Gene expression in eukaryotes is controlled by DNA sequences at promoter and enhancer regions, whose accessibility for binding by regulatory proteins dictates their specific patterns of activity. Here, we identify the protein Zbtb7a as a factor required for inducible changes in accessibility driven by transcription factors (TFs). We show that Zbtb7a binds to a significant fraction of genomic promoters and enhancers, encompassing many target genes of nuclear factor kappa B (NFκB) p65 and a variety of other TFs. While Zbtb7a binding is not alone sufficient to directly activate promoters, it is required to enable TF-dependent control of accessibility and normal gene expression. Using p65 as a model TF, we show that Zbtb7a associates with promoters independently of client TF binding. Moreover, the presence of prebound Zbtb7a can specify promoters that are amenable to TF-induced changes in accessibility. Therefore, Zbtb7a represents a widely used promoter factor that transduces signals from other TFs to enable control of accessibility and regulation of gene expression. Gene activation is driven by the binding of regulatory proteins to the specific DNA sequences that control each gene. However, these sequences are not always accessible for binding in every type of cell, and so differences in their accessibility can underlie the range of cell types in which particular genes can be activated. Although several cellular processes can alter the accessibilities of these sequences, it is still often unclear how these processes are directed to act at specific genes. We have discovered that the protein Zbtb7a binds near numerous gene-regulatory sequences throughout the genome and that it enables other DNA-binding proteins to trigger changes in their accessibility and to activate nearby genes. However, unlike many other factors that control gene activation, the binding of Zbtb7a alone does not seem to be sufficient to switch on gene expression; instead, its function is required for activation of genes that are independently bound by a specific set of transcription factors (TFs), and it could therefore be considered to “transduce” their gene-regulatory activities. The implication of this is that the presence or absence of Zbtb7a at any gene in a particular cell type may represent one of the aspects that can determine whether that gene is able to be activated or not. The expression of protein-coding genes is governed by the combined activities of activating and repressing transcription factors (TFs), which bind in a specific fashion to regulatory sequences in proximal promoter regions and at distal enhancers. The abilities of TFs to bind to their genomic target sequences are limited by the chromatin context, or “accessibility,” at these sites. Accordingly, regulation of the accessibility of promoter and enhancer sequences represents one of the major, critical controls of the specificity of gene expression ,2. HowevTFs differ in their requirements for binding to their target sites . In manyThe mechanistic steps underlying the activities of pioneer factors and similarly behaving TFs are still not well characterised . An intrSeparating regulation of accessibility from direct gene activation has been experimentally hard to approach because it is often difficult to distinguish whether changes in promoter accessibility represent downstream effects of the transcription process itself. However, we have previously shown that in mouse 3T3 fibroblasts, the principal activating NFκB subunit p65 is able to regulate target gene expression by 2 independent modes: either by direct transcriptional activation—dependent on its TA1 and TA2 domains, which interact with the Mediator complex—or indirectly, by controlling promoter accessibility for the binding of secondary TFs . We termCxcl2 gene and other NFκB targets in 3T3 fibroblasts (hereafter simply “fibroblasts”) upon stimulation of the NFκB pathway by tumour necrosis factor alpha . ForCxcl2 promoter as a reporter , we identified the protein Zbtb7a , which has previously been reported as an interaction partner of p65 [To search for factors that are generally required for TF-driven regulation of promoter accessibility and context-dependent gene activation, we used stable isotope labelling of amino acids in cell culture (SILAC) coupled with mass spectrometry (MS) to find proteins that could bind to the TA3 region but not to a functionally impaired mutant form . We exprr of p65 . Zbtb7a We validated the MS results by using the same GST fusion-protein baits to pull down a tagged form of Zbtb7a from transfected cells, followed by detection by western blotting . Even unDespite its high specificity for the functional TA3 region, Zbtb7a-derived peptides were detected by conventional MS with lower coverage than those of several other proteins that interact with the full C-terminus of p65, suggesting that the interaction between TA3 and Zbtb7a may be less structurally robust under the biochemical conditions used. Therefore, to confirm the intracellular interaction of Zbtb7a and the p65 C-terminus, independently of any biochemical extraction conditions, we used bimolecular fluorescence complementation (BiFC). We expressed Zbtb7a as a fusion protein with the N-terminal fragment of the fluorescent protein Venus (V1), together with p65 fused to the Venus C-terminus (V2). Cells expressing fusion proteins individually were nonfluorescent, and only background fluorescence could be detected from cells co-expressing Zbtb7a-V1 together with control protein fragments fused to V2. In contrast, co-expression of Zbtb7a and p65 fused to complementary Venus fragments yielded strong nuclear fluorescence, demonstrating their intracellular interaction . InteracCxcl2 gene in response to TNF-α stimulation  abolished the ability of p65 TA3 to induce expression of the endogenous mulation  but onlymulation , indicatFrom these experiments, Zbtb7a emerges as