Cells have developed many ways to cope with external stress. One distinctive feature in acute proteotoxic stresses, such as heat shock (HS), is rapid post-translational modification of proteins by SUMOs . While many of the SUMO targets are chromatin proteins, there is scarce information on chromatin binding of SUMOylated proteins in HS and the role of chromatin SUMOylation in the regulation of transcription.We mapped HS-induced genome-wide changes in chromatin occupancy of SUMO-2/3-modified proteins in K562 and VCaP cells using ChIP-seq. Chromatin SUMOylation was further correlated with HS-induced global changes in transcription using GRO-seq and RNA polymerase II (Pol2) ChIP-seq along with ENCODE data for K562 cells. HS induced a rapid and massive rearrangement of chromatin SUMOylation pattern: SUMOylation was gained at active promoters and enhancers associated with multiple transcription factors, including heat shock factor 1. Concomitant loss of SUMOylation occurred at inactive intergenic chromatin regions that were associated with CTCF-cohesin complex and SETDB1 methyltransferase complex. In addition, HS triggered a dynamic chromatin binding of SUMO ligase PIAS1, especially onto promoters. The HS-induced SUMOylation on chromatin was most notable at promoters of transcribed genes where it positively correlated with active transcription and Pol2 promoter-proximal pausing. Furthermore, silencing of SUMOylation machinery either by depletion of UBC9 or PIAS1 enhanced expression of HS-induced genes.HS-triggered SUMOylation targets promoters and enhancers of actively transcribed genes where it restricts the transcriptional activity of the HS-induced genes. PIAS1-mediated promoter SUMOylation is likely to regulate Pol2-associated factors in HS.The online version of this article (doi:10.1186/s13059-015-0717-y) contains supplementary material, which is available to authorized users. Heat shock (HS) induces many changes in cellular functions. Despite a general inhibition of gene expression at various levels  in response to acute heat stress or HS , specifiPost-translational modifications (PTMs) of proteins allow cells to react swiftly to changing environment, which is vital for survival in acute stress situations. One of the conserved PTM responses in various types of cell stresses is the modification by small ubiquitin-like modifier (SUMO) proteins . PTM of Recently, genome-wide ChIP-seq approaches have revealed cell status- and signaling-dependent alterations in the locus-selective occupancy of SUMOylated proteins and components of SUMOylation machinery –15. ConsTo address the role of chromatin SUMOylation in HS in a genome-wide manner, we studied chromatin binding dynamics of SUMO2/3 and PIAS1 by chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) using human K562 chronic myelogenous leukemia cells and VCaP prostate cancer cells. Chromatin binding of SUMO2/3 was further correlated with HS-induced changes in on-going transcription using global run-on sequencing (GRO-seq) and chromatin occupancy of RNA polymerase II (Pol2). Our results show that HS induces rapid and massive changes in chromatin SUMOylation pattern. HS-induced SUMOylation of chromatin-bound proteins correlates positively with transcriptional activity at promoters and enhancers. Promoter SUMOylation is dependent on active transcription, as inhibition of transcription initiation or elongation reduces the amount of SUMO2/3 at promoters. Mechanistically, SUMOylation seems to prevent hyperactivation of HS-induced transcription, as silencing of the SUMOylation machinery leads to significantly increased gene expression of HSPs in cells exposed to acute heat stress.To investigate HS-induced changes in chromatin SUMOylation, we exposed K562 cells to 43 °C for 30 min. The exposure induced a prominent accumulation of high molecular mass anti-SUMO2/3 immunoreactive protein forms, as judged by immunoblot analysis of whole cell lysates Fig. . The HS Analysis of SUMO2/3 peaks in K562 cells that were found in two independent biological replicates yielded in control conditions 30,605 SUMO2/3 peaks and in HS 19,869 SUMO2/3 peaks of which approximately 12,000 occurred in both conditions. In VCaP cells, the ChIP-seq analysis revealed 10,154 SUMO2/3 peaks in control cells, 25,382 in heat-shocked cells, and 19,132 in HS-recovered cells. In both cell lines, the majority of the SUMO2/3 peaks were found in intergenic, intron, and promoter regions in both conditions Fig. . The mosWe utilized the available ENCODE data of K562 cells  and analTaken together, the majority of the SUMO2/3 peaks is cell type-specific and located in introns and intergenic regions. Acute heat stress changed chromatin SUMOylation pattern rapidly: SUMO2/3 accumulated onto promoters and chromatin regions with active histone marks and it was lost from intergenic chromatin sites.Based on heat map and line profile analyses at the peak vicinities Fig. , the SUMZNF136 gene, Fig. Next, we compared the co-occurrence of SUMO2/3 peaks with K562 cell chromatin binding protein data from the ENCODE project , 18 and Some transcription factors in K562 cells showed a very high co-occurrence with SUMO2/3 peaks, suggesting that their regulation or chromatin binding is tightly linked to SUMOylation. The highest co-occurrence with SUMO2/3 was observed with RNA polymerase III (Pol3) and its associated transcription factor BRF1, and the situation prevailed in HS  markedly reduced HSF1 protein level Fig.  and inhiHSPA1A and UBB in Fig. s (spearman correlation coefficient) = 0.21, P value <0.0001) and HS , but not in control conditions  biological process terms related to protein folding, apoptosis, and heat response, while the HS-repressed genes were enriched in GO terms of gene expression, transcription, translation, and metabolic processes Table .Table 1ETaken together, HS triggered transcription-tied-up SUMOylation of promoters and enhancers. Promoter SUMOylation correlated positively with Pol2 promoter-proximal pausing and predicted transcriptional repression upon acute heat stress. Moreover, genes associated with the HS-enriched SUMO2/3 peaks shared several of the biological process categories of the HS-repressed genes.PIAS1 is the major PIAS family member in VCaP cells and known to bind chromatin . To get The above data suggest that HS induces activation and chromatin binding of PIAS1 where it functions as a SUMO ligase. To test this notion, we silenced PIAS1 using siRNA (siPIAS1) Fig.  and anals = 0.49, P <0.0001), HS , and recovery  conditions . These data show that promoter Pol2 and SUMO2/3 correlate in all assayed conditions, whereas the recruitment of PIAS1 onto Pol2-bound promoters is a transient response to acute heat stress.To analyze both SUMO2/3 and PIAS1 chromatin binding in the context of transcription, we mapped chromatin occupancy of Pol2 in control, HS and recovery conditions in VCaP cells using ChIP-seq. HS markedly increased Pol2 signal at promoters and at 3’-untranslated regions of HS-induced genes, such as those in the HSP gene cluster Fig. , indicatons Fig. . A correNext, we asked if the promoter SUMO2/3 signal is dependent on active transcription and whether it is a common feature of Pol2-paused promoters. To this end, we analyzed chromatin SUMO2/3 using ChIP-seq in cells treated with either DRB  that induces Pol2 promoter-proximal pausing or triptolide (TRP) that targets Pol2 to degradation . DRB treUBE2I (gene for UBC9) in VCaP cells with siRNA (siUBE2I) and used ChIP-seq to map the genome-wide binding of Pol2 in control (C) and HS conditions. A marked reduction of UBC9 protein level was seen in siUBE2I compared to siNON  were grown in RPMI1640 (Gibco) supplemented with 10 % fetal bovine serum, 1 % penicillin/streptomycin, and 2 mM l-glutamine. VCaP cells (American Type Culture Collection) were grown as previously described . For ChIAntibodies used for immunoblotting were anti-SUMO2/3 , anti-UBC9 , anti-PIAS1 , anti-tubulin , anti-laminB1 , anti-Pol2 , anti-histone H3 , and anti-HSF1 . Antibodies used for ChIP-seq were anti-SUMO2/3 , anti-PIAS1 , and anti-Pol2 .ChIP-seq was done with minor modifications from a published protocol . BrieflySamples were produced as two biological replicates essentially as described before . BrieflyUBE2I , PIAS1 (siPIAS1), or non-targeting control (siNON) siRNA  on 6-well plates using Lipofectamine RNAiMAX transfection reagent (Life Technologies). Four days after transfection, VCaP cells were heat shocked , total cellular RNA was collected , and cDNA was synthesized with oligo dT primers . Three biological replicates were analyzed using LightCycler 460 SYBR GREEN I reagent and LightCycler 480 PCR apparatus (Roche). GAPDH was used to normalize amounts of mRNA between samples, and non-stressed siNON sample to get the relative mRNA levels in each sample. Primer sequences are available upon request. Statistical significance of the changes in RT-qPCR data were analyzed using one-way ANOVA and Bonferroni’s multiple testing correction in GraphPad Prism.HSF1 was silenced from K562 cells as described before . Briefly7 reads for visualization and ChIP-seq signal comparison analysis. Signal matrixes for heat maps and line profiles were done in HOMER and visualized using imageJ [Sequenced raw reads were quality controlled using FastQC  and qualg imageJ  and R [4g imageJ  and asseg imageJ  using deg imageJ  and VENNg imageJ . Analysig imageJ .de novo transcript detection). Chromatin loci where two opposing strand transcripts with no more than two-fold difference in read count (±500 bp from the center) were identified no more than 1 kb apart from each other, were considered eRNA enhancers. Transcription and SUMO2/3 signal was measured from 1 kb window centered to eRNA enhancers. Kruskal-Wallis statistical test and Dunn’s post test were done in GraphPad Prism software to evaluate statistical significance of the changes in SUMO2/3 signals in different transcription categories (gene body transcription and eRNA enhancer transcription).GRO-seq reads were quality controlled using FastQC and FASTX-toolkit . Poly-A tails were trimmed, and smaller than 25 nt long reads and reads that mapped to rRNA were discarded. Remaining reads were mapped to reference human genome (hg19) using bowtie . Promoter (TSS to +250 bp) and gene body (+250 bp to TTS) transcription in was analyzed using HOMER. Gene body RPKM cutoff >0.5 was used to differentiate between transcribed and non-transcribed genes. Promoter-proximal pausing index (PPI) was calculated as ratio of RPKMs at promoter and gene body GRO-seq transcription. Differential expression (FDR <0.01 and fold change >2 as cutoffs) for transcribed genes was analyzed using edgeR . Gene On7 mapped reads. In SUMO2/3 co-occurrence analysis, K562 specific ChIP-seq datasets from transcription factor super-track (release 3 August 2013) were used [K562 cell specific hg19 mapped ChIP-seq and DNaseI data released in the ENCODE project were used in the analysis (ENCODE project consortium, 2011; full list of used datasets in Additional file ere used . VCaP SUere used  were anaChIP-seq and GRO-seq datasets are available with accession code: GSE66448 in GEO database.


Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl, and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5′ intergenic regions (5′ IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5′ IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PlcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5′ untranslated regions (5′ UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5′ UTR in B. cereus INRA C3 showed that the entire 331 bp 5′ UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5′ UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5′ IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5′ UTR downstream of the promoter, potentially acting as a nutrient-responsive roadblock repressor of toxin gene transcription. PlcR binding sites are highly conserved among all B. cereus sensu lato strains, indicating that this regulator does not significantly contribute to the heterogeneity in virulence potentials. The CodY recognition sites are far less conserved, perhaps conferring varying strengths of CodY binding, which might modulate toxin synthesis in a strain-specific manner. Bacillus cereus is an opportunistic pathogen and food contaminant that produces several toxins causing gastrointestinal illness in humans. B. cereus sensu stricto is closely related to B. thuringiensis and B. anthracis, which are regarded to be one species on the basis of high core-genomic relatedness , non-hemolytic enterotoxin (Nhe) and cytotoxin K (CytK). Hbl consists of a binding component B and the two lytic components L1 and L2. These proteins are encoded in one operon hblCDAB by the genes hblA, hblD, and hblC, respectively . However, enterotoxin expression is additionally regulated by nitrogen source availability and the general energetic cell status, sensed by the pleiotropic regulator CodY  by means of bioinformatics and hbl, nhe, and cytK strains representative for the seven phylogenetic groups were selected from a set of 142 B. cereus sensu lato strains (Table 1). Out of these, 29 nhe, 23 hbl, 3 cytK-1, and 15 cytK-2 5′ IGRs were examined. Enterotoxin promoter activity was studied experimentally in B. cereus INRA C3 (IV) and gel mobility shift assays were performed with both B. cereus INRA C3 and B. cytotoxicus CVUAS 2833 (VII).In order to compare intergenic regulatory regions upstream of the enterotoxin operons  strains ; Table 11 and sequence conservation was graphically represented as sequence logo2. The web-based program ORF Finder3 was used to detect potential open reading frames (ORFs) embedded in the 5′ UTRs of enterotoxin genes. The 5′ UTR RNA sequences were further analyzed for similarity to known RNA families with Rfam v.12.0  at 150 rpm or on LB agar plates at 37°C. B. cereus INRA C3 was grown in modified MOD , and added to the MOD medium to a final concentration of 20 mM. To obtain MOD + 1% CAAs or MOD + 1% tryptone, 10 g/l CAA, or 10 g/l tryptone were dissolved and autoclaved together with the MOD medium components . CGY medium was prepared in a volume of 900 ml ddH2O. After autoclaving 100 ml of filter sterilized glucose were added to a final concentration of 1%. The media compositions are listed in detail in Supplementary Table All cloning steps were performed in fied MOD  or CGY mfied MOD  at 30°C Bacilli were cultured in CGY medium supplemented with 1% (w/v) glucose as described previously , cell pellets were snap-frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted by resuspending the pellet in 1 ml TRIreagent (Sigma-Aldrich) followed by cell disruption using a Fastprep 24 instrument . DNase I digestion and RNA isolation were performed as previously described (hbl and nhe are listed in Supplementary Table eviously  to an ODescribed . DNA-freB. cereus reporter strains were constructed. The promoter region of interest was inserted into the E. coli/Bacillus shuttle vector pXen1 (luxABCDE by using primers comprising restriction sites for EcoRI and BamHI (Supplementary Table Pfu polymerase (Promega). The resulting plasmid was propagated in non-methylating E. coli INV110 and introduced in B. cereus by electroporation as described previously . Precultures grown for 16 h at 150 rpm were diluted 1:1000 in their respective medium supplemented with 5 μg/ml chloramphenicol, distributed into a 96-well microtiter plate  and incubated at 800 rpm and 37°C. Each condition was tested in quadruplicates and for each strain three independent biological replicates were analyzed. Cell density (OD600) and luminescence  were measured in hourly intervals for 19 h to detect the maximal promoter activity. Significance of differences between measured activities was calculated in the R free statistical software (version 3.1.1)4 using Welch’s t-test.Enterotoxin promoter activity of B. cereus INRA C3 and B. cytotoxicus CVUAS 2833 was performed in E. coli BL21(DE3) as a soluble N-terminal His6-tag fusion protein using the plasmid pET28b(+) as described previously (Heterologous expression of CodY from eviously . The prieviously  containi600 of 0.6 with 1 mM IPTG and cells were harvested after 5 h. Cell disruption and protein purification were performed as described (Protein expression was induced in LB medium at an ODescribed  with thecytK-2 (B. cereus INRA C3), PcytK-1 , parts of Phbl (B. cereus INRA C3) and Pnhe (B. cereus INRA C3 and B. cytotoxicus CVUAS 2833) were amplified by PCR. Primer pairs and fragments ranging from 241 to 568 bp are listed in Supplementary Table rrn was used as negative control, since it lacks any similarity to the CodY consensus sequence. Electrophoretic mobility shift assays were performed as described previously .To investigate the origin of differences in the regulation of enterotoxin expression, the 5′ IGRs of  strains  were comnsu lato  were selcytK-1 and cytK-2  were found to be relatively short (~100 bp) in comparison to the nhe and hbl 5′ IGRs. CytK-1 is exclusively found in rare B. cytotoxicus isolates, the most distant cluster of species within the B. cereus group . The nhe 5′ IGR is approximately 350 bp longer in B. pseudomycoides strains compared to that of all other B. cereus group strains investigated. In contrast, B. cytotoxicus (cluster VII) contains an nhe 5′ IGR which is ~50 bp shorter than the other 5′IGRs, thus lacking the second PlcR binding site (data not shown). B. weihenstephanensis WSBC 10204 (cluster VI) contains a 14 bp insertion upstream of the hbl ribosomal binding site . Strains of clusters III and IV, which harbor many pathogenic B. cereus isolates, lack 11 bp close to each ResD binding site within the hbl 5′ IGR. These missing regions might be used as an additional target to discern cluster III and IV from other B. cereus strains by molecular techniques. However, there is no correlation to high toxin production, since both high and low enterotoxigenic strains were found to harbor this deletions . The 5′ UTR is approximately 350 bp long and shows a high degree of sequence similarity to all other analyzed nhe 5′ UTRs.The TSSs of artcodon , while i of nheA . This mihbl operon in B. cereus ATCC 14579 was reported to localize 606 bp upstream of hblC . The TSS of the hbl cluster in B. thuringiensis Bt 407 is located 605 bp upstream of hblC . In the nhe and hbl promoter regions putative binding sites for an overlapping set of transcription factors were identified. Two PlcR binding sites are localized within the nhe 5′ IGR. PlcR 1 is 16 bp long and highly conserved, while the less conserved PlcR binding site 2 contains a 2 bp central insertion compared to the consensus sequence TATGNAN4TNCATA . Our analysis additionally revealed that nhe and hbl 5′ IGRs contain motifs which may be relevant for in vivo binding of CodY .While  control , the expnhe and hbl might encode small proteins or peptides. We could not detect any obvious ORFs upstream of nhe, but the 5′ UTR of hbl contains a putative ORF of varying size . This ORF appears in all clusters, but not in all investigated strains, and is thus not restricted to particular phylogenetic groups. A BLASTP analysis did not reveal any homology to proteins of known function, thus the expression and function of this ORF remains to be studied.There is increasing evidence that intergenic regions in prokaryotes code for unknown small proteins . ConsideListeria monocytogenes long 5′ UTRs were found to regulate virulence gene expression, e.g., the prfA 5′ UTR is a thermosensor allowing transcription of the transcriptional activator at 37°C and blocking it at lower temperatures . Nonetheless, the long 5′ IGRs could encode yet unknown functions or they may interact with different regulators to allow for differential expression of enterotoxin genes.Recently, several repeat regions that might encode novel riboswitches have been identified in . cereus , but nonB. cereus INRA C3 due to the presence of all three main enterotoxins nheABC, hblCDAB, and cytK-2 in this highly toxic strain . The promoter activities were assessed in the B. cereus INRA C3 background. The nhe operon was reported to contain two promoters and two PlcR binding sites  showed a strongly decreased activity . This strongly indicates the activating role of PlcR binding site 1, as confirmed by an 11-fold decrease of promoter activity . Thus, the highly conserved PlcR 1 site is responsible for PlcR-dependent nhe expression, while the less conserved PlcR 2 site contains a 2 bp central insertion . Deletion of the region containing the upstream-located promoter abolished transcription , indicating that this sequence is the active and essential promoter in B. cereus INRA C3. The luminescence intensity of a promoter fusion lacking both PlcR binding sites (Pnhe-s3) was similar to the intensity of the Pnhe-s2 construct, showing that the PlcR binding site 2 does not have an activating function by itself . A promoter fusion lacking the second putative promoter (Pnhe-Δ5′ UTR) resulted in an activity similar to the control plasmid pXen1 without any active promoter. This demonstrates that this fragment of the promoter region also contains one or more essential activating regulatory elements, which might include a ResD, cre, Fnr, PlcR 2 and a putative SinR binding site. In summary, transcription of the nhe operon strongly depends on the presence of the entire IGR, which underpins the necessity of a concerted interaction of all regulatory elements to regulate nhe expression.The functionality of the unusually long hbl 5′ UTR lux reporter fusions including partial or complete deletion of the 606 bp 5′ UTR were constructed and transferred into B. cereus INRA C3 . The full-length construct contains the highly conserved PlcR binding site 1 . Transcription of the hbl operon was previously shown to be PlcR-dependent (hbl-Δ5′ UTR) led to an increased promoter activity compared to the full-length construct . In contrast, deletion of the downstream half of the 5′ UTR (Phbl-Δ5′ UTR-down) resulted in luminescence levels comparable to the wild type situation. Therefore, the putative ResD, SinR, and Fnr binding sites do not influence promoter activity under our experimental conditions. Deletion of the upstream half of the 5′ UTR (Phbl-Δ5′ UTR-up) containing putative binding sites for CodY, ResD, SinR, and Fnr led to a stimulation of transcription, which was less pronounced compared to the deletion of the entire 5′ UTR. We conclude that the 268 bp region designated 5′ UTR-up  partially represses hbl transcription in B. cereus INRA C3. It has been shown previously that the deletion of the two-component system yvfTU leads to a reduced plcR, papR, nhe, nprB and plcB transcription but an increased hbl transcription  were used to compare the impact of a minimal medium (MOD) and a rich medium (CGY) on enterotoxin expression . In CGY the maximal promoter activity was reached during stationary phase, while peak activity was detected in the late exponential phase in defined MOD medium.Bioluminescent reporter strains containing the wild type 5′ IGRs of nhe and hbl enterotoxin promoter activity may show a trend to be slightly lower than in unsupplemented MOD . Free amino acids are also present in MOD, albeit in lower amounts, confirming that the depletion of free amino acids is a determining factor for enhanced enterotoxin promoter activity. When B. cereus INRA C3 was grown in MOD medium supplemented with 1% tryptone, promoter activities of Pnhe and Phbl were threefold and sevenfold lower than in MOD medium, respectively . These results point to an activation of enterotoxin promoter activity during unfavorable conditions, such as the absence of easily metabolizable amino acids. BCAAs and GTP activate the nutrient-sensitive repressor CodY , but no binding of CodY to Pnhe F4810/72 could be detected . Comparison with the consensus sequence  of around 700 nM. One potential repressor binding site with only one mismatch to the consensus sequence was found in Phbl-2 and Phbl-5 downstream of the promoter . Phbl-2 and Phbl-5 were both bound by CodY  with a KD of less than 250 nM. A gel retardation control experiment with the CodY-interacting inhA1 promoter region .Due to its size of over 600 bp Psequence  identifir region  also resnhe 5′ IGR of B. cereus INRA C3 (568 bp) and of B. cytotoxicus CVUAS 2833 (517 bp) at potential repressor binding positions with a KD of ~125 and ~330 nM, respectively . We additionally analyzed CodY affinity to the cytK promoter regions, but could neither detect specific interactions with PcytK-1 nor with PcytK-2 .In addition, CodY binds to a stretch of the B. cereus INRA C3 (our data), the B. cereus F4810/72 nhe 5′ IGR is not bound by CodY in vitro . The third potential binding site . These might influence CodY binding in vivo. Our results fit to the previously stated hypothesis that the difference in CodY binding strength is one of the determinant factors of the hierarchical CodY regulon expression . They show maximally one nucleotide mismatch to the B. subtilis consensus sequence . The sequence of the DNA-binding N-terminal domain of PlcR is conserved, while the regulatory C-terminal regions are variable to allow adaptation to changing conditions (In contrast, PlcR binding sites within the enterotoxin promoters reveal a much higher degree of conservation (nditions . TherefoB. cereus, PlcR is responsible for the general activation of enterotoxin transcription under unfavorable conditions (hbl-Δ5′ UTR still showed media-dependency (Supplementary Figure B. cereus strain.Being the major virulence regulator in nditions . In contnhe and hbl promoter activity under nutrient, especially amino acid limited conditions. While PlcR is the main virulence activator in B. cereus sensu lato, CodY may be used for a strain-specific fine-tuning of enterotoxin transcription via repression in response to specific environmental conditions. The unusually long promoter regions of nhe and hbl might be important for a concomitant interaction of several global regulators. The redox regulators ResD and Fnr, for instance, interact not only directly with their DNA recognition sites, but are also capable of interaction with each other (We present evidence for a high CodY-mediated ch other  and formch other .B. cereus is rarely consistent with transcriptional activity and is, moreover, highly strain-specific (However, the actual enterotoxin synthesis in specific . It mighspecific  to modiflux reporter experiments, and also with the design of the study. NJ was involved in compiling up the strain collection and critically revised the work. EF helped with the EMSA assays and critically revised the paper. SS was involved in the design of the study and wrote the paper together with M-EB. All authors worked on the manuscript. All authors finally approved the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.M-EB performed the experiments. VK helped with the construction of mutants and the


