that possess multiple copies of the GTnAC motif. For example, the Sven_2720 promoter region contains three separate copies of GTnAC separated by regions of thirty nine and thirty three base pairs. Further work is required to establish their possible role in facilitating GlnR binding.As discussed for in vivo and has shown that GlnR is able to associate with target promoters in both transcriptionally active and inactive forms.GlnR is the global nitrogen regulator in actinomycetes and plays a key role in regulating the assimilation and utilisation of nitrogen. This study has extended our knowledge of the GlnR regulon in streptomycetes. It has also indicated a possible link between GlnR and transcription of JadR1, the pathway specific regulator of the jadomycin B cluster, as well as secreted degradative enzymes and several proteins with functions relating to transport. Application of ChIP-chip has provided fresh insight into the DNA sequences to which GlnR binds S. venezuelae ATCC 10712 was the wild-type strain used throughout this study. All mutants were generated using PCR targeting. The ent et al. . For genGGGATCCGTCGACC-3' and the reverse primer was 5-CGACCGACCGACGGCGGGTCCGGCAGGTGGTGCGCGATGTGTAGGCTGGAGCTGCTTC-3.glnRII::aprR mutant, the forward primer was 5'-TCCGTTCGTTTCTTCGCGCGAAAGAGCTGAGACCTCATGATTCCGGGGATCCGTCGACC-3', and the reverse was 5'-TGGTGTCCAGGACGAGGGCGAAGGCGAACTGACGGATCATGTAGGCTGGAGCTGCTTC-3'.For the amtB levels were 5-TCCGCCGCCAACACCGGGTTCA-3 and 5-GGCGAGTGCCGGGGTCATCAGC-3, and for hrdB levels 5-CATGGCGGACCAGGCCCGAACC-3 and 5-CCTGGAGCATCTGGCGCTGCAC-3.Primers used for qRT-PCR to measure glnR promoter, using the primer pair 5-TATTATAAGCTTGTGGGCTATTCTCCT-3 and 5-CCTACCGGCAGGTCGCACTGTGGC-3. The amplicon was blunt-ended using Pfu polymerase (Invitrogen) and cloned into the StuI site of pIJ10500 , a modified version of the integrative pMS82 vector [Streptomyces codon usage-optimised triple FLAG epitope cassette, thereby creating a C-terminally tagged GlnR protein expressed from its native promoter in construct pIJ12248.FLAG-tagging of GlnR was achieved by amplifying a region from genomic DNA that contained the entire GlnR coding sequence excluding the stop codon, along with 264 bp of upstream sequence containing the 2 vector  containiS. venezuelae was grown in 30 ml batches of Evans defined minimal media [4Cl, unless stated otherwise. To transfer S. venezuelae between media types, the culture was transferred to a falcon tube and very briefly centrifuged to form a pellet. Media was decanted and the pellet resuspended in the alternative media. For growth on solid media, Evans was supplemented with agarose at a final concentration of 2% w/v.For all liquid culture experiments al media . The nit® Reagent (Sigma) and left at room temperature for 5 min before addition of 300 μl chloroform. The mixture was vortexed briefly then incubated at room temperature for 2 min. Samples were centrifuged for 10 min at 13000 rpm in a bench top microfuge. The aqueous phase, containing RNA, was harvested and RNA was purified using an RNeasy® Mini Kit (Qiagen) as per manufacturer's instructions, including the recommended on-column DNase digestion. The final elution step was carried out with 50 μl of nuclease-free water (Qiagen).At each experimental time point 10 ml of culture were centrifuged briefly to pellet the mycelium which was rapidly frozen under liquid nitrogen. Frozen pellets were ground using a pestle and mortar containing liquid nitrogen. The broken cells were mixed with 1.5 ml of TRIamtB and hrdB were designed using the Primer3 web-based tool [hrdB expression.Specific primers for sed tool . RNA  was used as the template for production of cDNA that was subsequently labelled and fragmented for hybridisation to Affymetrix th et al . Hybridi2 values and normalised per gene to the median.Expression data were imported into GeneSpring 9.0 (Agilent Technologies), normalised using the Robust Multichip Average algorithm (RMA), converted to logMicroarray data have been deposited in the ArrayExpress datatbase, under the accession number E-MEXP-2684.P < 0.01 or P < 0.05, and assuming variances to be equal. P values were corrected using the Benjamini and Hochberg false discovery rate multiple testing correction procedure.Two-way ANOVA was performed in GeneSpring using the parametric test option with a false discovery rate of S. venezuelae strains were grown to an OD600 of ~ 0.6; 10 ml of culture was briefly centrifuged to form a pellet, which was resuspended in 1.5 ml of SP buffer and sonicated on ice. Multiple 15 s bursts of sonication at 10 kHz were performed with 60 s intervals between bursts, until the suspension became clear. Cell debris was removed by centrifugation. Protein concentrations of cell fractions were determined using the Bio-Rad protein assay system using bovine serum albumin as a standard. In all cases, 5 μg of total protein was separated by SDS-PAGE (15% polyacrylamide). After transfer to a nitrocellulose membrane , the proteins were reacted with monoclonal ANTI-FLAG® M2 antibody (Sigma).S. venezuelae were grown to an OD600 of ~ 0.6 and formaldehyde (Sigma) was added to a final concentration of 1%. Cross linking was allowed to proceed for 30 min of continued incubation at 30°C. The addition of glycine, at a final concentration of 125 mM, halted the cross-linking. Cells were briefly centrifuged to form a pellet and washed twice with ice-cold PBS. The pellet was resuspended in 750 μl of lysis buffer  and incubated for 25 min at 25°C. Samples were placed on ice for 2 min after addition of 750 μl of IP buffer . Samples at this point resembled a slurry that was sonicated repeatedly at 10 kHz in 15 s bursts; sub-fractions were taken at frequent intervals, extracted twice with phenol/chloroform and run on agarose gels to check fragment size. Sonication was complete, typically after 7-8 cycles, when fragments were between 300 and 1000 bp in length, centred on 500 bp. Debris was removed from the sonicated extracts by centrifugation at maximum speed in a bench top microfuge for 10 min at 4°C and supernatant fluid was retained. At this point 25 μl of the extract was stored to be used as the control "total DNA" whilst the remainder, ~725 μl, was used in immunoprecipitation.Fifty millilitre cultures of ® M2 antibody (Sigma). Incubation was then continued overnight. A 1/10 volume of a fresh 50% Protein A-sepharose slurry was added and samples incubated for a further 4 h at 4°C before centrifugation for 5 min at 3500 rpm in a microfuge to harvest the bead-antibody-chromatin complex. This was washed once in 0.5x IP buffer and twice in IP buffer each for 15 min with gentle agitation. Elution of DNA was performed by addition of 150 μl of IP elution buffer  to the beads, as well as to 10 μl of the "total DNA" control, and incubation at 65°C overnight. Beads were pelleted by centrifugation and the supernatant harvested, treated with 2 μl of 10 mg/ml Proteinase K (Roche) and twice extracted with phenol chloroform. Finally, DNA was purified using the Qiagen QiaQuick according to manufacturer's instructions.Initial pre-clearance of the extracts was performed by incubation for 1 h at 4°C on a rotating wheel after addition of 1/10 volume of a 50% Protein A-sepharose slurry (Sigma) equilibrated in IP buffer. The mixture was centrifuged to pellet the beads, the cleared extract harvested and mixed with 5 μl monoclonal ANTI-FLAGS. venezuelae genome, were designed and produced by Oxford Gene Technologies . Labelling of control total DNA and immunoprecipitated DNA, with Cy5 and Cy3 respectively, as well as hybridisation, washing and scanning were performed by Oxford Gene Technologies according to their standard protocols (http://www.ogt.co.uk ) previously described in detail for E. coli [Custom made