a protein that interacts specifically with the functional form of the TA3 region of p65 and that is required for its context-dependent transcriptional activation. It is therefore a strong candidate as a cofactor that may be mechanistically involved in TF-dependent regulation of target promoter accessibility.Zbtb7a  belongs to the POZ-ZF family of proteins—characterised by linked POZ/BTB  and zinc-finger (ZF) domains—and has been previously reported to associate with promoters of several well-studied model genes (reviewed in ). To exaDe novo motif discovery at Zbtb7a-bound ChIP-seq peaks and promoters recovered the previously described Zbtb7a binding motif, confirming the ChIP specificity ,19; Fig , S2J FigThe large number of promoters bound by Zbtb7a is much higher than those bound by many conventional TFs and is more comparable to known transcriptional cofactors . Zbtb7a-To identify pathways and processes associated with possible regulation by Zbtb7a, we examined gene ontology (GO) annotations of genes with Zbtb7a-bound promoters. Zbtb7a target promoters displayed highly significant enrichments for multiple GO terms , promineWe used experimental depletion of Zbtb7a coupled with quantitative genome-wide DNase-I hypersensitivity mapping to examine the involvement of Zbtb7a in regulating promoter and enhancer accessibility.Zbtb7a-knockdown cells and performed all analyses within approximately 5 to 10 passages. For similar reasons, we also felt that (for these experiments) the knockdown approach is preferable to analysing cell lines from Zbtb7a-knockout mice because it avoids any indirect effects that may arise from the different developmental histories of distinct, independently derived cell lines.We depleted Zbtb7a in fibroblasts using stable shRNA . In agreUsing DNase-I hypersensitivity mapping, we could detect altered levels of accessibility at a significant fraction of Zbtb7a-associated promoters and enhancers upon knockdown of Zbtb7a . AccessiZbtb7a-knockdown cells. Indeed, for many motifs that are present within Zbtb7a-regulated regions, binding footprints are markedly reduced upon Zbtb7a depletion, indicative of Zbtb7a-dependent TF occupancy , these motifs nonetheless reflect targets of TFs that are relevant for gene regulation in fibroblasts. Many of these motifs are also enriched within the subset of regions exhibiting Zbtb7a-dependent accessibility, suggesting that these regions indeed include functional binding sites for TFs. We next mapped DNase-I cut sites across instances of each motif within regions exhibiting Zbtb7a-dependent accessibility: for many motifs, a clear and significant “footprint” of differential DNase-I sensitivity is apparent at these sequences, indicative of direct protein-DNA binding ,22; S3L . Thus, Zccupancy . ConsistTo verify whether Zbtb7a-depenedent regulation of accessibility is required for p65-driven recruitment of specific secondary TFs to NFκB target promoters, we performed ChIP for 2 previously characterised factors, Cebpb and JunD . In bothDespite the Zbtb7a-dependent regulation of accessibility at many promoters and enhancers, however, a major fraction of Zbtb7a-bound sites do not exhibit any detectable requirement for Zbtb7a for steady-state accessibility  or the TA3 region from p65 to the DNA-binding domain from yeast Gal4 and replaced the NFκB binding motifs in the Cxcl2 promoter with a Gal4 binding sequence . Similarly to the activities of p65 variants on the intact Cxcl2 promoter  was not detectably impaired by the absence of Zbtb7a. In contrast, however, the activity of Gal4-TA3 was completely abolished in Zbtb7a-knockout cells , so it was pertinent to assess the extent to which indirect, context-dependent activation transduced by Zbtb7a can additionally contribute to normal TF-driven gene expression levels. As already shown above , S1O FigTogether, our data show that Zbtb7a associates with a large set of genomic promoters and enhancers, where it acts as a prebound transducer, which is required for many TFs to induce changes in accessibility  varying according to the system studied . Our glonucleosome remodeling and deacetylase) complexes, which have been best characterised for their role in gene repression [The structural events that enable Zbtb7a to trigger changes in promoter accessibility remain to be determined. The POZ/BTB domain, present at the N-terminus of Zbtb7a, is a highly conserved interaction and dimerisation domain found in many metazoan DNA-binding proteins. Shortly after its original description ,29, it wpression  —therA key aspect of the “transducer” model of Zbtb7a function is our finding that its gene-regulatory activity is only triggered upon interaction with other TFs, exemplified by the TA3 region of p65. How could such an interaction enable Zbtb7a activity? One plausible possibility is suggested by the known ability the POZ and ZF domains of Zbtb7a to mutually interact . Our in Lastly, Zbtb7a has been strongly linked to cancer development and progression. Intriguingly, though, Zbtb7a has been found to behave both as an oncogene and as a tumour suppressor, depending on context . Based o−1 recombinant mouse TNF-α to stimulate NFκB pathway activation . For reporter assays, fibroblasts were transiently transfected by electroporation or using lipofectamine, and HEK-293 cells were transfected using CaPO4. Stably transduced fibroblast cell lines were generated with retroviral vectors using supernatants from transfected Ecotropic-Phoenix packaging cells. Reconstituted p65-knockout fibroblasts were infected with serial dilutions of retroviral supernatants and analysed by flow cytometry; samples with comparable expression levels of co-expressed Tomato protein were chosen and sorted for low and homogeneous Tomato levels, and the levels of expressed p65 variant proteins were assayed by intracellular staining using an antibody specific for the DBD of p65, present in all variants; all