Altering the ability of the MYC transcription factor to bind to individual genes can customize the global gene expression output of cells. Related research article Lorenzin F, Benary U, Baluapuri A, Walz S, Jung LA, von Eyss B, Kisker C, Wolf J, Eilers M, Wolf E. 2016. Different promoter affinities account for specificity in MYC-dependent gene regulation. eLife5:e15161. doi: 10.7554/eLife.15161Image High levels of MYC protein in cells can promote the growth of tumorsCancer develops when cells in the body gain mutations that enable them to divide rapidly and form a tumor. Some of these mutations can increase the expression of particular genes in cells. For example, a gene that encodes a transcription factor protein called MYC is upregulated in most types of tumor, where it supports several aspects of tumor development.Transcription factors can bind to promoter regions at the start of genes to regulate the first step in gene expression, a process called transcription. MYC can bind to virtually all promoters in the genome and trigger widespread increases in gene activity. However, it is not clear whether MYC binding directly increases the transcription of all the genes , or whetPrevious studies have shown that producing more MYC in cell lines does not increase the expression of all genes equally . Now, inLorenzin et al. – who are based at the University of Würzburg and the Max-Delbrück-Center for Molecular Medicine – manipulated the levels of MYC in a human cell line called U20S, which normally has less of this protein compared to many cancer cell lines. Upregulating MYC in U2OS cells to match the levels seen in cancer resulted in MYC molecules being bound to virtually all promoters.Further experiments indicated that MYC binding to any promoter can alter the activity of the corresponding gene, but that MYC is better at binding to some promoters than others. At normal levels of MYC, promoters where MYC binds weakly (i.e. those with a low binding affinity) will have few MYC molecules bound to them. However, under the same conditions, high-affinity promoters may already be saturated with MYC molecules. If cells make more MYC, the number of MYC molecules binding to the lower-affinity promoters would increase, but the high-affinity promoters would be largely unaffected. Therefore, the increases in MYC production observed in cancer cells can alter the transcription of some genes more than others. Because every cancer starts with a different pattern of gene expression and a different level of MYC, this may result in each tumor having a distinct transcription profile.What determines how strongly MYC binds to a promoter? Lorenzin et al. modeled MYC binding across the genome, taking into account that MYC is known to bind strongly to a DNA motif called the E-box , and lesBesides WDR5, MYC interacts with many other proteins and protein complexes during transcription . Do thesFinally, normal cells contain less MYC than the cells used by Lorenzin et al. and so the majority of promoters in a normal cell are not saturated with MYC molecules. In cancer cells, the genes with the highest affinity for MYC, such as the ribosomal protein genes, are saturated with MYC and are also the most sensitive to its inhibition or reduction. Understanding exactly how MYC binding influences gene expression promises to aid efforts to develop new therapeutic strategies that inhibit its cancer-promoting actions, while preserving its normal responsibilities in cells.


The rs539846-A risk allele alters a conserved RELA-binding motif, disrupts RELA binding, and is associated with decreased BMF expression in CLL. These findings are consistent with rs539846 influencing CLL susceptibility through differential RELA binding, with direct modulation of BMF expression impacting on anti-apoptotic BCL2, a hallmark of oncogenic dependency in CLL.Chronic lymphocytic leukemia (CLL) is an adult B cell malignancy. Genome-wide association studies show that variation at 15q15.1 influences CLL risk. We deciphered the causal variant at 15q15.1 and the mechanism by which it influences tumorigenesis. We imputed all possible genotypes across the locus and then mapped highly associated SNPs to areas of chromatin accessibility, evolutionary conservation, and transcription factor binding. SNP rs539846 C>A, the most highly associated variant  reduces enhancer activity and RELA binding in CLL•BMF expressionrs539846-A confers lower  Kandaswamy et al. find that SNP rs539846 underlies the 15q15.1 chronic lymphocytic leukemia risk locus. Follow-up data demonstrate that rs539846 resides within a transcriptional enhancer and alters RELA binding at a conserved site. The rs539846-A risk allele results in reduced RELA-mediated enhancer activity and lower expression of BCL-2-modifying factor. BMF) at 15q15.1 being highly robust (Although genome-wide association studies (GWASs) frequently have identified statistically significant associations within non-coding regions of the genome, the underlying causal variant has been elucidated in only a few instances. GWASs of chronic lymphocytic leukemia (CLL) have identified 31 risk loci, with the signal annotating B cell lymphoma 2 (BCL2)-modifying factor , mapped to the third intron of BMF and was in high linkage disequilibrium  with the published SNP, rs8024033. We verified the fidelity of imputed rs539846 genotypes by Sanger sequencing in a subset of 176 CLL cases, demonstrating >95% concordance.A previous GWAS reported an association between rs8024033 at 15q15.1 and CLL risk . To refi−7 . The leaBMF locus, we repeated association testing conditional on rs539846 genotypes, observing no significant variants  with rs539846, based on epigenetic data from lymphoblastoid cell lines  and priSince causal SNPs that drive GWAS associations may function by altering transcription factor binding, we examined whether 15q15.1 candidate risk SNPs disrupt predicted JASPAR motifs. This revealed that rs539846 alters a highly conserved base within a putative RELA-binding motif  B. EncyclNo other candidate CLL risk variant at the 15q15.1 locus showed the unique combination of evolutionary conservation, active enhancer localization, and disruption of a transcription factor-binding motif, thus re-affirming that rs539846 is the single best causal SNP candidate.We next performed luciferase reporter assays to determine the effect of rs539846 on enhancer activity. MEC1 cells transfected with constructs containing the risk A allele demonstrated a significant reduction in normalized luminescence compared to the C allele  dataset, revealing a correlation between RELA and BMF expression in CLL  experiments in MEC1 cells, where knockdown of RELA was accompanied by a significant reduction in BMF mRNA  data from 30 CLL cases, finding no evidence of aberrant splicing. We also found no differences in the splicing levels of known BMF exons between the rs539846 risk allele and non-risk allele homozygotes  mutation status; expression levels of CD38, ZAP70, and CLLU1; as well as somatic genomic abnormalities . We found no association between the rs539846 genotype and these features in a subset of UK-GWAS and ICGC study cases  is a sub-unit of the necrosis factor kappa B (NF-κB) protein complex. This transcription factor complex regulates expression of genes involved in biological processes, such as proliferation, survival, and inflammation. NF-κB signaling is constitutively active in CLL , while h disease .BMF is transcriptionally regulated by RELA, in keeping with the somewhat counter-intuitive observation that levels of pro-apoptotic BMF are high in CLL .−4), and those evaluated to be of non-European ancestry (using HapMap version 2 populations as a reference) were excluded.We made use of data from two published CLL GWASs: (1) UK-CLL-1 , a scan 2 > 0.2 with rs539846). To validate imputed rs539846 genotypes, we performed Sanger sequencing in 176 CLL GWAS cases. Primers are listed in GWAS data were imputed using 1000 Genomes Project phase 1 integrated release 3 , and UK1IGHV (>90% tumor cell content), also were examined (http://www.blueprint-epigenome.eu). We also used HaploReg (r2 > 0.2 in 1000 Genomes EUR reference panel) annotate transcription factor-binding sites or enhancer elements. We assessed sequence conservation using GERP (+ and CD20+) and T cell  lineages , histone modification, and transcription factor ChIP-seq data from the ENCODE project . In addiexamined . DetaileHaploReg  to examiing GERP  and Phasing GERP . We searlineages .We made use of publicly available Hi-C data on GM12878 cells , based oMEC1 (human CLL) cells were grown in Iscove’s modified Dulbecco’s medium (Life Technologies) supplemented with 10% fetal calf serum.http://mnlab.uchicago.edu/4Cpd/), we identified a viewpoint adjacent to SNP rs539846. Primary and secondary restriction enzymes were DpnII and HindIII . Primers are listed in 4C-seq libraries were prepared as described , using tluc2 promoter vector using Gateway technology (Life Technologies). Reporter constructs were introduced into MEC1 cells by nucleofection, using program X-01 on the Amaxa Nucleofector I (Amaxa Biosystems). Typically, 5 × 106 cells were resuspended in 100 μl Cell Line Nucleofector Solution V and mixed with 3 μg reporter plasmid DNA and 60 ng internal control plasmid (pRL-SV40). Transiently transfected cells were grown for 24 hr before assaying with the Dual-Luciferase Reporter Assay System (Promega) and the Fluoroskan Ascent FL plate reader (Labsystems). Relative luciferase activity was calculated as the ratio of luminescence from the experimental reporter to that of the control reporter. Each transfection experiment was repeated three times and statistical significance was calculated using the Student’s t test.Allele-specific fragments of a 591-bp region spanning rs539846 were amplified from human genomic DNA using primers detailed in Nuclear protein was extracted from MEC1 cells using NE-PER nuclear and cytoplasmic extraction kits (Thermo Fisher Scientific). Infrared dye DY-682-labeled (Eurofins Genomics) and unlabeled (Life Technologies) complementary oligonucleotides flanking rs539846 (5′-GAGGGGACTTT[C/A]CCTCCCCAAAC-3′ and 5′-GTTTGGGGAGG[G/T]AAAGTCCCCTC-3′) were annealed to generate double-stranded EMSA probes. Each 20 μl binding reaction contained 50 fmol labeled target DNA, 1× binding buffer . The reaction mix was incubated in the dark for 30 min at room temperature. Competition assays were performed by adding 200-fold molar excess of unlabeled probes to the binding reaction. Super-shift EMSAs were conducted by adding 2 μg RELA antibody (Santa Cruz Biotechnology) to the binding reaction and incubating for 15 min prior to the addition of labeled probe. Post-incubation, 2 μl 10× Orange loading dye (LI-COR Biosciences) was added to the reaction mix, and the DNA-protein complexes were resolved by electrophoresis on a 6% DNA retardation gel (Life Technologies) in 0.5× Tris-borate-EDTA (TBE) at 4°C. Gels were imaged using the Odyssey Fc Infrared Imaging System (LI-COR Biosciences).BMF and RELA transcript levels in the ICGC dataset  and normalized to GAPDH. The experiment was repeated three times. Primer sequences are detailed in siRNA targeting RELA and a control siRNA  were obtNOTCH1 mutation, and SF3B1 mutation statuses were determined in a subset of UK-CLL-1 patients who were participants in the LRF CLL4 Trial (IGHV mutation status was determined as per BIOMED-2 protocols (Logistic regression was used to test the association between the rs539846 genotype and prognostic factors. Trisomy 12, 13q14 deletion, 6q21 deletion, 11q23 deletion, 17p13 deletion, CD38 expression, ZAP70 expression, CLLU1 expression, L4 Trial , as prevL4 Trial . IGHV murotocols  in a subrotocols . In accorotocols , we clasR.S.H. designed the study and drafted the manuscript with contributions from all other authors. R.K., G.P.S., H.E.S., and J.B.S. performed the experiments. P.J.L., H.E.S., R.K., G.P.S., and G.M. performed the bioinformatics analysis. D.C. and J.M.A. performed sample recruitment. R.S.H. obtained financial support. In Spain, S.B., J.I.M.-S., D.M.-G., I.S., X.S.P., and J.G.-A. performed data analysis. C.L.-O. and E.C. supervised the data analysis.


RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3’ RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3’ end RNA processing and/or degradation at specific targets.The exosome and its nuclear specific subunit Rrp6 form a 3’-5’ exonuclease complex that regulates diverse aspects of RNA biology including 3’ end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3’ end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3’ end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of  Saccharomyces cerevisiae, termination of RNAPII-transcribed RNAs ≤1000 bases requires the NNS complex , processing by the exosome, and the nuclear specific catalytic subunit, Rrp6. It has been shown that Rrp6 interacts directly with Nrd1, but whether or not Rrp6 is required for NNS-dependent termination is unclear. Loss of Rrp6 function may result in extension (or inhibition of termination) of NNS-dependent transcripts, or Rrp6 may only function after the fact to carry out RNA 3’ end processing. Here, we performed in-depth differential expression analyses and compare RNA-sequencing data of transcript length and abundance in cells lacking RRP6 to ChIP-exo analysis of RNAPII localization. We find many transcripts that were defined as unterminated upon loss of Nrd1 activity are of similar length in rrp6Δ, and expression levels of downstream genes are significantly decreased. This suggests a similar transcription interference mechanism occurs in cells lacking either Nrd1 or Rrp6. Indeed we find increased RNAPII located downstream of its termination site at many know Nrd1-regulated transcripts. Overall, our findings clearly demonstrate that Rrp6 activity is required for efficient NNS termination in vivo.RNAPII is responsible for transcription of protein-coding genes and short, regulatory RNAs. In  Saccharomyces cerevisiae . I. IRRP6 rrrp6Δ, and our dataset confirms these findings. Previously published data by Kim et al. has shown a large extension for snR13 in SEN1 and NRD1 mutant strains, with the snR13 transcript extended across the coding region of TRS31 .,62.rrp6Δas snR71 . With stanscript . We see wnstream . This isnotation . Interesnotation , suggestRRP6. ChIP-exo analysis of RNAPII (Rpb3-FLAG) clearly show that RNAPII terminates downstream of its normal stopping point in WT cells at specific snRNAs leading to mislocalization and causing transcription interference at downstream genes  and persistence through the coding region (arrow #2) in rrp6Δ (HRP1 RNA levels relative to WT (SRG1-SER3 region. SRG1 (SER3 regulatory gene 1) is a known non-coding RNA whose transcription down-regulates expression of SER3 [SRG1 RNA is bound by both Nrd1 and Nab3 just prior to the SER3 transcribed region, which could then terminate SRG1 transcription to prevent interference with SER3 [rrp6Δ cells with increased RNAPII occupancy in the SER3 coding region  targets was also observed at RG1-SER3 .. The RNin rrp6Δ .. RNA-Seve to WT .. Anothe of SER3 . SRG1 RNith SER3 ,47. SRG1ith SER3 . ChIP-exg region .. An inc6Δ cells . Our datsruption .. These cis through transcript extension across the sense gene promoter [FMP40 transcript has been previously shown by several groups to be regulated by an Nrd1-terminated antisense transcript initiating at the 3’ end of FMP40 [FMP40 antisense transcript  is readily detectable in our RNA-sequencing data from WT cells and it is significantly upregulated in rrp6Δ cells as has been previously reported , supporting the hypothesis that Nrd1-termination is not as efficient in rrp6Δ mutants. To determine the length of the antisense transcript in rrp6Δ and compare to an nrd1-ts mutant, we performed northern blot analysis with strand specific oligonucleotide probes to FMP40 and its antisense transcript YPL222C-A  aresignificantly longer than CUTs, our data clearly shows that specific NNS-dependent transcripts rely on Rrp6 for proper termination while others do not. For instance, snr71 transcripts show a requirement for Rrp6, Nrd1, and Ssu72 for termination, while snR13 does not require Rrp6  at many locations and the transcripts observed following deletion of the nuclear specific 3’-5’ exonuclease subunit Rrp6 genome-wide. The tight coupling of the NNS pathway with exosome function has been previously characterized; however our findings are the first to demonstrate on a large scale that Rrp6 function is required for termination through the NNS pathway at specific transcripts. We have also shown that deletion of  strains . Previou as well ,58. The  as well . Future rrp6Δ, the majority of which (76%) were upregulated. These data will serve as a valuable resource to determine the diverse roles of the NNS pathway and Rrp6 in transcriptome-wide gene expression regulation. Our dataset also shows that the increased dynamic range and resolution provided by RNA-Seq and ChIP-exo gives a distinct advantage in precise mapping of the transcripts that accumulate following deletion of RRP6 [In this study, we have performed multiple biological replicates (n = 4) to provide highly accurate differential expression analysis for all classes of annotated transcripts. In addition, we have included differential expression analysis of 1215 antisense transcripts that were significantly changed in  of RRP6 ,11,48. U of RRP6 . This cl of RRP6 ,74,75. Brrp6Δ cells we observed proper termination for snR13 although termination was defective in other NNS-pathway components  [RRP6 deletion Rpb3–3XFLAG strains were produced by amplification of the RRP6 knockout cassette from the yeast knockout collection RRP6 deletion strain and transformation into the wild-type Rpb3-FLAG strain. RRP6 knockout was confirmed by PCR genotyping. RNA was extracted using the hot acid phenol method. Briefly, strains were grown in 100ml YPD medium to an OD600 of 0.8. Cells were collected by centrifugation, washed, and resuspended in 10ml AE buffer  in a Nalgene phenol-resistant 50ml tube. 800μl 20% SDS and 10ml cold acid phenol were added to each sample and mixed well by vortexing. Samples were incubated at 65°C for 10 minutes with vortexing every minute then cooled on ice for 5 minutes. Samples were centrifuged for 15 minutes at 10,000 rpm. The top phase was transferred to a pre-spun 50ml 5 PRIME Phase Lock Gel tube (Ref # 2302870). 13ml chloroform was added and well mixed before centrifuging for 10 minutes at 3000 rpm. The top phase was poured into a new phenol-resistant tube, and 1/10 volume sodium acetate at pH 5.2 and equal volume room temperature isopropanol was added. The precipitated RNA was collected by centrifuging for 45 minutes at 12,000 rpm. The pellet was washed with 70% ethanol, allowed to dry in a fume hood, and resuspended with molecular biology grade water. The Ambion DNase-turbo kit was used to degrade any contaminating DNA. The quality of the samples was determined with an Agilent Bioanalyzer before preparation of the sequencing libraries.All yeast strains used are isogenic to BY4741. systems) . The Rpbsystems) . RRP6 derrp6Δ strains were grown to an OD600 = 0.8–1 prior to crosslinking with formaldehyde . Immunoprecipitation was performed with 50uL of anti-FLAG agarose (Sigma). Subsequent sample processing steps including exonuclease treatment and sequencing library preparation were performed as previously described [Chromatin IP followed by exonuclease treatment was performed using the protocol described by Rhee and Pugh with the following modifications . Rpb3-FLescribed .FMP40 and YPL22C-A followed by scanning using a phosphorimager .30ug of total RNA was loaded per lane on a 1.5% and separated by electrophoresis at 120 volts for 1 hour at 4°C. The RNA was transferred to Bio-Rad Zeta-Probe blotting membranes by capillary overnight. Transfer efficiency was determined by Methylene Blue staining. DNA oligonucleotide probes listed in Standard methods were used for RNA-seq library construction, EZBead preparation and Next-Gen sequencing, based on Life Technologies SOLiD5500xl system. Briefly, five microgram of total RNA per sample  was applied in library preparation. rRNA was first depleted using the standard protocol of RiboMinus Transcriptome Isolation Kit for yeast , and rRNA-depleted RNA was concentrated with the PureLink RNA Micro Kit  using 1 volume of Lysis Buffer and 2.5 volumes of 100% ethanol. Following the rRNA depletion, whole transcriptome library was prepared and barcoded per sample using the standard protocol of SOLiD Total RNA-seq Kit . Each barcoded library was quantified by quantitative PCR using SOLiD Library Taqman qPCR Module , and pooled in equal molarity. Fifty microliters of 500 pM of pooled library was used in subsequent EZBead preparation, which involves bead emulsion, bead library amplification, and bead enrichment using Life Technologies EZ Bead E120 System (Cat# 4465571). Approximately six hundred million enriched beads then were deposited onto each lane of a 6-lane SOLiD5500xl flow chip. And finally sequencing by ligation was carried out using standard single-read, 5’-3’ strand-specific sequencing procedure (75b-read) on SOLiD5500xl Sequencer.Saccharomyces cerevisiae reference genome sacCer3 using in-house mapping pipelines that utilizes bfast-0.7.0a [The libraries were sequenced on Solid 5500XL. The resulting 75 bp solid reads were mapped to t-0.7.0a . Brieflyt-0.7.0a . Differet-0.7.0a . All rawrrp6Δ were used for subsequent differential expression analysis to generate the final dataset in Following data alignment, snRNA transcripts were manually inspected individually using the Integrative Genomics Viewer ,85. For http://gostat.wehi.edu.au/cgi-bin/goStat.pl) to search for the top 30 most over-represented GO terms and to obtain p-values to indicate the significance of enrichment .(A) RNAs annotated as NUTs, a classification based on the dependence of Nrd1 for appropriate termination are shown as aqua dots while all other transcript annotations are shown as black dots. (B) Western blot analysis of whole cell extracts prepared from Rpb3-FLAG WT and (TIF)Click here for additional data file.S2 Figet al 2013 (top two panels) and our RNA-Seq data (bottom two panels) at snR3-STR2 region, forward strand reads only. The location and transcription direction of all annotations within this region are diagrammed below. Processed length of snRNAs and mRNAs are in black, snRNA-extended regions are in green (labeled “ET”), NUTs are in blue, CUTs are in gray, and arrows indicate annotated transcript state site and direction of transcription. The dotted green line marks the 3’ end of the extended snR3 annotation in rrp6Δ. (B) Average normalized read counts ± standard deviations for transcripts in this region that are not significantly changed in rrp6Δ versus wild-type (n = 4). The colors of the bars correspond to the color representing the annotation.