microarray slides, consisting of 44,000 60-mer oligonucleotide probes covering the entire  E. coli .S. venezuelae carrying an empty vector instead of the FLAG-tagged GlnR construct. The ChIP-chip data have been deposited in the ArrayExpress database, under the accession number E-MEXP-2933.Cy3/Cy5 ratios were calculated for each probe. Ratios were then plotted against genome position. A peak in the plot representing a protein binding site was scored as present when firstly, two consecutive probes gave a ratio that was 2.5 standard deviations above the mean calculated across all probes in at least two of the six experiments, and secondly when this peak was absent in the control sample, derived by immuno-precipitation of a culture of ChIP: chromatin immuno-precipitation; qRT-PCR: quantitative reverse-transcriptase polymerase chain reaction; IP: immunoprecipitation; PBP: periplasmic binding protein; NRPS: non-ribosomal peptide synthetase.STP carried out all experimental work, participated in design of the study, analysed data and drafted the manuscript. GC programmed analytical tools for interpretation of ChIP-chip and aided the analysis of sequence data. MB participated in design and coordination of the study and helped to draft the manuscript. MM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.amtB-glnK-glnD, glnA and glnII over the wild-type, glnR and glnRII mutant time coursesMicroarray expression profiles of . Expression is the average of three biological replicates and normalised intensity is plotted on a log2 scale. Diagram adapted from GeneSpring 9.0 (Agilent).Click here for fileFull list of genes induced >2 upon nitrogen starvation, that are also repressed by ammonium.Click here for fileglnR mutant strainGenes induced >2 fold by nitrogen starvation and repressed by ammonium in the wild-type strain, but non-responsive in the .Click here for file


Interventions on macrophages/foam cells to redirect intracellular cholesterol towards efflux pathways could become a very valuable addition to our therapeutic arsenal against atherosclerosis. However, certain manipulations of the cholesteryl ester cycle, such as the inhibition of ACAT1, an ER-resident enzyme that re-esterifies cholesterol, are not well tolerated. Previously we showed that targeting perilipin-2 (PLIN2), a major lipid droplet (LD)-associated protein in macrophages, prevents foam cell formation and protects against atherosclerosis. Here we have assessed the tolerance of PLIN2-deficient bone marrow derived macrophages (BMM) to several lipid loading conditions similar to the found during atherosclerosis development, including exposure to modified low-density lipoprotein (mLDL) and 7-ketocholesterol (7-KC), a free cholesterol (FC) metabolite, in media with or without cholesterol acceptors. BMM isolated from mice that do or do not express PLIN2 were tested for apoptosis (TUNEL and cleaved caspase-3), ER stress (CHOP induction and XBP-1 splicing), and inflammation (TNF-α and IL-6 mRNA levels). Like in other cell types, PLIN2 deficiency impairs LD buildup in BMM. However, while most stress parameters were elevated in macrophages under ACAT inhibition and 7-KC loading, PLIN2 inactivation was well tolerated. The data support the safety of targeting PLIN2 to prevent foam cell formation and atherosclerosis. Lipid-laden macrophages or foam cells are fundamental to the formation and progression of atherosclerosis Perilipin, Adipose differentiation-related protein and Tail-interacting protein of 47 kDa In vivo, PLIN2 mRNA is upregulated in carotid endarterectomy specimens and in atherosclerosis-studded arteries of apoE−/− mice compared to control healthy arteries LDs consist of a core of neutral lipid, mainly CE and triglycerides, coated by a monolayer of phospholipids, FC and proteins All animal experiments were conducted following a protocol approved by the Institutional Animal Care and Use Committee at Albany Medical College (protocol number 901397). All efforts were made to minimize suffering.BMM isolated from femurs of PLIN2-deficient mice in C57BL/6J background Fifteen µg of protein were resolved by SDS-PAGE and transferred onto a PVDF membrane. The antibodies used for Western blot included a rabbit anti-PLIN2 , a rabbit anti-GADD153 , a rabbit anti-cleaved caspase-3 , and a rabbit anti-GAPDH (Sigma-Aldrich). RNA was isolated with TRIzol (Invitrogen), digested with DNase I and purified using the Absolutely RNA miniprep kit (Agilent Technologies). Reverse transcription of 500 ng of total RNA was performed with SuperScript III (Invitrogen). Primer sequences were designed with Primer3 software For oil red O (ORO) staining BMM were fixed with 3.7% paraformaldehyde, washed with phosphate buffered saline (PBS) and stained for 30 minutes with ORO . Nuclei were stained with hematoxylin (Vector Laboratories). TUNEL staining was performed with the DeadEnd Fluorometric TUNEL system (Promega), and cell nuclei were stained with DAPI (Sigma-Aldrich). Cell number and TUNEL-positive cells in six to ten randomly selected fields per sample were quantified blindly by two independent investigators.t-test was used to compare WT and PLIN2-deficient macrophages within each treatment group. Differences were considered significant at p<0.05. Results are shown as mean ± SEM.Statistical significance of the effect of the three independent variables included in this study  was determined by three way ANOVA using the SigmaStat software. Pairwise comparisons were performed by the Holm-Sidac method. In addition, a two-tailed Student's in vitro, and this allowed us to obtain enough cells to simultaneously compare the response between WT and PLIN2-deficient macrophages under multiple culture conditions. Because the role of PLIN2 on LD formation in BMM had not been studied, first we asked whether this is similar to the previously described in other primary macrophages and monocyte/macrophage cell lines BMM can be expanded Cholesterol loading can induce macrophage apoptosis +/+ macrophages and 10.6% in PLIN2−/− macrophages. In general, the rate of apoptosis increased when the macrophages were cultured without FBS, ranging between 1.5 and 4% in the control, acLDL and ACATi groups. Again, acLDL treatment did not induce apoptosis in PLIN2-deficient macrophages and in this case the rate of apoptosis in acLDL+ACATi-treated cells was similar in both genotypes (6.1% in PLIN2+/+ macrophages and 6.0% in PLIN2−/− macrophages). 7-KC treatment also increased the number of TUNEL positive cells to a comparable level in both genotypes: 21.7 and 22.4% in WT and PLIN2-deficient macrophages, respectively. Three way ANOVA analyses showed that FBS depletion significantly increased apoptosis. Among the treatments, the effect of 7-KC was highly significant. While comparisons between the acLDL+ACATi treatment and each one of the other treatment groups yielded p values<0.05, these values did not reach the critical level to be considered significant in this analysis. The PLIN2 genotype did not influence the results (p = 0.650). The ANOVA results are summarized in t-test analyses did not show significant differences between WT and PLIN2-deficient macrophages under any condition.Apoptosis was assessed by two commonly used methods: TUNEL to detect DNA fragmentation and Western blot to detect cleaved caspase-3. The TUNEL results are summarized in −/− macrophages than in their WT counterparts, although this difference was not statistically significant Click here for additional data file.Table S2Three way ANOVA analysis of the TUNEL data.(DOCX)Click here for additional data file.Table S3Three way ANOVA analysis of the TNF-α mRNA levels.(DOCX)Click here for additional data file.Table S4Three way ANOVA analysis of the IL-6 mRNA levels.(DOCX)Click here for additional data file.