cell lines used expressed similar levels of p65 variants to the level of p65 in wild-type fibroblasts (see 13C6 plus arginine 15N4) amino acids.Mouse 3T3 fibroblasts used in these experiments were derived from wild-type, p65-knockout, p53-knockout, and p53-/Zbtb7a-double-knockout mice and were treated with 5 ngmlasts see . Zbtb7a asts see . All celCxcl2, Saa3, and Gem promoters: approximately 1 kb genomic sequences upstream of the TSS (Cxcl2: mm9 chr5:91331907-91332973(+); Saa3: mm9 chr7:53970968-53971803(-); Gem: mm9 chr4:11630585-11631714(+)). The Cxcl2 promoter was modified in some experiments , as well as a consensus motif within the Gfp coding sequence; a 5x tandem repeat of the Gal4 UAS sequence was cloned into the Gem promoter reporter plasmid (Zbtb7a was stably knocked down using hairpins directed against either GCACAACTACGACCTGAAGAA or GAAGCCCTACGAGTGTAACAT (within the Zbtb7a CDS), cloned into the retroviral vector pSirΔ-U6CPuro (which drives hairpin expression from the mouse U6 promoter and confers resistance to puromycin). For control experiments, a hairpin directed against GGCACAAGCTGGAGTACAACT (derived from the Gfp CDS) was used. The full-length Zbtb7a coding sequence was expressed in fibroblasts using pMY-ires-Tomato. The Zbtb7a ZNF domain (aa 343–569) was expressed in vitro from pCDNA3. For production of GST fusion proteins, bait protein coding sequences  were cloned downstream of the GST coding sequence in pGEX-4T-1.All p65 variants were derived from the coding sequence of mouse p65 and were expressed using pMY-ires-Tomato  or pCDNA3. p65 DBD: amino acids (aa) 1–305; p65 TA3: aa 1–440,475–519; p65 TA1&2: aa 1–305,441–549; p65 TA3 mutant: substitution of aa 361–370 in TA3 to alanine residues. The positions of other truncations, deletions, and mutations within p65 are summarised in nts Figs  and 5C bmodified  by disru plasmid . Zbtb7a Polyclonal antibodies against p65 , Cebpb , JunD , and the HA epitope  as well as monoclonal antibodies against p65  and Zbtb7a  were from Santa Cruz biotechnology.Zbtb7a CDS or a control region (within the Gfp CDS) and were selected for stable transduction using puromycin. For both hairpins targeting Zbtb7a, Zbtb7a mRNA levels were reduced by 50% to 60%, Zbtb7a protein levels were reduced by around 80%, and TNF-α-induced expression of the TA3-responsive p65 target gene Cxcl2 was reduced more than 10-fold. Zbtb7a mRNA and protein levels, and expression of Cxcl2, were unaffected in control cells using the hairpin targeting Gfp.p65-knockout 3T3s were infected using retroviral knockdown vectors targeting either of 2 regions within the Tbp. For microarray analysis, RNA samples were prepared and processed using Qiagen RNeasy purification kits. Sample processing and microarray hybridisation was performed using standard procedures .For analysis of the expression of individual genes, cDNA was prepared from total cellular RNA by reverse transcription using random hexamer primers, and gene expression was assayed using quantitative real-time PCR with gene-specific fluorescent probes. Gene expression was normalised to the expression level of 7/ml. Chromatin in the supernatant was fragmented to a size range of approximately 300 to 700 bp using a tip sonicator, and insoluble debris was removed by centrifugation. Chromatin was diluted 10-fold in DB  and precleared with 27 μl/ml protein-A sepharose (for ChIP using rabbit IgG) or protein-G sepharose (for mouse and hamster IgG) for at least 1 hour at 4°C. Precleared chromatin was incubated with antibodies at a concentration of 2μg/ml overnight at 4°C, immunoprecipitated for 30 minutes using 10 μl/ml protein-A or -G sepharose, and sequentially washed 6 times with ice-cold NaCl wash buffer  followed by 3 times with ice-cold TE . Immunoprecipitated chromatin was released by incubation at room temperature in buffer EB , and cross-links were reversed by overnight incubation at 65°c. ChIP and input DNA were both purified using Qiagen MinElute PCR purification kits. Recovery at individual promoters was determined using quantitative real-time PCR with amplicon-specific fluorescent probes and was normalised by parallel measurement of input DNA samples. For ChIP-seq, DNA was refragmented as required to achieve a mean size of 300 bp using a water-bath sonicator. Sequencing library preparation was performed using standard procedures , and samples were sequenced using single-end reads.For ChIP, fibroblasts were washed in PBS and fixed at room temperature with 2 mM di-succinimidy-glutaraldehyde (DSG) for 45 minutes, washed extensively in PBS, and fixed again with 1% formaldehyde for 15 minutes. Cells were then washed extensively in ice-cold PBS, and nuclei were released by incubation for 5 minutes in ice-cold L1 buffer  followed by 5 minutes of centrifugation at 1,000 g. Nuclei were lysed in L2 buffer  at a concentration of 5 × 106/ml. Nuclei were released by the addition of an equal volume of buffer A plus 0.2% NP40 and incubation for 8 minutes at 4°C, then washed once in buffer A, and pelleted by centrifugation for 5 minutes at 500 g. Separate pellets of 5 × 106 nuclei were resuspended in 600 μl of 37°C digestion buffer (buffer A plus 6 mM CaCl2 plus 75 mM NaCl) and digested with varying amounts of DNaseI  for 150 seconds at 37°C. Digestion reactions were stopped by the addition of 700 μl of stop solution  and incubation for 15 minutes at 55°C, followed by addition of 200 μg proteinase K and incubation for an additional 2 hours at 55°C. Digestions were emulsified with an equal volume of phenol/chloroform/IAA, and the aqueous phase containing digested DNA was collected after centrifugation. NaCl was added to a final concentration of 0.8 M, and digested DNA samples were loaded onto a 10% to 40% sucrose gradient and separated by overnight centrifugation at 90,000 g. Fractions were collected and analysed by agarose gel electrophoresis and staining with SYBR green, and