(A) Mapped reads obtained from our in-house alignment see  section (TIF)Click here for additional data file.S3 Figrrp6Δ. Decreased NNS-termination at select snRNAs results in longer transcribed region, extending over transcription start site of downstream convergent gene, GeneX. The hypothesized resulting increased localization of the transcription machinery interferes with initiation at the TSS of GeneX (indicated with a red ‘X’). (B) Strand-specific northern blot analysis using a 5’ end labeled DNA oligo probe specific to the processed region of snR11 directly comparing rrp6Δ to mutants known to be defective in Nrd1-dependent termination. The 26S and 18S ribosomal RNAs are shown as a loading control (bottom). (C) Graphical representation of strand-specific RNA-seq reads mapped to snR11-ICY1 region. Reads mapped to the positive strand are on top in red, while reads mapped to the negative strand are on the bottom in blue. The location and direction of transcription for all analyzed annotations are diagrammed below the graphs to scale. Processed length of snRNAs and mRNAs are in black, snRNA-extended transcripts, including pre-snRNAs and termination read-through products, are in green (labeled “ETs”), NUTs are in aqua, CUTs are in gray, SUTs are in dark blue, SRTs are in purple, and bent arrows indicate direction of the TSS. The dotted black lines mark the transcription start sites (TSS) of CMC4. (D) Rpb3-FLAG localization as determined by ChIP-exo sequencing reads mapped to the same region and aligned to (C). Wild-type normalized read counts are in black, and rrp6Δ are in orange. Arrows indicate areas of interest.(A) Diagram showing proposed mechanism where down-regulation of GeneX results from faulty termination of snRNA in (TIF)Click here for additional data file.S4 FigHRP1 region. Wild-type reads are in black, and rrp6Δ are in orange. The location and direction of transcription for all analyzed annotations are diagrammed below the graphs to scale. Length of mRNAs including untranslated regions are in black. (B) Graphical representation of strand-specific RNA-seq reads mapped to HRP1 transcribed region. Reads mapped to the positive strand are shown in red. No reads were mapped to the reverse strand at HRP1.(A) Rpb3-FLAG localization as determined by ChIP-exo sequencing reads mapped to the (TIF)Click here for additional data file.S5 FigSRG1-SER3 region. Wild-type normalized read counts are in black, and rrp6Δ are in orange. The location and direction of transcription for all analyzed annotations are diagrammed below the graphs to scale. Length of mRNAs including untranslated regions are in black, CUTs are in gray, and SRTs (Ssu72 regulated transcripts) are in purple. Note that SRG1 and SER3 annotations are overlapping. (B) Graphical representation of strand-specific RNA-seq reads mapped to SRG1-SER3 transcribed region. Reads mapped to the positive strand are shown in red while reverse reads are shown in blue.(A) Rpb3-FLAG localization as determined by ChIP-exo sequencing reads mapped to the (TIF)Click here for additional data file.S1 Table2rrp6Δ/WT ratio (i.e. fold change), as well as p-values and false discovery rate (FDR), all calculated from four replicates by the EdgeR program as discussed in the methods. Class abbreviations are ORF-T: open reading frame transcript, AST: antisense transcript, NUT: Nrd1-unterminated transcript, SRT: Ssu72-restricted transcript, SUT: stable unannotated transcript, CUT: cryptic unstable transcript, sn/snoRNA-ET: Extended region of an sn/snoRNA. Transcript name listed is the systematic name where possible. “AS_” preceding the name designates antisense transcripts, NUTs, CUTs, SUTs, and SRTs are listed as the number provided by their original publications [Table includes differential expression data expressed in logications , 46, 59.(PDF)Click here for additional data file.S2 TableTable includes GO identification number (“Best GOs”), number of hits from GOStat analysis of our down-regulated protein coding gene list matching the GO ID (“Count”), total number of genes assigned the GO ID , p-value for the GO term, and the descriptor for the GO ID. Information calculated using GOstat, as described in the methods.(PDF)Click here for additional data file.S3 Table2rrp6Δ/WT ratio (i.e. fold change) as well as p-values and false discovery rate (FDR), all calculated from four replicates by the EdgeR program as discussed in the methods. Table also includes standard gene names (or acronyms) for clarity. Significantly downregulated transcripts are in red-shaded cells whereas upregulated sn/snoRNAs are in green-shaded cells.Table includes differential expression data expressed in log(PDF)Click here for additional data file.S4 Table2rrp6Δ/WT ratio (i.e. fold change), as well as p-values and false discovery rate (FDR), all calculated from four replicates by the EdgeR program as discussed in the methods. Significantly downregulated sn/snoRNAs are in red-shaded cells whereas upregulated sn/snoRNAs are in green-shaded cells.Table includes differential expression data expressed in log(PDF)Click here for additional data file.S5 TableOligonucleotide sequences for each strand specific probe used for end labeling and northern blot analysis.(PDF)Click here for additional data file.


The rearrangement of nucleosomes along the DNA fiber profoundly affects gene expression, but little is known about how signalling reshapes the chromatin landscape, in three-dimensional space and over time, to allow establishment of new transcriptional programs.Using micrococcal nuclease treatment and high-throughput sequencing, we map genome-wide changes in nucleosome positioning in primary human endothelial cells stimulated with tumour necrosis factor alpha (TNFα) - a proinflammatory cytokine that signals through nuclear factor kappa-B (NF-κB). Within 10 min, nucleosomes reposition at regions both proximal and distal to NF-κB binding sites, before the transcription factor quantitatively binds thereon. Similarly, in long TNFα-responsive genes, repositioning precedes transcription by pioneering elongating polymerases and appears to nucleate from intragenic enhancer clusters resembling super-enhancers. By 30 min, widespread repositioning throughout megabase pair-long chromosomal segments, with consequential effects on three-dimensional structure (detected using chromosome conformation capture), is seen.Whilst nucleosome repositioning is viewed as a local phenomenon, our results point to effects occurring over multiple scales. Here, we present data in support of a TNFα-induced priming mechanism, mostly independent of NF-κB binding and/or elongating RNA polymerases, leading to a plastic network of interactions that affects DNA accessibility over large domains.The online version of this article (doi:10.1186/s13059-014-0536-6) contains supplementary material, which is available to authorized users. The arrangement of nucleosomes along the chromatin fibre profoundly affects genome function ,2. For eNucleosome positioning can be mapped genome-wide at single-nucleosome resolution using micrococcal nuclease digestion followed by sequencing (MNase-seq) ,12. We aWe focused on spatial and temporal changes in chromatin architecture during the critical window when ‘immediately-early’ proinflammatory genes become active: 0, 10 and 30 min post-stimulation. In agreement with the idea that nucleosomes reposition in coincidence with (and/or as a result of) transcription factor binding at cognate sites , we did HUVECs grown to confluence were serum-starved (to promote synchrony), stimulated with TNFα for 0, 10 or 30 min, and treated with MNase to release mononucleosomes. The purified DNA  and selng sites  and confng sites , were amng sites ,26,27. B2-fold ; constitutively expressed genes (±0.01 log2-fold) provided controls  and were required to change by at least ±0.6 logNFKBIA, showed nucleosomes already repositioned by 10 min, and changes in nucleosome occupancy became more pronounced at 30 min, when density decreased throughout the locus as NF-κB binding increased . By contrast, profiles on a typical down-regulated gene, LIN37, became heightened and more defined . This held true for other up- or down-regulated genes, whilst those of constitutively expressed loci varied little  read profiles along a typical immediate-early up-regulated gene, Global changes in genic nucleosome occupancy were assessed using ‘metagene’ analyses, by aggregating profiles from all up- or down-regulated genes. In up-regulated genes, the first few nucleosomes downstream of the promoter became more precisely positioned (most likely as transcription start site (TSS)-proximal nucleosomes form well-positioned arrays ), and ocThe transcriptional activation of five long genes of >100 kbp has been studied in detail in this experimental model –20. FollEXT1, 41, 38 and 24 peaks were called at 0, 10 and 30 min, respectively; Additional file EXT1 fell by 12% after 30 min; Additional file To simplify analysis, we initially applied the PeakPredictor algorithm  to our MALCAM and 221-kbp SAMD4A . Genes were divided into 5-kbp non-overlapping windows, and changes in each window scored relative to (background) levels of nucleosome repositioning occurring in transcriptionally inert genomic segments (see Methods). This revealed a decrease in nucleosome occupancy (hereafter termed depletion), which was evident throughout 186-kbp We next examined whether NF-κB binding was enriched in kilobase pair-long genomic segments displaying reduced MNase-seq signal. ChIP-seq collected 10 min post-stimulation showed sparse binding of p65 . TheSAMD4A and constitutively expressed ΕDN1 as viewpoints. For the SAMD4A TSS, we showed previously that stimulation induces development of new contacts throughout the genome , while 3C-seq, p65 ChIP-seq and total (ribo-depleted) RNA-seq data generated here can be accessed at the SRA archive under accession number [SRA: SRP044729].