In other metal sensing MerR family regulators, such as MerR, CueR, and ZntR the cognate regulator binds to a promoter with an unusually long spacer between the −35 and −10 sequences, and activates transcription of resistance genes as a consequence of binding the appropriate metal. Cysteine residues in these regulators are essential for metal ion coordination and activation of expression from their cognate promoter. In this study we investigated the interaction of PbrR with the promoter for the structural pbr resistance genes, PpbrA, effects on transcriptional activation of altering the DNA sequence of PpbrA, and effects on Pb(II)-induced activation of PpbrA when cysteine residues in PbrR were mutated to serine.The pbrA promoter, which has a 19 bp spacer between its −35 and −10 sites. Using β-galactosidase assays in C. metallidurans, we show that when PpbrA is changed to an 18 bp spacer, there is an increase in transcriptional activation both in the presence and absence of Pb(II) salts up to a maximum induction equivalent to that seen in the fully-induced wild-type promoter. Changes to the −10 sequence of PpbrA from TTAAAT to the consensus E. coli −10 sequence (TATAAT) increased transcriptional activation from PpbrA, whilst changing the −10 sequence to that of the Tn501 mer promoter (TAAGGT) also increased the transcriptional response, but only in the presence of Pb(II). Individual PbrR mutants C14S, C55S, C79S, C114S, C123S, C132S and C134S, and a double mutant C132S/C134S, were tested for Pb(II) response from PpbrA, using β-galactosidase assays in C. metallidurans. The PbrR C14S, C79S, C134S, and C132S/C134S mutants were defective in Pb(II)-induced activation of PpbrA.Gel retardation and footprinting assays using purified PbrR show that it binds to, and protects from DNase I digestion, the PThese data show that the metal-dependent activation of PbrR occurs by a similar mechanism to that of MerR, but that metal ion coordination is through cysteines which differ from those seen in other MerR family regulators, and that the DNA sequence of the −10 promoter affects expression levels of the lead resistance genes. Escherichia coli, and CadA from Staphylococcus aureus plasmid pI258, can export Pb(II) as well as Zn(II) and Cd(II) )pbrA promoter, and study the effects on transcription of shortening the 19 bp spacer between the −35 and −10 sequences, and altering the −10 sequence of PpbrA; and 2) to investigate the importance of cysteine residues in PbrR activation of PpbrA in response to Pb(II) ions. To this end each of the cysteine residues in PbrR  were individually changed to serine residues and a double mutant  was created. The effects of these mutations on in vivo transcriptional activation in response to Pb(II) were determined in C. metallidurans using β-galactosidase assays.The objectives of this study were to 1) Characterize the interaction between PbrR and the Escherichia coli strains were grown in LB broth -dATP for gel retardations. Further gel retardation and footprinting experiments used a 296 bp PpbrA PCR product, amplified from pMOL1139 using the primers pbrBstEII and pbrNruI   to make d with γ3P-dATP foribed by , using td with γ3P-dATP foE. coli K-12 TG2. The 1144 bp pbrR-PpbrA-ΔpbrA DNA fragment described above was cloned into pMa5/8 [EcoRI and BamHI sites to make pMaPbrR/PpbrA. Gapped duplex mutagenesis of each of the cysteine residues in pbrR was as previously described [pbrRC14S, pbrRC55S, pbrRC79S, pbrRC114S, pbrRC123S, pbrRC132S, pbrRC134S, or pbrRC132S, C134S  was cloned from pMOL1139, into the HindIII site of pUK21, by rendering the vector and insert blunt-ended using T4 DNA polymerase. The cloned PpbrA DNA fragment was sub-cloned as an EcoRI - BamHI fragment into pMa5/8 for site directed mutagenesis. The −10 sequence of PpbrA was mutated as described above using the primers conpbr and merpbr (Table pbrA −10 sequence from TTAAAT (wild type) to TATAAT (consensus) or TAAGGT (mer-like). The mutant PpbrA promoters were cloned into pMU2385 using EcoRI and BamHI, creating plasmids pMUPpbrA, pMUPpbrA(con) and pMUPpbrA(mer) in which the pbrA promoter regulates expression of the lacZ gene. After DNA sequencing, the activity of these mutant promoters was assayed in C. metallidurans CH34.A 266 bp pbrR-PpbrA-ΔpbrA DNA fragment from pMapbrR/PpbrA was used to construct the −1 promoter mutant of PpbrA, using the primers -1CentreEco and -1CenterBam to introduce the −1 deletion, and primers -1EcoPbr and -1BamPbr as flanking primers . Addition of PbrR to the end-labelled 296 bp PpbrA PCR product retarded this fragment, and addition of Pb(II) to PbrR and PpbrA increased the amount of PbrR required to retard the PpbrA DNA fragment  . We had originally identified a candidate Pr operon  and studiewed in ).pbrA PCR product described above was also used to determine the PbrR binding site on the promoter by DNase I protection assay. Figure pbrA. The region protected by PbrR on PpbrA includes the −35 and −10 sequences as well as the 19 bp spacer containing an imperfect dyad symmetrical sequence between them, and is consistent with DNAse I protection results for MerR, CueR and ZntR [The 296 bp Pand ZntR ,23,24,40pbrA promoter protected by PbrR are shown in Figure pbrA promoter has a −35 sequence (TTGACT) that is identical to those for PmerT from Tn501 and PzntA from E. coli K-12  has a 4/6 identity to the consensus E. coli −10 sequence (TATAAT) and the spacing between the −35 and −10 sequences is 19 bp, as is the case with other MerR family regulatory regions except ZntR .pbrA promoter, and the effects of altering the DNA sequence of the PbrR binding site or −10 sequence of PpbrA were investigated using pMUPbrR/PpbrA −1 in C. metallidurans AE104. The PpbrA −1 mutant . Changes made in PpbrA so that it resembled the Tn501 merT promoter −10 sequence resulted in promoter activity remaining repressed in the absence of Pb(II), but strongly induced in its presence to expression levels 5-fold higher than the wild-type pbrA promoter   (20 μM) which was sufficient to activate expression from PpbrA, without affecting growth of the Pb(II) sensitive AE104 strain. β-galactosidase assays of wild type and cysteine mutant PbrR responses to Pb(II) in C. metallidurans AE104  induced activation of transcription from PpbrA, consistent with the result for the single C134S mutant.pMUPbrR/Pmer operon from a divergent promoter: Pmer. MerR activates expression of the structural genes at the PmerT operator/promoter (o/p) site, which has an unusually long spacer of 19 bp between the −35 and −10 sequences of the promoter   repression and induction, rather than a constitutive up-regulation seen in the −10 consensus promoter mutant. These data emphasize the importance of individual nucleotides within the promoter in affecting promoter strength, and indicate that PpbrA is suboptimal for maximum induction of the structural pbr genes. It is possible that this may represent a mechanism for fine-tuning of expression of the pbr structural genes.PbrR from r Figure  (4). Ppbs Figure . Assays In vivo assays of the activity of cysteine to serine mutant PbrR proteins in C. metallidurans AE104 (which