fractions containing fragments <600 bp (with an average size of around 200 bp) were pooled and used for sequencing. Sequencing library preparation was performed using standard procedures , and samples were sequenced using single-end reads.For mapping of DNase hypersensitive sites, fibroblasts were washed twice in ice-cold PBS, pelleted by centrifugation for 5 minutes at 500 g, and resuspended in ice-cold buffer A  at a concentration of 5 × 10Fibroblasts were transfected with reporter plasmids, and HEK-293 cells were cotransfected with BiFC plasmids; the fluorescence intensity of GFP-expressing cells was quantified by flow cytometry after 24 to 48 hours.E. coli and purified to approximately 90% purity (based on inspection of coomassie blue-stained gels) using glutathione sepharose. For pull-downs of in vitro expressed proteins, Zbtb7a fragments were expressed using T7-coupled transcription and translation  in the presence of 35S-methionine. Expressed proteins were incubated with immobilised bait proteins in NETN buffer  at 4°C, washed 3 times in ice-cold NaCl wash buffer , and bound proteins were directly analysed by SDS-polyacrylamide gel electrophoresis (PAGE). Gels were stained with coomassie brilliant blue R-250, dried, and exposed using a storage phosphor screen for up to 48 hours to detect 35S-labelled proteins. The intensity of the coomassie blue signal was used as a loading reference for bait proteins. For pull-downs of cellular proteins, nuclear extracts were prepared from HeLa S3 cells or 3T3 fibroblasts as described [GST fusion protein baits were produced in BL21-codon plus escribed  and incu10 greater than 50. Zbtb7a ChIP-seq datasets exhibited consistently higher backgrounds and lower peak heights when compared to p65 ChIP-seq datasets, likely reflecting differences in ChIP efficiency; nevertheless, high-confidence Zbtb7a peaks exhibited strong correlations between biological replicate datasets  and between parallel datasets generated from untreated and TNF-α-treated cells (Pearson’s r > 0.91), and the Zbtb7a binding sequence motif could be identified at 66% of Zbtb7a peaks, arguing that the signal is highly specific. DHS datasets were normalised  by scaling the coverage so that the number of read-starts (corresponding to DNase cut sites) mapping under predicted DHS peaks was equal for each sample. Note that this conservative approach is valid if the mean accessibility of all DHS regions genome wide is assumed to be approximately equal in all samples; it will underestimate DHS changes between samples if the mean accessibility changes consistently at all, or a large fraction of, DHS sites.Sequencing reads were aligned to the mouse reference genome (mm9) using bowtie . SubsetKnown and de novo enriched TF motifs at ChIP-seq peaks and promoter subsets were identified using Homer . To analTo determine the overlap of ChIP-seq data with particular genomic features , promoteP < 0.05 using unpaired, two-tailed Student t tests between groups.Promoters with Zbtb7a-regulated promoter accessibility , S3G FigP < 0.05 using unpaired, two-tailed Student t tests between groups. Where more than 1 member each TF family exhibited significant enrichment for Zbtb7a-regulated accessibility, the family member exhibiting the greatest fraction of high-confidence target promoters with Zbtb7a-regulated accessibility is shown in To identify genes whose expression is controlled by TFs belonging to the AP1, Tead, Runx, Cepb, and NF1 families , we retrNumerical values underlying figures are reported in https://www.ncbi.nlm.nih.gov/geo/) through GEO series accession number GSE97468, and sequencing data are available from the NCBI sequence read archive (https://www.ncbi.nlm.nih.gov/sra/) through the SRA study accession numbers SRP103286, SRP103318, and SRP103319.Microarray datasets generated in this study are available from the NCBI gene expression omnibus Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 FigCamp gene (top), or the control non-NFκB target Hprt gene (bottom), in TNF-α-treated p65-knockout fibroblasts expressing p65 variants. Grey signal indicates coverage of recovered DNA fragments; black signal indicates the density of individual DNase-I cut sites. Lower tracks indicate predicted p65 binding peaks and RefSeq genes. (D–F) Identification of functional TA3 subregions. Variants of p65 TA3 were used to reconstitute p65-knockout fibroblasts and assayed by their ability to activate mRNA expression of the endogenous, TA3-responsive Cxcl2 gene after treatment with TNF-α. Numbers indicate the amino acid positions in full-length mouse p65. (D) Truncations and deletions of p65 TA3 indicate that removals of regions overlapping positions 344–389 result in >50% loss of activity. (E) Replacement of contiguous 10 amino acid regions within p65 TA3 with alanine residues indicates that positions 361–370 and 370–379 are each required for TA3 activity. The replacement mutant 361–370 >10A (third bar) was used as a loss-of-function mutant in the remainder of this study. (F) Expression of subregions of p65 TA3 indicate that the region spanning positions 342–390 (sixth bar) is sufficient to confer 43% of TA3 activity . Inclusion of additional adjacent downstream regions  is able to further augment TA3 activity. We refer to positions 342–390 of p65 as the “minimal TA3” region. Error bars indicate SEM. (G–I) Conservation and primary structure of the p65 TA3 region. (G) Top: cartoon of mouse p65 indicating approximate amino acid boundaries of DNA-binding domain, TA3 region , TA2 domain (“2”), and TA1 domain (“1”). Middle: percentage amino acid identities between mouse and human p65 sequences in each region. Bottom: selected species in which genes encoding orthologous regions can be detected using the tblastn search algorithm; the TA3 region is found in most placental mammals but not in more distantly related species. (H) Multiple alignment of p65 protein sequences from