After six months of selective culture, pEHZ-LDLR-LDLR was recovered from FH-iPSC-LDLR and transfected into Ldlr-deficient CHO-a7 cells, which then exhibited feedback-controlled LDLR-mediated endocytosis. To quantify endocytosis, FH-iPSC ± LDLR were differentiated into mesenchymal cells (MC), pretreated with excess free sterols, Lovastatin, or ethanol (control), and exposed to DiI-LDL. FH-MC-LDLR demonstrated a physiological response, with virtually no DiI-LDL internalization with excess sterols and an ~2-fold increase in DiI-LDL internalization by Lovastatin compared to FH-MC. These findings demonstrate the feasibility of functionalizing genetically deficient iPSC using episomal plasmids to deliver physiologically responsive transgenes.Acquiring sufficient amounts of high-quality cells remains an impediment to cell-based therapies. Induced pluripotent stem cells (iPSC) may be an unparalleled source, but autologous iPSC likely retain deficiencies requiring correction. We present a strategy for restoring physiological function in genetically deficient iPSC utilizing the low-density lipoprotein receptor (LDLR) deficiency Familial Hypercholesterolemia (FH) as our model. FH fibroblasts were reprogrammed into iPSC using synthetic modified mRNA. FH-iPSC exhibited pluripotency and differentiated toward a hepatic lineage. To restore LDLR endocytosis, FH-iPSC were transfected with a 31 kb plasmid ( Induced pluripotent stem cells (iPSC) derived from patients are a unique, potential alternative for replacing diseased tissue because of their virtually unlimited supply, differentiation potential, and tolerance by the immune systemFamilial Hypercholesterolemia (FH) is one example of a monogenic autosomal dominant disorder that affects low-density lipoprotein cholesterol (LDL) metabolism3LDLR physiologically controlled by 10 kb upstream genomic regulatory control sequence and (b) the minimum number of required Epstein-Barr Virus (EBV) replication and retention sequences (Epstein-Barr Nuclear Association 1 (EBNA1) and origin of plasmid replication (oriP))We used FH as a model system for interrogating the LDLR functionalization of FH-iPSC. We restored FH-iPSC LDLR-mediated endocytosis using a novel 31 kb plasmid containing (a) wild-type OCT4, SOX2, KLF4, c-MYC, and LIN28in vitro spontaneous differentiation and in vivo teratoma formation, with both assays demonstrating formation of the three germ layers . Karyotyping  were examined to verify their inherent LDLR dysfunction using an LDL internalization assay . After pyotyping  and DNA FH patients benefit from a variety of pharmacological and biomechanical interventions principally targeting liver hepatocytes. Thus, we wanted to determine if FH-iPSC could differentiate towards a hepatic lineage using a modified version of an established five-stage hepatic differentiation protocol that mimics key developmental embryologic cues 11. At thDuring each round of FH-HLC production, we noticed the presence of differentiating cells that took on an increasingly granular, vacuolar appearance  that looked lipid-like – an observation consistent with that of Fattahi and colleaguesin vivo (−/− × LDLR−/− double-knockout mice for 2 weeks. We labeled the explanted constructs with Albumin-488 and UEA-1-Cy5 (a lectin that binds to human endothelium). Fluorescence confocal microscopy showed that FH-HLC had engrafted and expressed Albumin in the presence of SVF, whereas FH-HLC did not survive in the absence of SVF. Together, these findings suggest that FH-iPSC can differentiate down a hepatic lineage and, given the proper stromal and vascular support, survive and engraft in vivo.We then asked if FH-HLC could engraft in vivo . We prevLDLR transgene expression for treating FH has not yet achieved a cure1719pEHZ-LDLR-LDLR to impart physiological responsiveness by 10 kb of up-stream genomic control sequences821FH-iPSC retain the same genetic dysfunction as their source patient fibroblasts and therefore offer limited therapeutic value with regards to restoring LDLR-mediated endocytosis. Since constitutive pEHZ-LDLR-LDLR physiological restoration of receptor-mediated endocytosis in the Ldlr-deficient cell line CHO-a7  its function is independent of genomic integration.pEHZ-LDLR-LDLR had an effect on the inherent pluripotence of our FH-iPSC. Therefore, we assessed expression of TRA-1–60 on transfected cells cultured for over a year under antibiotic selection pressure. Like their non-transfected FH-iPSC counterparts , differences in internalization between the two groups (FH-MC: 188.57 ± 74.33 vs. FH-MC-LDLR: 438.82 ± 180.57 fluorescence units/mg protein (FU/mg); mean ± SEM) reflecting the cellular lipid requirement due to the lipoprotein-deficient media environment. This difference was amplified when the cells were exposed to the additional stimulus of Lovastatin. Transgene-expressing cells displayed a statistically significant increase (p = 0.035) in specific internalization versus their non-transfected counterparts (FH-MC: 321 ± 61.7 FU/mg vs. FH-MC-LDLR: 526.15 ± 21.18 FU/mg). As expected, free sterols suppressed DiI-LDL internalization to a statistically equivalent level regardless of transgene presence  since free sterol internalization is receptor independentp = 0.00004) and significant with regards to the ethanol control (p = 0.02). Presenting these differences another way, transfected FH-MC-LDLR demonstrated 1.63- and 2.33-fold increases in specific DiI-LDL internalization over FH-MC in the presence of Lovastatin and ethanol, respectively. These findings suggest that the pEHZ-LDLR-LDLR plasmid functionally restores LDL receptor-mediated endocytosis without direct genetic manipulation in FH-iPSC-derived progeny.To facilitate the quantification of Here, we present a proof-of-concept strategy with broad implications for generating and functionally restoring genetically deficient iPSC utilizing transient reprogramming and non-viral, episomal vectors with genomic transgene control, respectively. The significant findings of this project are that (a) an episomal transgene plasmid can be maintained in an extra-chromosomal position within iPSC, (b) the plasmid does not diminish iPSC pluripotency, and (c) genomic control of plasmid transgene expression can restore physiologic functionality to genetically deficient cells.We used modRNA to reprogram FH-fibroblasts into FH-iPSC. modRNA is reported to yield one of the highest reprogramming efficiencies, approaching 2% in normal fibroblasts1229LDLR in E1-deleted adenovirusLDLR gene therapy with constitutive transgene expression driven by hepatocyte-specific promoters (such as α1-antitrypsin or thyroid binding globulin) did not detect cytotoxic lipid accumulations31LDLR expression with hepatocyte-specific promoters may provide some unidentified mechanism for protecting against lipid accumulationWith regards to genetic correction, attempts to resolve LDLR deficiency via gene therapy approaches have primarily relied on viral vectors3132333119LDLR genetic deficiency. The inclusion of genomic sequences allows for precise physiological control of the transgene and enhances long-term LDLR expressionpEHZ-LDLR-LDLR, is maintained as an episome by the inclusion of the EBV retention and replication sequences EBNA1 and oriPtrans regulatory protein is the only EBV protein required for retained episomal functionEpisomal plasmids containing genomic DNA expression control sequences are an alternative approach to addressing the LDLR expression is tightly controlled by a feedback mechanism within the cellpEHZ-LDLR-LDLR plasmid restored receptor-mediated endocytosis in FH fibroblasts and persisted long-term in non-dividing hepatocytes in vivopEHZ-LDLR-LDLR restores physiologically sensitive receptor-mediated endocytosis in FH-iPSC and that this function is retained after differentiation into various progeny.As previously mentioned, LDLR expression.We also demonstrate the effectiveness of large plasmid transfection into reprogrammed iPSC. Although transfection of the source FH fibroblasts before reprogramming is an option, it is unknown if  a large 31 kb episomal plasmid would affect reprogramming or if long-term plasmid retention would be compromised. Directly transfecting the FH-iPSC derivatives was also an option, but the cells would still require the time and culture for antibiotic selection, both of which are problematic with hepatocytesin vitro with antibiotic selection until a minimally proliferative state is reached prior to implantation, or (b) generate a liver-tissue mimic with stromal and vascular support that facilitates selection and phenotype stabilization prior to implantation.A major issue to address with our approach is plasmid retention, a common problem with using episomal viral vectors for direct gene therapy to the liverThough our approach specifically targets the LDLR dysfunction in FH, a strategy using episomes with genomic control of transgenes can be tailored to other biological conditions or clinical deficiency with potential for amelioration by iPSC intervention, further enhancing its clinical potential in tissue engineering applications.All animal procedures were conducted in compliance with University of Louisville School of Medicine IACUC-approved protocols and NIH guidelines. All recombinant DNA work was approved by the University of Louisville Institutional Biosafety Committee and performed in accordance with NIH guidelines.et al.FH-iPSC were plated to confluence in Matrigel-pretreated wells of a 6-well tissue culture plate or 6 Permanox chamber slides (Sigma-Aldrich). Each well of the 6-well plate  and each Permanox slide (used for immunocytochemistry) corresponded with one of the six stages of differentiation . To generate HLC, we used a modified version of the protocols and medias proposed by Song FH-HLC derived from transfected FH-iPSC (FH-HLC-LDLR) were starved overnight in 5% lipoprotein-deficient serum supplemented with 2 μM of Lovastatin, excess sterols (12 μg/ml cholesterol and 0.6 μg/ml 25-hydroxysterol), or ethanol (vehicle control). The following day, the starvation media was replaced with 5 μg/mL DiI-LDL in DMEM-HG for 4.5 hours. Cells were thoroughly washed with di-cationic PBS prior to imaging via an Olympus IX81 fluorescence microscope.Ldlr-deficient CHO-a7 cells  via Lipofectamine 2000 to demonstrate non-integration and preservation of plasmid functionality after rescue.Plasmid rescue was performed on transfected FH-iPSC-LDLR using methods described by Lufino and colleaguesMesenchymal cells were generated as previously described125Iodinated-LDL. Specific receptor-mediated internalization was determined by subtracting the internalization of [DiI-LDL + 50-fold excess of unlabeled LDL (Alfa Aesar)] from the internalization quantified with DiI-LDL alone. Cells were then lysed with 250 μl 0.1 N NaOH . Fluorescence was quantified using a 96-well plate reader (BioTek), with the excitation and emission set to 514 nm and 550 nm, respectively. Fluorescence units were normalized to total protein content, which was determined via a DC Protein Assay kit (Bio-Rad).FH-MC±LDLR were plated in gelatin-coated wells of a 24-well tissue culture plate. We utilized an established protocolt-test and confirmed via one-way ANOVA using SigmaPlot (Systat Software).Experiments were conducted in triplicate with duplicated data points per experiment. Values are presented as means ± S.E.M. Significance was determined using a Student’s Additional procedures can be found in the How to cite this article: Ramakrishnan, V. M. et al. Restoration of Physiologically Responsive Low-Density Lipoprotein Receptor-Mediated Endocytosis in Genetically Deficient Induced Pluripotent Stem Cells. Sci. Rep.5, 13231; doi: 10.1038/srep13231 (2015).