lacks pMOL30) have shown that C14, C79 and C134 are essential for PbrR Pb(II) sensing and activation of PpbrA . Pb(II) has been shown to have a preference for binding to cysteine residues in a tri-coordinate Pb(II)-thiol conformation [C. metallidurans genomic island 1 coordinates Pb(II) via 3 (possibly 4) cysteine coordination [S. aureus pI258 cadmium resistance repressor CadC is dependent on three cysteine residues [C134 in PbrR Rmet_5496) is also essential for Pb(II) response and is part of a CVC (CXC) motif which is often found in PbrR regulators associated with orthologs of PbrABC, but not in the PbrR homologues PbrR2 (PbrR691 Rmet_2302) and PbrR3 (PbrR710 Rmet_3456), or CadR  dination , and theresidues . There aresidues ,54. StruWe cannot exclude the possibility that the PbrR C79S and C134S mutants we have made may have altered DNA-binding features, which may account for loss of Pb(II) response. However, mutants in the MBD of other MerR family regulators do not, but mutants in the helix-turn helix domain of these regulators do ,46.C. metallidurans  or u or uC. ms (PbrR: ) withouts  using different amino acids to other divalent metal ion-activated MerR regulators, but further work is required to determine whether Pb(II) coordinates other residues in PbrR.Our experiments show that the mechanism of transcriptional activation by PbrR appears to be essentially identical to that of MerR family regulators that have been characterized. PbrR contains three cysteine residues that are necessary for Pb(II)-induced transcription from the Tp, Trimethoprim; Ap, Ampicillin; Km, Kanamycin.The authors declare that they have no competing interests.JLH and DJJ carried out the experimental studies. JLH drafted the manuscript. NLB conceived and coordinated the study. All authors read and approved the manuscript.


The transcription factor Pax8 is essential for the differentiation of thyroid cells. However, there are few data on genes transcriptionally regulated by Pax8 other than thyroid-related genes. To better understand the role of Pax8 in the biology of thyroid cells, we obtained transcriptional profiles of Pax8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysisin vivo. Moreover, both factors modulate Pax8-dependent transcriptional activation of the sodium iodide symporter (Nis) gene promoter. We ultimately combined putative and novel Pax8 binding sites with actual target gene expression regulation to define Pax8-dependent genes. Functional classification suggests that Pax8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes.Exhaustive analysis of Pax8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of Pax8 in transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify Pax8 partners, including proteins involved in tertiary DNA structure (CTCF) and chromatin remodeling (Sp1), and these direct transcriptional interactions were confirmed Our study provides novel insights into the role of Pax8 in thyroid biology, exerted through transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new transcriptional partners of Pax8, which functionally cooperate with Pax8 in the regulation of thyroid gene transcription. Besides, our data demonstrate preferential location of Pax8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism that represents a novel role of Pax8 in the transcriptional output of the thyrocyte. Gene regulation has been the subject of intense investigation over the past decades, mainly focusing on detailed characterization of a particular gene or gene family. However, genome-wide mapping of protein-DNA interactions and epigenetic marks is essential for a full understanding of transcriptional regulation. A precise map of binding sites for transcription factors (TFs), core transcriptional machinery, and other DNA-binding proteins is necessary to decode the gene regulatory networks and their contribution to developmental processes and human disease . In factTg), Thyroperoxidase (Tpo), and the Sodium Iodide Symporter (Nis), thus regulating their expression. Nevertheless, despite the key relevance of these TFs for thyroid biology, few studies have described additional loci that are transcriptionally regulated by the above mentioned TFs, nor have sequences been described to which these factors bind in enhancers, silencers, or boundary elements that could potentially regulate the transcription of genes over large distances.To elucidate the mechanism(s) operating in the establishment and maintenance of cell-specific differentiation, we used thyroid epithelial cells as a model system. These cells are the largest cell population of the thyroid gland and express different TFs called Nkx2.1, Foxe1, Hhex and Pax8, which define the thyroid differentiated phenotype . It is wPax8 knockout mice, it seems to be responsible for the formation of the follicles of polarized epithelial thyroid cells [PAX8 and congenital hypothyroidism in humans underlines an important function of this transcription factor in thyroid pathologies [Among these thyroid TFs, Pax8 is a member of the paired box-containing proteins and is expressed in the thyroid and kidney, and in the central nervous system during development . It playid cells . Also, thologies . In ordein vivo Pax8 binding sites that were significantly associated with CpG islands or high GC content sequences. Interestingly, immunoprecipitated peaks were mainly located along intronic regions and grouped in distal positions with respect to transcriptional start sites. Consensus sequence screening of these areas suggested Pax8 interaction with several core transcriptional elements , transcription factors belonging to the AP1 family, and trans-elements factors involved in high order chromatin structure (CTCF) and remodeling (Sp1). Co-immunoprecipitation and reporter assays demonstrated both physical binding and transcriptional cooperation between CTCF/Sp1 and Pax8. Combining sequencing and expression array data, we ultimately provided insights into Pax8-transcriptional networks in the differentiated thyroid that predict its involvement in relevant biological processes and pathways.The ChIP-Seq strategy allowed us to identify a large number of novel Nis and Tpo promoters  [Nis , whose transcription status is tightly regulated by Pax8 [Nis upstream enhancer  , showed  by Pax8 , showed Association of Pax8 enriched regions with annotated genomic features indicated that Pax8 tends to localize within intronic regions (82%); only a few peaks revealed binding to coding (6%) or 5'-UTR regulatory regions (2%) Figure . In addiin silico tools, we observed a significant overrepresentation of Pax-related binding sites, including sites for Pax8 and members of its own subfamily (Pax2 and Pax5)  Figure . The Paxigure TPO,12 and fenhancer  ) showed significant association with Pax8 IP peaks  overlapped with potential sites for both transcription regulators (data not shown), we performed transfection experiments in HeLa cells using a reporter construct containing the Nis promoter [To validate the interactions between Pax8 and the TFs described before, we performed co-immuprecipitation assays in PCCl3 cells using specific antibodies for Pax8, Sp1 