different mammalian species with the mouse minimal TA3 sequence. Only aligning protein sequences detected using the blastp search algorithm are shown. (I) Primary structure of the p65 TA3 region. Red arrows represent a series of 6–8 amino acid degenerate repeats with the indicated consensus sequence VPAPAP(AS), which surround a conserved, 20–amino acid region of unique sequence at position 355–374, represented by a green box. Truncations or deletions that reduce the number of repeats progressively diminish the activity of TA3 , whereas mutations or deletions within the unique region abolish its activity . Note that the replacement mutant 361–370 >10A within the unique region, used as a loss-of-function mutant throughout the study, retains all the surrounding repeats, which may contribute to its residual, reduced interaction with Zbtb7a  or human HEK-293 cells (right) cotransfected with the indicated p65 C-terminal regions fused to the DBD of Gal4. Note that the increase in fold expression of the GFP reporter is affected both by the strength of the mouse Cxcl2 promoter and by the relative transfection efficiencies in each of the 2 cell types; nevertheless, the mouse TA3 region consistently displays an even greater activity in human HEK-293 cells than mouse fibroblasts, when compared with the activity of the full p65 C-terminus . Error bars indicate SEM. (K) Unique peptides detected by nonquantitative MS. Table lists unique peptide counts for proteins detected after GST pull-down using GST-p65 (left), GST-TA3 (left centre), GST-TA3 loss-of-function mutant , and GST alone (right). The upper box highlights proteins preferentially detected in pull-downs using the full p65 C-terminus and includes known interactors (such as CBP/p300 and components of the Mediator complex). The central box highlights proteins with increased coverage in pull-downs using TA3 than in those using TA3mut . The lower box highlights proteins that were consistently detected in pull-downs using TA3mut and/or GST alone and that thus represent likely promiscuously binding contaminants. (L) Validation of the interaction between endogenous p65 and Zbtb7a proteins by co-immunoprecipitation from TNF-α-treated unmodified fibroblasts using anti-p65, followed by immunoblotting to detect Zbtb7a. Arrowhead indicates Zbtb7a; dotted lines indicate where membrane was cut after transfer to separate Zbtb7a from the heavy chain of the immunoprecipitating antibody (hc). (M) Measurement of in vivo Zbtb7a interaction with the isolated TA3 region of p65 using BiFC. BiFC fluorescence values represent the GMFI of cells co-expressing the indicated proteins tagged with V1  or V2  fragments of Venus fluorescent protein, expressed as a percentage of the GMFI of cells expressing Zbtb7a-V1 plus V2-p65. Error bars indicate SEM. (N) Pull-down of in vitro transcribed and translated, 35S-methionine-labelled Zbtb7a ZF fragment using purified GST alone or GST-TA3. Left: coomassie blue staining of input and pull-down to indicate amounts and purities of bait proteins; right: autoradiograph to detect pulled-down Zbtb7a ZF. No interaction was detected between the TA3 region and full-length Zbtb7a or the BTB domain of Zbtb7a  DNase-I hypersensitivity levels induced by p65 DBD, p65 or by p65 TA3, at TA3-responsive or control (non-NFκB target) promoters, in TNF-α-treated p65-knockout fibroblasts. DNase-I hypersensitivity levels represent the mean normalised cut frequencies in 200 bp windows and are shown as the differences to the levels observed in p65-knockout fibroblasts, at individual promoters (A), or the mean levels across all promoters in each group (B). (C) Genome browser example tracks of DNase-I hypersensitivity surrounding the promoters of the TA3-responsive h Zbtb7a . (J) Actg domain ). (O) Ex(PDF)Click here for additional data file.S2 FigCamp (A) and Col4a1/2 (B) loci, indicating the p65 ChIP signal and locations of predicted p65 binding peaks (green). (C–F) Overlaps of Zbtb7a ChIP-seq peaks with p65 target promoters and ChIP-seq peaks. Number of overlaps between Zbtb7a ChIP-seq peaks and p65 target promoters ± 500 bp (C), all p65-associated promoters ± 500 bp  and all genome-wide p65 ChIP-seq peaks . Curves indicate the distribution of overlaps expected by chance ; grey bars (in panel E) indicate empirically determined distribution of overlaps, using 900 randomisations of the 2 peak datasets retaining interpeak distances; line indicates the observed number of overlaps and calculated P value. (G) Co-association of p65 and other TFs at promoters ± 500 bp. The ϕ2 statistic  is indicated for pairwise comparisons of overlaps at promoters of p65 and each of 108 encode ChIP-seq datasets for TFs and other factors that are available for mouse genome assembly mm9, plus the set of Zbtb7a peaks from this study. The co-association of p65 with Zbtb7a ranks fourth among all 109 datasets analysed. Similar results were obtained using the Jaccard statistic to quantify co-association . Note, however, that encode datasets are derived from diverse cell types, which may contribute to reduced data overlap.  