TpnC41C, a Troponin C gene expressed in the tubular jump muscles, but not in the fibrillar flight muscles. We identified a 300-bp promoter fragment of TpnC41C sufficient for the fiber-specific reporter expression. Through an analysis of this regulatory element, we identified two sites necessary for the activation of the enhancer. Mutations in each of these sites resulted in 70% reduction of enhancer activity. One site was characterized as a binding site for Myocyte Enhancer Factor-2. In addition, we identified a repressive element that prevents activation of the enhancer in other muscle fiber types. Mutation of this site increased jump muscle-specific expression of the reporter, but more importantly reporter expression expanded into the indirect flight muscles. Our findings demonstrate that expression of the TpnC41C gene in jump muscles requires integration of multiple positive and negative transcriptional inputs. Identification of the transcriptional regulators binding the cis-elements that we identified will reveal the regulatory pathways controlling muscle fiber differentiation.Most animals express multiple isoforms of structural muscle proteins to produce tissues with different physiological properties. In Drosophila, the adult muscles include tubular-type muscles and the fibrillar indirect flight muscles. Regulatory processes specifying tubular muscle fate remain incompletely understood, therefore we chose to analyze the transcriptional regulation of  MHC-IId Myosin heavy-chain gene in mice is partially compensated for by sustained expression of MHC-IIa, the mutant animals nevertheless show severe muscular defects, including growth inhibition and abnormal muscle contraction characteristics [Skeletal muscle performance in higher animals is dictated in large part by the composition of their muscle fibers. A preponderance of fast fibers is found in muscles with high performance and rapid fatigue, and slow fibers predominate in muscles adapted for endurance (reviewed in ). These eristics ,3. In aderistics .Sox6, which otherwise represses some slow fiber genes [While research over the past 30 years has identified factors required for skeletal muscle specification and development, there is still much to learn concerning how individual fiber types are specified. Clearly, the differential presence of muscle protein isoforms must arise from differential gene expression between fiber types. In zebrafish, fast and slow fiber types are physically separated from one another, making this a suitable model system for elucidating mechanisms of fiber specification. At the molecular level, the transcription factor PRDM1a represses the fast fiber phenotype by binding to the promoters of fast fiber-specific genes, and promotes slow fiber fate by suppressing er genes . For faser genes , in manyer genes . These sexd/hth or salm in tubular muscles can specify flight muscle fate [Recent research in Drosophila has demonstrated that mechanisms directing myoblasts to different fiber types indeed have evolutionarily-ancient components. The adult Drosophila thorax contains two distinct fiber types: the fibrillar indirect flight muscles that power flight, and the tubular muscles including the large tergal depressor of the trochanter (TDT), or jump muscle. These muscles differ significantly in their physiological characteristics , and in cle fate ,12. ThesMyocyte enhancer factor-2 (Mef2) is essential for formation of all adult muscles [By contrast, the regulatory pathways that promote TDT fate have yet to be defined.  muscles , 14, how muscles , 15, 16.Troponin C at 41C (TpnC41C) gene. TpnC41C is one member of a family of five TpnC genes encoded by the Drosophila genome. TpnC41C is expressed exclusively in the tubular muscles of the adult head and thorax, and is the predominant TpnC expressed in that tissue [To address mechanisms of tubular muscle gene expression, we sought to identify and characterize the enhancer for a gene expressed exclusively in the tubular muscle, the Drosophila t tissue ,18. By dTpnC41C in the TDT is regulated by a 300-bp promoter element that interacts with at least three different regulatory proteins. Two of these proteins, including MEF2, promote increased expression of TpnC41C in the jump muscles; a third factor binds to the enhancer to repress TpnC41C enhancer activity in the flight muscles. We therefore propose a mechanism for expression of this gene that requires the integration of both positive and negatively-acting factors to achieve proper tissue-specific expression.In this paper, we demonstrate that expression of TpnC41C promoter corresponding to genomic sequence 2R:50700048–5070661 was amplified using the primers 5’-AGGGTGTATAAGCTTAGG-3’ and 5’-GCTAAATAAACAATTGAAGAC-3’ and cloned into the pGEM-T Easy vector (Promega Corp). Next, the fragment was subcloned upstream of the cytoplasmic lacZ reporter gene of pCHAB [lacZ reporter gene of pnLacZattB , to analyze reporter expression in cell culture and in Drosophila transgenic animals.Drosophila genomic DNA was isolated according to Huang et al  with minof pCHAB . Note thGGATAA was inserted in the full-length promoter fragment using primers 5’-GATGAATATTGTGTATCTAAGGATAAAACTCTG-3’ and 5’-TTAGATACACAATATTCATCAAAATTCTTTA-3’, and the mutated fragment was subcloned into pCHAB. Mutation of the R1 site in the promoter sequence (GATGAATATTGTGTA) was inserted into pBS-TC41C-E using the oligonucleotides 5’-TTATAAAAATAAAGAATTTTTCGTCCGCGGTGTGCTCTAAAAATAAAA-3’ and 5’-AAAATTCTTTATTTTTATAAACCGTACATT-3’; next, the mutated promoter was sub-cloned into the pnLacZattB vector to create pTC41C-ER1.For mutagenesis of the MEF2 binding site in the 614bp promoter and the site designated R1 in the 300bp TC41C-E, we used the GeneTailor Site-Directed Mutagenesis System (Invitrogen). The MEF2 mutation CTAACCTGTCTAGATTGCCCTATAATACGTTCCAA-3’ and 5’-ACGTATTATAGGGCAATCTAGACAGGCAAAAATTGCAATTC-3’ for SM3 site; oligonucleotides 5’-TATTGTGTATTCTGCAGCGGAACTCTGAGTATTTT-3’ and 5’-AAAATACTCAGAGTTCCGCTGCAGAATACACAATA-3’ for the MEF2 site; and oligonucleotides 5’-CGCGAATTCGAGTATCAGAAGGGCGAG-3’ and 5’-GTACGGCCGATACGATGATAGCTCTGC-3’ as common flanking primers for both mutations. Mutated enhancer PCR fragments were inserted into the EcoRI-EagI sites of pnLacZattB to create pTC41C-EMEF2, pTC41C-ESM3 and pTC41C-EMef2/SM3 plasmids. Construction of the MEF2 expression plasmid pPac-Mef2 was published elsewhere [Mutations of the sites designated MEF2 and SM3 in TC41C-E  were generated by gene SOE-ing  using thlsewhere .y w and w1118 flies were used as controls. Drosophila transgenic lines expressing cytoplasmic lacZ controlled by the TpnC41C full-length promoter and its deletion variants were generated by transposon-mediated transgenesis [lacZ controlled by the 300-bp enhancer and its mutant derivatives were generated by site-dependent phi-integrase mediated recombination of pTC41C-E, pTC41C-EMef2, pTC41C-ESM3, pTC41C-EMef2/SM3 and pTC41C-ER1 [All fly stocks were maintained at 25°C, and gene and chromosome symbols are as described at FlyBase.org. sgenesis . TransgeC41C-ER1 .6 cells/ml. For co-expression of Drosophila MEF2 protein and β-galactosidase controlled by full-length TpnC41C-lacZ and its deletion variants, cells were plated into 24-well plates at 5X105 cells per well and incubated with DNA and Cellfectin II Reagent (Invitrogen) in serum-free Schneider’s Drosophila Medium for 18 h at 24°C. Total plasmid DNA concentration per well was 0.5 mg/ml, with a transcription factor (MEF2) to reporter (TpnC41C-lacZ) plasmid ratio of 1:9. Next, an equal volume of serum-containing culture medium was added, and cells were allowed to grow for an additional 24 h at 24°C. After this time, growing media was removed from the wells, cells were lysed, and lysates were used in β-galactosidase assays.Drosophila S2 cells were cultivated in flasks with Schneider’s Drosophila Medium (Invitrogen) supplemented with 10% FBS , at 24°C to a density of 2-4X10We analyzed the amount of expressed β-galactosidase reporter protein by measuring its activity in a colorimetric reaction using the All-in-One β-Galactosidase Assay reagent (Pierce). For β-galactosidase activity in cell culture, cells were lysed in 100 μl of M-PER buffer (Pierce) and incubated for 20 min at room temperature with shaking. Next, 20 μl aliquots of lysates were mixed with equal amount of β-galactosidase reagent, and reporter activity was measured at 405 nm in a Multiskan FC plate reader (Thermo Scientific). The resulting activity was normalized to the total protein concentration in the lysates, measured with the Bio-Rad Protein Assay Reagent.For β-galactosidase activity in transgenic flies, ten dissected TDT muscles were homogenized in 100 μl of PBTx. All insoluble debris were sedimented by centrifugation, and 30 μl aliquots of the supernatant were mixed with 10 μl of the β-galactosidase reagent. The reactions were analyzed immediately by taking ten measurements every 2 minutes in the Multiskan FC plate reader at 405 nm. Resulting activity was calculated as a change in the light absorbance per second. The results were expressed as a percent of β-galactosidase activity relative to that observed in transgenic control muscles.TpnC41C putative MEF2 binding site, plus ten nucleotides on each side, was used as a binding DNA probe. Double-stranded oligonucleotides corresponding to this region included 5’-GG overhangs necessary for incorporation of radioactive 32P-dCTP in a Klenow Exo- enzymatic reaction (New England Biolabs). For the competitor-containing reaction we used 100X excess of unlabeled DNA probe. The mutated competitor had a two-nucleotide substitution in the MEF2 binding site. Binding reactions were carried out in binding buffer  supplemTpnC41C promoter for new protein binding sites, we designed 50-bp overlapping probes that completely covered its sequence. Nuclear protein extracts from w1118 pupae were prepared for binding reactions according to Kawasaki et al [m1-m6 were designed to define the 15-bp binding site.To explore the ki et al , and 2μgTpnC41C promoter region (bait), containing either the native or the mutated MEF2 binding site. Baits were amplified using the biotinylated forward primer 5’-Biotin-ATTATAGCGTTCGCTCTTG-3’ and the non-biotinylated reverse primer 5’-AATTTTATCCACAAGACAG-3’, and plasmids pTC41C-E and pTC41C-EMef2 as templates. For the binding reactions, we scaled up the pupal nuclear extract preparation to obtain ~ 5mg of total protein per sample. The nuclear extract buffer was further exchanged with binding buffer described above using Amicon Ultra Centrifugal Filters (Millipore), and incubated with 250 μl of salmon sperm DNA (10mg/ml) at 4°C for 30 min to block non-specific DNA-protein binding. Next, the nuclear extract was incubated with magnetic beads carrying baits at 4°C for 2 h. After incubation, beads were washed twice with binding buffer containing protease inhibitors, and twice with 20mM Tris-HCl, pH 8.0, 2mM CaCl2, and bound proteins were digested with trypsin directly on the beads as described in Belozerov et al [Pull down experiments were performed using Dynabeads MyOne Streptavidin T1 magnetic beads (Invitrogen) and double-stranded 170-bp biotinylated ov et al . The proCryosectioning of adult flies, immunostaining and histochemical staining for β-galactosidase were performed as described before . AntibodTpnC41C, we searched the DNA sequence immediately upstream of this gene for regions that are conserved across multiple Drosophila species. We found that sequence in the immediate proximity to the transcription start site has 76% nucleotide identity across five species of the Melanogaster subgroup to which D. melanogaster belongs (To determine how the tissue-specific activity of the -2 (MEF2), that ma-2 (MEF2).in vitro translated MEF2 bound to this