and CTCF. As shown in Figure promoter . As showWe used whole genome expression arrays to identify Pax8-regulated genes by comparing expression profiles of Pax8-silenced PCCl3 cells with both scrambled siRNA-treated and wild type (wt) PCCl3 cells. This last condition was included to consistently integrate both expression array signals and global cis-regulatory sequencing studies into the same experimental conditions. Misinterpretation of expression data due to compensatory effects via Pax8-related paralogues (Pax2 and Pax5) is ruled out, given that both transcription factors are not expressed in thyroid cells.Pax8-PCCl3 vs. wt PCCl3, 3,035 and 3,354 probes were down and up-regulated in the Pax8-silenced condition, respectively   for both comparisons included 633 down and 565 up-regulated targets , KEGG pathway , and cell component analysis  (data not shown).Gene lists were ranked based on t-statistics for gene set enrichment analysis. The most significant GO terms and adjusted in silico tool to extract Gene Ontology (GO) terms overrepresented in our down- and up-regulated set of differentially expressed genes. Considering down-regulated probes for each comparison, we observed an enrichment in biological processes related to a wide variety of DNA, RNA, and protein processes , cell response to chemical and stress stimuli, immune response, and p53 and insulin-related pathways (phosphatidyl inositol system and metabolism)  representing 54 loci were associated with genes showing a significant peak around +/− 1 kb from a TSS. This number of genes represents 6.4% (54 out of 849 genes) of the significantly associated genes identified by expression arrays  on chromosome 5, and Mlph (melanophilin) and Rab17 on chromosome 9 , a well-known tumour suppressor gene involved in the maintenance of genomic stability and related to breast carcinoma development [Cdh16 , a member of the cadherin protein family acting as a morphogenic factor for tissue development [Rab17 (a member of the RAS oncogene family) and Myo5b (myosin VB), genes previously described to be involved in epithelial vesicle trafficking in highly polarized cells [Dab2ip (DAB2 interacting protein), a tumour and metastasis suppressor gene which encodes a Ras GTPase-activating protein [Dio1 , an essential gene for thyroid hormone action given that it codes for an oxidoreductase involved in thyroid hormone activation by converting the prohormone T4 into bioactive 3,5,3'-triiodothyronine (T3) [Tmod1 (tropomodulin 1), a gene encoding a protein which inhibits actin filament elongation and that is consequently involved in cytoskeleton structure regulation and cell morphology [We performed experimental validations using independent immunoprecipitated and siRNA silenced PCCl3 samples, as well as their corresponding controls. Among the genes included for this validation were: elopment ; Cdh16   and Tmodrphology . As show+ transport and differentiation in epithelial cells, respectively [In silico analysis of significant IP peaks located along promoter areas of these loci demonstrated Pax8 potential binding sites in 3 out of 4 genes (data not shown). Globally, these findings underscore the efficiency and accuracy of ChIP-Seq and expression array technologies to define a Pax8-dependent gene network, which allowed us to identify biological functions of Pax8 in thyroid cells.In addition, Pax8 silencing by means of transient transfection of siRNA was significantly associated with decreased expression levels of these potential targets . The transcriptional output of Pax8 during thyroid development is unknown but essential, given that thyroid follicular precursors are not formed in Pax8 null mouse embryos, which ultimately impairs the formation of follicle structures and thyroid hormone biosynthesis [Despite the known relevance of the transcription factor Pax8 for adult thyrocyte physiology, few data have been published concerning Pax8 target genes other than key thyroid-related genes . MTE constitutes a core promoter element (~20-30 nt downstream of the TSS) associated with RNA polymerase II-mediated transcription ,45. Furtan genes . WhereasNis transcription through interaction along the NUE element [loci rather than representing a global transcription phenomenon in thyroid cells.2Apart from the classical view of TFs interacting with promoter regions, TFs could activate gene expression by interacting with common lineage-specific TFs and/or binding to distal regions (enhancers). Synergistic effects of Pax8 and AP1 proteins have been shown to occur in the regulation of  element , and AP1 element . Accordi element , indicatin vivo interactions between Pax8 and CTCF or Sp1 in thyrocytes. These novel partners were further demonstrated to modulate the effect of Pax8 on the transcription of the NIS gene, thus confirming that these interactions are functionally relevant. Evidence has been accumulating concerning the role of CTCF in the establishment of intra-chromosomal loops which ultimately mediate protein-protein contacts between distal complexes and the general transcription machinery [Functional studies described in the present paper confirmed physical achinery ,50. On tachinery . Interesachinery ,53. TakiDIO1 after abolishing Pax8 . Moreover, gene expression profiling in normal versus malignant thyroid tissues demonstrated a downregulation of DIO1 and DIO2 [Pax8 has been mainly associated to thyroid differentiation and development through its transcriptional role in key thyroid-related genes ,55. At tand DIO2 , which cCDKN2BCCNB1 and CCNB2, among others)  and cell cycle processes , highlighting the novel finding of  tumours . This filoci involved in cell motion/adhesion, notably the Pax8 effect on NCAM1  transcription. NCAM1 and other components of adherens junctions, such as cadherins, have been described to be essential for maintaining cell polarity and epithelial integrity [Cdh16 by PAX8, but also defined additional PAX8-dependent genes that could be essential for thyroid cell polarity . In this regard, germline mutations in MYO5b have been associated with disruption of epithelial cell polarity in MVID (MIM251850) [Recent reports have associated the transcription factors Pax2 and Pax5 with increased capabilities for cell motility and adhesion in human cancer ,59. In pntegrity . Interesntegrity , and itsntegrity ,62. We hM251850) . This roState-of-the-art cis-regulatory sequencing studies have been combined with mRNA silencing and expression arrays to further characterize the functional relevance of TF-interacting DNA regions and thus to define their transcriptional output. In our study, we describe Pax8 as a master regulator of key cellular processes for thyrocyte biology, including cell cycle regulation, DNA repair, replication and metabolism, and cell polarity, and define a large set of genes whose expression is modulated by Pax8. However, only a minor fraction (6.4%) of the Pax8 binding sites identified are close to TSSs and correlate with altered mRNA expression, in agreement with studies carried out on other TFSs (1-10%) ,36. ThisTgTpo, and Nis, as well as the thyroid-specific transcription factors Nkx2.1, Foxe1, and