Genomic context of Zbtb7a peaks. (H) Fraction of CGIs that are associated with predicted Zbtb7a ChIP-seq peaks. (I) GC content (left) and ratio of observed/expected CG dinucleotides (right) at all promoters ± 250 bp and at Zbtb7a-associated promoters. (J) The Zbtb7a binding motif is found at both CGI- and non-CGI-associated Zbtb7a ChIP-seq peaks. DNA sequence motifs matching the described binding specificity of Zbtb7a—enriched among promoter-overlapping (top) and enhancer-overlapping (bottom) Zbtb7a ChIP-seq peaks—were identified by de novo motif prediction. Promoters and enhancers were divided according to their overlap (left) or nonoverlap (right) with annotated CGIs. De novo motif prediction at Zbtb7a peaks overlapping CGI promoters and enhancers was performed using Zbtb7a-negative CGI promoters and enhancers as a control set to minimise the influence of GC content and CG dinucleotiodes on motif prediction. (K) Number of peaks overlapping promoters for Zbtb7a and other ChIP-seq datasets. Encode mm9 ChIP-seq datasets were manually curated as either TF , transcriptional cofactor , Ctcf, or Pol-II. Violins indicate the distribution of the number of overlaps of predicted peaks in each dataset with promoter regions; the top line indicates overlaps of Zbtb7a ChIP-seq peaks with promoter regions. Dots in violins indicate mean values. (L) Zbtb7a-associated promoters include those of genes that are nonexpressed under steady-state conditions in fibroblasts. Mean Zbtb7a ChIP signals across promoters with overlapping Zbtb7a ChIP-seq peaks that are expressed in fibroblasts at the indicated mRNA levels (upper panels) and at promoters without any overlapping predicted Zbtb7a peak . (M) Enrichment of selected GO annotations among genes whose promoters are associated with Zbtb7a peaks  or with increased or decreased mRNA expression in control fibroblasts, compared to Zbtb7a-knockdown fibroblasts (second and third columns). Dots indicate statistically significant enrichments . The most significantly enriched annotations among genes with Zbtb7a-associated promoters are shown, of which some are also enriched among genes exhibiting Zbtb7a-regulated expression. Bars indicate level of enrichment compared to all annotated genes. Additional details of statistical analysis are provided as Supporting information, and numerical values underlying figures are reported in  Genome browser example tracks across the same 1 Mb and 25 kb genomic intervals shown in (PDF)Click here for additional data file.S3 FigZbtb7a coding region. (B) Efficiency of stable mRNA knockdown by hairpins targeting Zbtb7a in p65-knockout fibroblasts, assayed by expression of GFP coded by an independent mRNA engineered to contain the same Zbtb7a-derived target sequences. Fibroblast cell lines with higher levels of knockdown (up to 90%) could be generated, but they exhibited reduced proliferation and could not be efficiently expanded . Based on the consistency of the inhibition of Cxcl2 mRNA expression mediated by both hairpins, we used hairpin 1 for all subsequent experiments in this study. (D) Endogenous Zbtb7a mRNA levels in p65-knockout fibroblasts after stable knockdown of Zbtb7a, assayed by quantitative PCR. Error bars indicate SEM. (E) Zbtb7a protein levels in p65-knockout fibroblasts (left), upon ectopic expression of an epitope-tagged form of Zbtb7a (centre), or after stable knockdown of Zbtb7a (right), assayed by immunoblotting for Zbtb7a (top), or Gapdh as a control (bottom). Quantitation of the immunoblot signal indicates that Zbtb7a protein levels are depleted by around 70% to 80% in knockdown cells. (F–J) Genome browser example tracks of DNase-I hypersensitivity (F–I) across the same genomic loci shown in Neto2 gene promoter (Zbtb7a-bound and Zbtb7a-dependent reduced accessibility), in control or Zbtb7a-knockdown fibroblasts, and indicating the Zbtb7a (black) and p65 (green) ChIP signals and the locations of predicted Zbtb7a- and p65 binding peaks. (K) Zbtb7a-associated promoters that do or do not exhibit ongoing regulation of accessibility by Zbtb7a in fibroblasts display comparable levels of Zbtb7a. Mean Zbtb7a ChIP signals across promoters with overlapping Zbtb7a ChIP-seq peaks (upper panels) and at promoters without any overlapping predicted Zbtb7a peak (bottom panel). Promoters are divided according to whether or not they exhibit Zbtb7a-driven increased or decreased accessibility, as in Zbtb7a. The Ip10+4kb region represents a control unbound location, and the Ip10 promoter exemplifies a non-TA3-driven, non-Zbtb7a-dependent gene promoter. ChIP signal is expressed as the percent recovery of input DNA. Error bars indicate range of duplicate samples. Statistical analysis is provided as Supporting information, and numerical values underlying figures are reported in supporting (A) Target sequences for knockdown hairpins within the roblasts ). Error (PDF)Click here for additional data file.S4 FigP < 0.05 and with an affymetrix signal difference of ≥0.5 (indicated as “+++” / “−−−”) or ≥0.2 (indicated as “+” / “−”). (B) Zbtb7a-associated promoters that do or do not exhibit ongoing regulation of expression by Zbtb7a in fibroblasts display comparable levels of Zbtb7a. Mean Zbtb7a ChIP signals across promoters with overlapping Zbtb7a ChIP-seq peaks (upper panels) and at promoters without any overlapping predicted Zbtb7a peak (bottom panel). Promoters are divided according to whether or not they exhibit Zbtb7a-driven increased or decreased gene expression, as in panel A. (C) Zbtb7a dependence of DNase-I hypersensitivity levels at Zbtb7a-negative promoters and at Zbtb7a-associated promoters of genes with expression that is not Zbtb7a regulated, or with Zbtb7a-driven increased or reduced expression. Zbtb7a-dependent DHS levels represent differences in mean cut frequencies within ±600 bp surrounding the TSS, compared to Zbtb7a-knockdown fibroblasts. Dots in violins indicate mean values. (D) DNA sequence motifs that are enriched at Zbtb7a-repressed promoters (defined as genes with increased expression in Zbtb7a-knockdown fibroblasts). Motif enrichment analysis was performed separately using all promoters, Zbtb7a-independent promoters, and Zbtb7a-dependent promoters as background sets, and motifs exhibiting consistent enrichment are shown. Known TF families with specificities matching each motif are indicated; additional letters of matching known motifs that are not present in the identified de novo motif logos are indicated in grey. *Note that the 2 most enriched motifs (resembling the specificities of the Nrf1 and Egr TFs) are similar to each other . (E) Zbtb7a regulates both gene activation and gene repression. Percentages of genes exhibiting differential expression in Zbtb7a-deficient cells, in publicly