sequence, and the binding was sensitive to mutations in the MEF2 consensus binding site and therefore sequence-specific . We found that specific . This inTpnC41C promoter, containing the MEF2 site, as a bait to isolate nuclear proteins prepared from pharate adults. In parallel, we mutated the MEF2 site in the context of the 170-bp fragment and used it as a negative control for the pull-down experiment. The purified nuclear protein samples were then analyzed using LC-MS/MS analysis to identify factors interacting with the control and mutant DNA fragments. All Drosophila proteins identified in this approach are listed in TpnC41C promoter fragment. We note that a number of other proteins are co-purified in this approach, including a number of muscle structural proteins that are unlikely to interact with DNA. Further optimization of this approach should identify additional factors interacting specifically with the TpnC41C promoter, and reduce the background of non-specific proteins.To determine if this sequence could interact with MEF2 isolated from intact animals, we used a 170-bp fragment of the in vivo. We co-transfected Drosophila S2 cells with a Mef2 expression plasmid and full-length TpnC41C-lacZ. This resulted in activation of lacZ expression, evident from the increased β-galactosidase activity in cell lysates compared to control co-transfections containing an empty expression plasmid plus full-length TpnC41C-lacZ. A similar co-transfection experiment with Mef2 expression plasmid and mutated TpnC41C-lacZ, in which the MEF2 site was altered, had a significant reduction in β-galactosidase activity compared to the control fragment plus MEF2  (To determine more accurately the requirements of the MEF2 and SM3 sites for ctively) .TpnC41C promoter, driving most of its activity in the jump muscle, additional factors contribute to the positive regulation of TpnC41C transcription.Our data demonstrate that although MEF2 is seemingly the most significant regulator of the TpnC41C is expressed only in the jump muscles. We therefore sought to identify additional regulatory elements in the TpnC41C promoter, by performing EMSA using nuclear extracts prepared from developing pupae. By using a series of 30- and 50-bp DNA probes, collectively overlapping the entire length of the cloned TpnC41C promoter, we identified a 30-bp region that produced a reproducible protein-DNA complex when combined with nuclear extracts . It is -factors .TpnC41C was to use the promoter to purify DNA binding factors from nuclear extracts. Using this approach, we identified MEF2 as a factor bound to the wild-type sequence, that was not bound to a MEF2-mutated version of the promoter. This provided strong support for our data suggesting that MEF2 was a regulator of TpnC41C. On the other hand, while we hoped that this approach might identify additional regulators of the TpnC41C promoter, relatively few additional DNA-binding factors interacted with the sequences used as bait, and a number of muscle structural proteins were also detected .We also note that mutation of the repressor site, termed R1, causes an increase in promoter activity in the jump muscles, suggesting that the repressor may be expressed strongly in non-tubular muscles, but also at low levels in the TDT. One candidate repressor may be the zinc finger transcription factor Spalt-major, that is detected in the flight muscles and functions there to suppress jump muscle fate , and thaTpnC41C-lacZ reporter is active in the flight muscles, albeit at very low levels, and that mutation of R1 simply enables higher levels of reporter expression in both muscle types. Although in our experiments we never observed specific expression from the wild-type TpnC41C-lacZ reporter in the flight muscles, a recent paper reported low levels of TpnC41C in IFMs [An alternative interpretation of the effects of R1 mutation is that the R1 repressor site represents a location at which promoter activity is globally repressed, and jump muscle-specific gene expression is achieved through over-riding such repression. Such an interpretation would imply that the  in IFMs . This faS1 Table(XLSX)Click here for additional data file.


Dynamic transcriptional regulation is critical for an organism’s response to environmental signals and yet remains elusive to capture. Such transcriptional regulation is mediated by master transcription factors (TF) that control large gene regulatory networks. Recently, we described a dynamic mode of TF regulation named “hit-and-run”. This model proposes that master TF can interact transiently with a set of targets, but the transcription of these transient targets continues after the TF dissociation from the target promoter. However, experimental evidence validating active transcription of the transient TF-targets is still lacking.de novo synthesis of RNAs made in response to TF nuclear import. To do this, we introduced an affinity-labeled 4-thiouracil (4tU) nucleobase to specifically isolate newly synthesized transcripts following conditional TF nuclear import. Thus, we extended the TARGET system (Transient Assay Reporting Genome-wide Effects of Transcription factors) to include 4tU-labeling and named this new technology TARGET-tU. Our proof-of-principle example is the master TF Basic Leucine Zipper 1 (bZIP1), a central integrator of metabolic signaling in plants. Using TARGET-tU, we captured newly synthesized mRNAs made in response to bZIP1 nuclear import at a time when bZIP1 is no longer detectably bound to its target. Thus, the analysis of de novo transcripomics demonstrates that bZIP1 may act as a catalyst TF to initiate a transcriptional complex (“hit”), after which active transcription by RNA polymerase continues without the TF being bound to the gene promoter (“run”).Here, we show that active transcription continues after transient TF-target interactions by tracking de novo transcripts produced by transient TF-target interactions allowed us to capture new models for genome-wide transcriptional control.Our findings provide experimental proof for active transcription of transient TF-targets supporting a “hit-and-run” mode of action. This dynamic regulatory model allows a master TF to catalytically propagate rapid and broad transcriptional responses to changes in environment. Thus, the functional read-out of The online version of this article (doi:10.1186/s12864-016-2410-2) contains supplementary material, which is available to authorized users. The ability of plants to mount rapid responses to changes in nutrient signals is key to their survival. In response to extracellular signals, a cell must regulate the expression of thousands of genes within a short time. For example, plant roots mount a broad transcriptional response within just three minutes of exposure to external nitrate treatment . In thisde novo transcription of “transient” targets after TF dissociation is still lacking.Recently, a dynamic mode of TF regulation named “hit-and-run” was described for the master regulator Basic Leucine Zipper 1 (bZIP1) in propagating nitrogen nutrient signals . In thisde novo synthesis of mRNAs in response to conditional import of a TF into the nucleus , allows affinity-based capture of ized RNA , 10. Wheized RNA –13, and ized RNA . In our TARGET (Transient Assay Reporting Genome-wide Effects of Transcription factors), which can identify primary TF targets from either TF-regulation (by transcriptomics) or TF-binding (by ChIP-Seq) events assayed in the same cell samples [TARGET system to include 4tU-labeling (TARGET-tU pronounced TARGET “two”), which enabled us to capture de novo RNA synthesis induced by the conditional nuclear import of a TF-of-interest  tag and the cytoplasmic heat shock protein (HSP90). Treatment with dexamethasone (DEX) disrupts the GR-HSP90 complex, allowing nuclear import of the TF. This conditional nuclear localization of the TF in the presence of 4tU enables the incorporation of labeled UTP into actively transcribed TF-targets , a central integrator of metabolic signaling by carbon and nitrogen in plants [We applied the n plants , 18–22. cis-element analysis of the promoter regions of these target genes identified the known bZIP1-binding site  terms associated with the known functions of bZIP1 including, regulation of transcription as well as primary and secondary metabolic processes . In ourde novo transcribed targets captured from 4tU-labeled RNA, and known bZIP1 targets identified from total mRNA studies [TARGET-tU, and previously reported bZIP1 targets including stably-bound bZIP1 targets (Class II) and transiently-bound bZIP1 targets (Class III) , were previously identified as bZIP1 induced primary targets based on steady state mRNA [TARGET-tU and the original TARGET methods [TARGET-tU captured a large number of repressed bZIP1 targets ; Class IIIA  = 46 genes (p = 6.8e−50); Class IIIB  = 27 genes (p = 2.2e−20), confirming that bZIP1 is a dual-mode regulator (activator and repressor). Also consistent with previous findings, Class I “poised” bZIP1 targets, to which bZIP1 is bound but are not regulated [Overall, we find significant overlaps between  studies  both perets Fig.  that havTARGET approach based on steady-state mRNA [TARGET-tU study which captures only actively transcribed targets, 5 h after bZIP1 nuclear import  are performed in comparable experimental setups , which belongs to an important TF family for early response to nitrate signaling in Arabidopsis [THA1 (THREONINE ALDOLASE 1) and MCCA (3-METHYLCROTONYL-COA CARBOXYLASE), which encode enzymes involved in the catabolism of amino acids [NLP3, THA1 and MCCA) within 1 and 5 min of the TF nuclear entry . First, we compared this new replicate to the previous binding data from Para et al. [−10) from the two independent ChIP-Seq experiments  for 20 min, and 10 μM dexamethasone (Sigma-Aldrich) for 5 h at room temperature  was extracted from protoplasts using an RNeasy Micro Kit with RNase-free DNaseI Set (QIAGEN) and quantified on a Bioanalyzer (Agilent).The presence of a thiol group in 4tU-labeled fractions enables conjugation with biotin-HPDP . In detaThe biotinylated RNA was fractionated from unlabeled RNA using streptavidin magnetic beads (NEB). Pull-downs were performed as previously described . The beaPull-down RNA from 4tU-bZIP1 and 4tU-EV fractions was converted into cDNA, amplified and labeled with Ovation Pico WTA System V2 (NuGEN) and Encore Biotin Module (NuGEN), respectively. The labeled cDNA was hybridized, washed and stained on an ATH1-121501 Arabidopsis Genome Array using a Hybridization Control Kit, a GeneChip Hybridization, Wash, and Stain Kit, a GeneChip Fluidics Station 450 and a GeneChip Scanner (Affymetrix).http://www.bioconductor.org/packages/2.11/bioc/html/gcrma.html). Genes significantly differentially expressed between 4tU-bZIP1 and 4tU-EV fractions were identified as a union of differentially expressed genes determined by either ONE-way ANOVA or Rank Product analysis [www.virtualplant.org; [The raw probe intensities were normalized using the GC-robust multiarray averaging package  and sigThe set of genes in Fig. De novo motif discovery was performed on 1 kb regions upstream of the transcription start site of the genes actively induced by bZIP1 based on TAIR10 annotation and submitted to the MEME program [http://stan.cropsci.uiuc.edu/tools.php, [ program . Known mols.php, ). The frNLP3 (AT4G38340), THA1 (AT1G08630) and MCCA (AT1G03090) in Fig. The chromatin immunoprecipitation sequencing (ChIP-Seq) time-course dataset of bZIP1 binding was previously deposited in the NCBI Sequence Read Archive SRX425878; ). Briefl25878; . A second replication of the ChIP-Seq experiment (see Additional file The transcriptomics data sets supporting the results of this article are available in the Gene Expression Omnibus  reposito