Pax8 [PCCl3 cells are a continuous line of thyroid follicular cells derived from Fischer rats that express the thyroid-specific genes and Pax8 . They weand Pax8 . For traand Pax8 .Nis promoter (pNIS-2.8) which contains the NUE region with two Pax8 binding sites was cloned in our laboratory [The 2,854-bp DNA fragment of the rat boratory . Full leboratory -68.6 cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature. Cross-linking was stopped by the addition of glycine to a final concentration of 125 mM, and cells were washed twice with PBS. The cell pellet was resuspended consecutively in ChIP lysis buffers [ChIP samples were prepared from PCCl3 cells as follows: cultures of 10 × 10 buffers  and soniNis upstream enhancer element (NUE) and Tpo promoter sequences [Gad1 (glutamate decarboxylase 1) and Afm , and a region of the Nis locus that does not bind Pax8. PCR reactions were assembled in triplicate with SYBR Green ER qPCR Supermix  and run on an Applied Biosystem 7500 Real Time PCR system. The enrichment of target sequences in ChIP material was calculated relative to the Afm negative control, and normalized to their relative amplification in non-IP DNA.Before sequencing, Pax8-IP DNA (IP) was used to confirm enrichment of target DNA fragments . Briefly, DNAs were blunted with a combination of T4 DNA polymerase, Klenow polymerase, and T4 PNK. Then, a single 3′-end “A” base was added using Klenow exo (3′-to-5′ exo minus). Adapters provided by Illumina were ligated to the ends of the modified DNA before size selection of 200-bp fragments via polyacrylamide gel electrophoresis followed by extraction. The isolated DNA samples were used as the template for amplification by 18 cycles of PCR, and used for cluster generation on the Illumina Genome Analyzer II. Amplified products were column-purified with the QIAquick PCR Purification Kit  and assayed for quantity and quality with the Agilent 2100 Bioanalyzer .http://genome.ucsc.edu), the sequence reads were directionally extended to 300 bp, and for each base pair in the genome the number of overlapping sequence reads was determined and averaged over a 10 bp window. All sequencing data can be downloaded from Gene Expression Omnibus (GEO) under accession number GSE26938.The 50 bp sequence reads were aligned to the rat genome  using the MIRO pipeline  allowing 2 mismatches, and aligned tags were converted to BED format and used for identification of binding sites. In order to visualize the data in the University of California Santa Cruz genome browser  was used with default parameters to determine enriched Pax8 binding regions using non-IP DNA as control. PeakAnalyzer software  was asseThe distance correlation analysis was done with the GenomeInspector tool of the Genomatix suite , within +/−10Kb from the middle of the CpG islands and simple nucleotide repeats. CpG islands and simple nucleotide repeat coordinates were obtained from the UCSC Genome Browser .We used two approaches for motif search in the Pax8 binding sites defined by MACS. The first method, based on MEME-ChIP  and focuPolyclonal antibodies (1 μg) were bound to Dynabeads  and incubated with 200 μg of nuclear proteins extracted as described  from PCCP < 0.05.HeLa cells were transiently transfected using calcium phosphate with 1 μg of pNIS-2.8 reporter alone or in combination with 0.5 μg of expression vectors for Pax8, Sp1 and CTCF as indicated in the text. The Renilla luciferase-encoding pRL-CMV vector (50 ng) was used to correct for transfection efficiency. Forty-eight hours after transfection cells were harvested, lysed, and analyzed for firefly and renilla luciferase activities by the Dual-Luciferase reporter assay system . Promoter activity was determined as the ratio between firefly and renilla luciferase and represented as relative luciferase activity. The results were expressed as the mean ± SD of three independent experiments, each performed in triplicate. Data were analyzed with GraphPad Prism . Statistical significance was determined using an Anova one-way test, and differences were considered significant at a Pax8 PCCl3), and scrambled siRNA-treated (siScramble PCCl3) vs. Pax8-silenced PCCl3 cells. Given that each comparison was performed using quadruplicates and dye-swaps (Cy3 and Cy5 fluorochromes), our experimental design included sixteen independent competitive hybridizations . For this purpose, we generated three different conditions to finally establish two main comparisons: wild type vs. Pax8-silenced PCCl3 cells (siPax8 siRNA conditions (10 ng siRNA /ml) . Pax8 silencing was tested by means of western blotting using a polyclonal Pax8 mouse antibody  at different time points (24 and 48 hours) after transfection  following the manufacturer’s recommended protocol. RNA quality was evaluated with the Agilent 2100 Bioanalyzer and later amplified and labelled by using the Low RNA Input Linear Amplification Kit PLUS, Two-Color . Briefly, for each sample 2 μg of total input RNA were amplified in two rounds of amplification following the manufacturer’s instructions. First strand cDNA synthesis and amplification reactions were carried out using random and T7 primers, respectively. During the 2-hour in vitro transcription, Cy3- or Cy5-labeled CTP was incorporated into each amplified RNA (cRNA). Products of the reaction were then purified using RNAeasy mini spin columns . Hybridization and slide and image processing were carried out according to the manufacturer’s instructions . In each experiment, 825 ng of contrasting cRNA samples were fragmented at 60°C for 30 min and hybridized at 65°C for 17 hr. The slides were scanned at a 10 μm resolution using the Agilent G2565BA Microarray Scanner . Signal quantification was carried out with Feature Extraction 9.1 software , using default analysis parameters for Agilent’s whole rat genome 44 K gene expression arrays. Array data were normalized using loess and quantile methods for normalization within and between arrays, respectively. Differential expression analysis was done using Bioconductor’s limma package. At a later stage, we used the annotate package and the data base rgug4131a.db to obtain the annotations of the rat genome from Agilent. Genes that showed adjusted p-values <0.005 were considered differentially expressed both in wild type vs. siPax8 cells and in siScramble PCCl3 vs. siPax8 PCCl3 cells. Functional analysis of Gene Ontology (GO) terms was carried out using the FatiGO tool and gene set enrichment analysis was performed using FatiScan [Transient transfections of PCCl3 cells were performed using Lipofectamine 2000 , both for scrambled and for FatiScan ,77. All loci, which showed significant results for both expression profiling and ChIP-Seq, independently of the peak location along the considered gene. After performing an independent Pax8-chromatin IP, we obtained genomic DNA from IP, non-IP, and input samples, which were further used to amplify specific fragments contained in IP peaks , which were confirmed as negative controls by the ChIP-Seq results.Technical validations were performed by means of real-time PCR to verify the Pax8-dependency of 7 Pax8 siRNA, scrambled siRNA, and wild type PCCl3 cells). Expression level changes were defined in 7 down-regulated genes and in 4 up-regulated genes by means of real-time PCR for fragments specifically amplifying transcripts of interest .http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=tjwrbocecmewahm&acc=GSE26938Nis upstream enhancer element (NUE) [Tpo promoter sequences [Nis locus were visualized in the UCSC genome browser. Significant immunoprecipitated peak corresponding to MACS program included the Nis upstream enhancer (NUE), previously described to be regulated by Pax8 (underlined red letters).