available datasets derived from diverse human and mouse cell types. Genes with reduced expression in Zbtb7a-deficient cells (“Zbtb7a-dependent gene activation”) are shown in green, and those with increased expression in Zbtb7a-deficient cells (“Zbtb7a-dependent gene repression”) are shown in red. In all cases, differentially expressed genes were scored as those with >2-fold change in the means of all replicates and P < 0.05 calculated by Student t test; however, the relative proportions of activated and repressed genes were also comparable across a range of other expression criteria. (F) Zbtb7a dependence of DNase-I hypersensitivity levels at promoters of genes with Zbtb7a-independent expression, or with increasing Zbtb7a dependency of expression levels, divided into equal quartiles . Zbtb7a-dependent DHS levels are expressed as the differences (left panel) or percentage reduction (right panel) in mean cut frequencies within ±600 bp surrounding the TSS, compared to Zbtb7a-knockdown fibroblasts. Lines in boxplots indicate median values; whiskers extend to the most extreme data within 1.5× the IQR from the box; outliers are not shown. Significance of difference in Zbtb7a dependence of DHS between least (Q1) and most (Q4) Zbtb7a-dependent promoters P = 2.3 × 10−14. Additional details of statistical analysis are provided as Supporting information, and numerical values underlying figures are reported in (A) Fractions of promoters of genes with changed mRNA expression in Zbtb7a-knockdown fibroblasts. Left: promoters without any associated Zbtb7a peak (“Zbtb7a-negative”); right: promoters with associated Zbtb7a peaks. Green/red slices indicate promoters with increased/reduced mRNA expression in control fibroblasts compared to Zbtb7a-knockdown fibroblasts, at (PDF)Click here for additional data file.S5 FigZbtb7a mRNA levels, reflecting expression of both the endogenous Zbtb7a gene (in control cells) as well as that driven by the Zbtb7a cDNA transgene (in cells with restored Zbtb7a expression). (B) Expression of the control housekeeping genes Taf1 and Hprt. (C–G) Expression of target genes of cJun (C), Tead2 (D), Runx2 (E), Cebpd (F), and NFκB (G) identified by microarray analysis (see Ptx3) are shown separately for unstimulated cells and for cells stimulated by TNF-α treatment. Statistical analysis is provided as Supporting information, and numerical values underlying figures are reported in Zbtb7a-knockout and congenic-control fibroblasts are both derived on a p53-knockout background, to prevent premature senescence . mRNA leysis see . Express(PDF)Click here for additional data file.S6 FigSaa3 (F) and Gem (G) genes, in transfected p65-knockout fibroblasts expressing the p65 minimal TA3 region. Red bars in (F) represent expression from an Saa3 promoter with a targeted mutation to disrupt the single consensus Zbtb7a binding motif. The cloned Gem promoter (G) does not contain a consensus Zbtb7a motif , and the reporter plasmid is appended with the Gal4-UAS. Blue bars in (G) represent artificial recruitment of Zbtb7a to the cloned promoter by expression of Zbtb7a fused to the DBD of Gal4. White bars in (G) represent cells expressing of the Gal4DBD alone as a control. Error bars indicate SEM. Statistical analysis is provided as Supporting information, and numerical values underlying figures are reported in (A) Outline of cellular reconstitution system used to analyse functions of p65 variants under conditions of normal expression levels in fibroblasts. (B) Expression levels of p65 variants in reconstituted p65-knockout fibroblasts. Flow cytometry analysis of p65-knockout (upper left) and normal (lower left) fibroblasts, after intracellular staining using antibodies recognising the p65 DBD, which is present in all p65 variants used. Centre panel: before sorting, transduced fibroblasts express a range of p65 levels and include cells with strong p65 overexpression; right panels: after sorting for low levels of co-expressed Tomato protein, cells express each p65 variant protein at similar levels to that of endogenous p65 in normal fibroblasts. (C–E) Function-impairing mutations within p65 TA3, which disrupt its interaction with Zbtb7a, reduce p65 TA3-driven changes to target promoter accessibility. Panels C, D: induced DNase-I hypersensitivity at p65 target promoters in TNF-α-treated fibroblasts expressing p65 TA3 or the loss-of-function mutant form of p65 TA3 (“TA3 mutant”). DNase-I hypersensitivity levels are shown at individual promoters as the differences to the levels observed in non-reconstituted p65-knockout fibroblasts (C) or as the differences between fibroblasts reconstituted with p65 TA3 mutant and with p65 TA3 (D). (E) DNase-I hypersensitivity changes induced by p65 TA3 or p65 TA3 mutant, at distinct groups of promoters in TNF-α-treated fibroblasts. DHS differences represent the mean changes in cut site frequencies at within a range of ±600 bp surrounding the TSS, compared to non-reconstituted p65-knockout fibroblasts. Lines in boxplots indicate median values; whiskers extend to the most extreme data within 1.5× the IQR from the box; outliers are not shown.  GFP reporter expression from plasmids containing 1 kb promoter sequences from the TA3-responsive (PDF)Click here for additional data file.S7 FigCamp gene (A), the control non-NFκB-regulated Hprt gene (B), in p65-knockout fibroblasts (top), in p65-knockout fibroblasts reconstituted with p65 (middle), and in p65-knockout fibroblasts reconstituted with p65 and with simultaneous knockdown of Zbtb7a (bottom), after TNF-α treatment. Lower tracks indicate predicted Zbtb7a and p65 binding peaks, and RefSeq genes.  