(A). Pax8 chromatin immunoprecipitation validation performed to confirm enrichment of target DNA fragments by means of real-time PCR. Sequences belonging to the nt (NUE)  and Tpo equences , previouClick here for fileChIP-Seq MACS data: Excel file containing genomic coordinates of 13,151 rat genomic regions significantly immunoprecipitated according to the MACS ChIP program.Click here for fileChIP seq peaks used for MEME/TOMTOM consensus motif analysis: Genomic coordinates of the 500 most significant ChIP peaks used to verify Pax8-dependent immunoprecipitation and to delineate the consensus Pax8 DNA binding site.Click here for fileDNA binding motifs overrepresented in Pax8 IP peaks. Genomatix matrices significantly associated to Pax8 MACS peaks. Overrepresentation (genome) column represents overrepresentation values of these matrices in our IP peaks compared to their presence along the rat genome, and Z-Score (genome) column indicates association value of Pax8-immunoprecipitated DNA for each considered DNA matrix.Click here for fileExpression data of wt and scrambled conditions vs. siPax8 conditions and association with ChIP-Seq data.Common probes (+/− 1kb) Excel sheet includes expression data for the 78 probes common to both expression comparisons  and belonging to genes showing a significant IP peak within 1kb of a TSS. Additional Excel sheets include significant probes for each array comparison.Click here for fileFatiScan gene set enrichment analysis. Excel file containing Gene Ontology (GO) terms commonly overrepresented in both expression array comparisons (datasheet “Common GO terms") and their adjusted p-values. This file also contains datasheets showing GO terms statistically significant for each individual comparison (WT or SCR sign GO biol. process).Click here for fileFatiScan gene set enrichment analysis for scrambled and wild type conditions vs. siPax8 conditions. FatiScan image showing enriched GO terms in siScramble vs. siRNAPax8 and wt vs. siRNAPax8 comparisons, respectively.Click here for fileFatiScan gene set enrichment analysis for scrambled and wild type conditions vs. siPax8 conditions. FatiScan image showing enriched GO terms in siScramble vs. siRNAPax8 and wt vs. siRNAPax8 comparisons, respectively.Click here for fileSignificant biological processes among underexpressed probes. FatiGO images showing overrepresented biological processes among common downregulated (n=633) probes for both expression array comparisons.Click here for fileSignificant biological processes among overexpressed probes. FatiGO images showing overrepresented biological processes among common upregulated (n=565) probes for both expression array comparisons.Click here for fileOverrepresented KEGG pathways among under- and overexpressed probes. KEGG pathways enriched among common downregulated (n=633) and upregulated (n=565) probes for both expression array comparisons.Click here for fileloci which were detected to be significantly deregulated in expression arrays (p<0.005).UCSC genome browser images showing significant Pax8 IP peaks for closely positioned Click here for fileOligonucleotides used for immunoprecipitation validation prior to performing high throughput sequencing (including positive and negative Pax8 immunoprecipitation controls), or for experimental validation of ChIP-Seq.Click here for filePax8 vs. PCCl3-wt and PCCl3-siPax8 vs. PCCl3-siScramble) included four different biological replicates that were cross-labelled with either Cy3 or Cy5.Schematic representation of experimental design followed for whole genome rat expression arrays. Both comparisons (PCCl3-siClick here for filePax8 conditions (siPax8) versus control conditions, including the no transfection condition (wt) and siScramble PCCl3-transfected cells (siScramble). Time points include 24 and 48 hours.Immunoblot demonstrating Pax8 downregulation in siClick here for fileOligonucleotides used for experimental validation of expression arrays.Click here for file


Among CREs, enhancers are abundant and predominantly act in driving cell-specific gene expression. Krüppel-like factors (KLFs) are a family of eukaryotic transcription factors. Several KLFs have been demonstrated to play important roles in hematopoiesis. However, transcriptional regulation of KLFs via CREs, particularly enhancers, in erythroid cells has been poorly understood.Mapping of DNase I hypersensitive sites (DHSs) is a powerful tool to experimentally identify In this study, 23 erythroid-specific or putative erythroid-specific DHSs were identified by DNase-seq in the genomic regions of 17 human KLFs, and their enhancer activities were evaluated using dual-luciferase reporter (DLR) assay. Of the 23 erythroid-specific DHSs, the enhancer activities of 15 DHSs were comparable to that of the classical enhancer HS2 in driving minimal promoter (minP). Fifteen DHSs, some overlapping those that increased minP activities, acted as enhancers when driving the corresponding KLF promoters (KLF-Ps) in erythroid cells; of these, 10 DHSs were finally characterized as erythroid-specific KLF enhancers. These 10 erythroid-specific KLF enhancers were further confirmed using chromatin immunoprecipitation coupled to sequencing (ChIP-seq) data-based bioinformatic and biochemical analyses.Our present findings provide a feasible strategy to extensively identify gene- and cell-specific enhancers from DHSs obtained by high-throughput sequencing, which will help reveal the transcriptional regulation and biological functions of genes in some specific cells. CREs, including promoters, enhancers, silencers, insulators and locus control regions (LCRs) etc., are abundant in the human genome . Gene expression intensity was calculated by normalizing the read counts to RPKM according to the gene length and total mapped reads, and genes with RPKM < 0.01 were removed.mRNA-seq was originally designed to explore the dynamic transcriptomes during human erythroid differentiation and development  and DNA contamination was removed using the TURBO DNA-free™ Kit . DNA-free RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit  according to manufacturer’s instruction. Primers were designed using Primer 5  and the NCBI Gene Expression Omnibus (GEO) data repository under accessions GSE29692 and GSE32970. DHSs were identified using an algorithm developed by the University of Washington , 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) [Bio-11-dUTP  and TdT  were used to label the 3′-OH of single-stranded oligos  and complementary oligos were annealed as per the manufacturer’s instructions. Nuclear proteins of K562, HeLa, and HEK293 cells were extracted using the rapid micro-preparation method  . Protein.9, 10 mMhttp://genome.ucsc.edu/cgi-bin/hgFileUi?db=hg19&g=wgEncodeUwDnase), or through the NCBI Gene Expression Omnibus (GEO) data repository . The other data sets supporting the results of this article are included within the article and the additional files.DNase-seq data are available through the UCSC Genome Browser  or putative erythroid-specific (arrow and column in blue) DHSs. KLF loci were arbitrarily defined as extension from 70 kb upstream of the TSSs to 20 kb downstream of the poly (A) sites. Erythroid-specific or putative erythroid-specific DHSs were respectively marked with green and blue arrowheads and named with Roman numbers. Figure S2. Chromatin profiles of KLF genes without erythroid-specific or putative erythroid-specific DHSs. Figure S3. Distribution statistics of the identified erythroid-specific or putative erythroid-specific DHSs and enhancers in the genomic regions of KLFs. A. Statistics of the distribution of identified erythroid-specific or putative erythroid-specific DHSs relative to KLF genes and TSSs. In total, 18 (78%) and five (22%) DHSs are localized to the intergenic and intronic regions, respectively; 15 (65.2%) DHSs are located far distal (>10 kb) to TSSs, four (17.4%) DHSs are located distal (2-10 kb) to TSSs, and four (17.4%) DHSs are located in proximal (<2 kb) promoter regions. DHS KLF1-III contains TSS . B. Statistics of the distribution of the identified enhancers under the control of minP in K562 cells relative to KLF genes and TSSs. C. Statistics of the identified erythroid KLF enhancer distribution relative to the KLF genes and TSSs. D. Statistics of the identified erythroid-specific KLF enhancer distribution relative to KLF genes and TSSs.Chromatin profiles of Click here for fileTable S2. Position, length, GC content, chromatin accessibility (DNase I hypersensitivity), and references of ten KLF promoters used in enhancer assays. Table S3. Activities of KLF promoters measured by the luciferase reporter assay. Table S4. Primers used for measuring the expression patterns of KLFs in real-time PCR. Table S5. Primers used for amplification of erythroid-specific DHS fragments inserted in luciferase reporter constructs. Table S6. Primers used for amplification of KLF promoters. Supplementary References.The relative position and length of 23 identified erythroid-specific or putative erythroid-specific DHSs. Click here for file


They can be classified into two types by the degree of imprecision of their transcription start sites: peak promoters, which initiate transcription from a narrow genomic region; and broad promoters, which initiate transcription from a wide-ranging region. Eukaryotic transcription initiation is suggested to be associated with the genomic positions and modifications of nucleosomes. For instance, it has been recently shown that histone with H3K9 acetylation (H3K9ac) is more likely to be distributed around broad promoters rather than peak promoters; it can thus be inferred that there is an association between histone H3K9 and promoter architecture.Transcription promoters are fundamental genomic Here, we performed a systematic analysis of transcription promoters and gene expression, as well as of epigenetic histone behaviors, including genomic position, stability within the chromatin, and several modifications. We found that, in humans, broad promoters, but not peak promoters, generally had significant associations with nucleosome positioning and modification. Specifically, around broad promoters histones were highly distributed and aligned in an orderly fashion. This feature was more evident with histones that were methylated or acetylated; moreover, the nucleosome positions around the broad promoters were more stable than those around the peak ones. More strikingly, the overall expression levels of genes associated with broad promoters (but not peak promoters) with modified histones were significantly higher than the levels of genes associated with broad promoters with unmodified histones.These results shed light on how epigenetic regulatory networks of histone modifications are associated with promoter architecture. Recent progress in high-throughput technologies has made it possible to collect a variety of "omics" data on transcripts and on the epigenetic behaviors of the histones that are often associated with these transcripts -5.Cap analysis of gene expression (CAGE) is a high-throughput method that enables large-scale identification of transcription start sites (TSSs) of eukaryotic species. This method measures gene expression levels simultaneously with TSS identification by counting the sequenced 5' ends of full-length cDNAs, termed CAGE tags ,6. With In eukaryotic species, chromosomal DNAs is packed into nucleosomes, each of which comprises approximately 147 base pairs wrapped around a histone protein octamer consisting of two copies of each of the four core histones, H2A, H2B, H3, and H4 . Two bioH3K9ac has recently been frequently observed around broad promoters . . 5]. AceTo compare the distributions of nucleosomes that had H3K4me3, were located upstream of TSSs (positions from -150 to -100 bp), and were associated with either broad promoters or peak promoters in cases where the downstream genes showed similar expression levels, we selected 1788 genes associated with broad promoters and 138 associated with peak promoters that had expression levels in the range of 250 to 750 . Frequencies of the characteristic sequence patterns associated with promoters are shown by bar charts. The y-axis shows the proportions of broad and peak promoters that have TATA boxes (A) and CpG islands (B).Click here for fileSupplemental figure 2. Figure S2. Distributions of Sp1 sites around transcription start sites (TSSs). Distributions of nucleosome regions and Sp1 sites around TSSs associated with broad (A) and peak (B) promoters are shown. Nucleosome position is defined as the center position of the nucleosome. The x-axis shows genomic positions with respect to TSSs (from -500 bp to 500 bp). Sp-1 sites were obtained by the FANTOM 4 project.Click here for fileSupplemental figure 3. Figure S3. Distributions of the binding sites of two transcription factors (MAZ and PU.1) around transcription start sites (TSSs). Distributions of nucleosome regions and transcription factor binding sites around TSSs associated with broad  and peak  promoters are shown. Nucleosome position is defined as the center position of the nucleosome. The x-axis shows genomic positions with respect to TSSs (from -500 bp to 500 bp). Both MAZ and PU.1 sites were obtained by the FANTOM 4 project.Click here for fileSupplemental figure 4. Figure S4. Distributions of expression levels of genes selected for comparison of broad and peak promoters associated with similar downstream gene expression. The box plots represent the distributions of the microarray intensities of the gene sets that were selected from among those associated with broad and peak promoters and that had similar expression levels (from 250 to 750).Click here for file