Induced DNase-I hypersensitivity at control promoters, in TNF-α-treated control or Zbtb7a-knockdown fibroblasts expressing p65  Genome browser example tracks of DNase-I hypersensitivity surrounding the promoters of the TA3-responsive (PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Symbiodinium spp., from coral tissues) and subsequent coral mortality, devastating reefs worldwide. Genetic variation within and between Symbiodinium species strongly influences the bleaching tolerance of corals, thus recent papers have called for genetic engineering of Symbiodinium to elucidate the genetic basis of bleaching-relevant Symbiodinium traits. However, while Symbiodinium has been intensively studied for over 50 years, genetic transformation of Symbiodinium has seen little success likely due to the large evolutionary divergence between Symbiodinium and other model eukaryotes rendering standard transformation systems incompatible. Here, we integrate the growing wealth of Symbiodinium next-generation sequencing data to design tailored genetic engineering strategies. Specifically, we develop a testable expression construct model that incorporates endogenous Symbiodinium promoters, terminators, and genes of interest, as well as an internal ribosomal entry site from a Symbiodinium virus. Furthermore, we assess the potential for CRISPR/Cas9 genome editing through new analyses of the three currently available Symbiodinium genomes. Finally, we discuss how genetic engineering could be applied to enhance the stress tolerance of Symbiodinium, and in turn, coral reefs.Elevated sea surface temperatures from a severe and prolonged El Niño event (2014–2016) fueled by climate change have resulted in mass coral bleaching (loss of dinoflagellate photosymbionts,  Symbiodinium is considered one of the most important dinoflagellate genera given its role as the essential photosymbiont of many tropical reef invertebrates, notably reef-building corals  strongly influence coral gene expression and bleaching susceptibility  and a reporter gene (GUS) (Symbiodinium was reported (bar) and a reporter gene (GFP) were introduced to type A1, B1, and F1 strains using glass beads. Whilst cells transiently exhibited improved herbicide resistance and suggestive GFP signal, transformations were not validated through DNA, RNA, or protein analysis  to drive transcription of antibiotic resistance genes (ne (GUS) ; howeverreported . Plasmidanalysis . Furthero divide . Of thesSymbiodinium (and other dinoflagellates), we recommend a new approach that capitalizes on the recent surge in “omics” breakthroughs . By evaluating the rapidly increasing supply of next-generation sequencing (NGS) data, we propose a genetic engineering framework for Symbiodinium that may markedly advance our understanding of these important dinoflagellates. Furthermore, genetic manipulation of Symbiodinium in order to reduce coral bleaching has been hypothesized as a strategy to facilitate coral management as reefs continue to rapidly deteriorate under climate change , particularly the assembly of the first Symbiodinium genomes and transcriptomes, direct correlation between Symbiodinium transcriptional and physiological states, and discovery of genes from viruses actively infecting Symbiodinium cells. Furthermore, NGS of Symbiodinium has revealed genetic elements that may allow for transformation of Symbiodinium. In the following sections, we detail how unique Symbiodinium promoters, specific Symbiodinium genes underpinning important phenotypes, and a viral internal ribosomal entry site recognized by Symbiodinium ribosomes could be integrated to build expression constructs for Symbiodinium.Fundamental components of Symbiodinium , branch point (YTNAY), and acceptor for the dinoflagellate spliced leader (AG) (Symbiodinium promoters and terminators (as opposed to promoters and terminators from other organisms) would likely improve expression and stability of transgenes introduced into Symbiodinium. By chance, the CAMV 35S  promoter happens to contain all of the described Symbiodinium promoter elements, and the CAMV 35S  and nos (plant) terminators both contain the dinoflagellate polyadenylation signal; this may have contributed to their ability to drive transgene expression in Symbiodinium previously  . Additioder (AG) . Hence, eviously .Symbiodinium nuclear genes that can be genome-mapped to uncover strong, endogenous promoters and their corresponding terminators. These promoters and terminators can be isolated from purified genomic DNA (gDNA) through PCR and incorporated into custom DNA expression constructs for Symbiodinium . Among the most highly expressed transcripts in Symbiodinium transcriptomes are genes for peridinin-chlorophyll a-binding protein, caroteno-chlorophyll a-c-binding protein, major basic nuclear protein 2, dinoflagellate viral nucleoprotein, and glyceraldehyde-3-phosphate dehydrogenase  genes are believed to minimize thermally induced oxidative damage to photosynthetic apparatuses and leakage of harmful reactive oxygen species from type C1 Symbiodinium cells—determinants of coral bleaching  reverse transcribed from purified mRNA, since gDNA introns may prevent proper expression in constructs .Recent transcriptomic studies have been fundamental in the discovery of ral host . For insleaching ; howevervariants . A Fe-soSymbiodinium could also greatly advance investigations of ecological processes central to coral reef health. For instance, documenting competition between Symbiodinium types, transmission and acquisition of Symbiodinium types by the coral host, and shuffling of Symbiodinium types within host tissues , and 2A peptides are staple regulatory elements incorporated in expression constructs since they have evolved to be recognized by eukaryotic machinery for efficient and stable foreign gene expression . Symbiodiodinium . A putatcriptome , and sucFigure 2). Conversely, in monocistronic constructs, the selectable marker gene often maintains expression, while the gene of interest becomes transcriptionally repressed over time if it does not increase fitness of the cell  can be combined with the backbone of a standard cloning plasmid; added into an artificial, replicating minicircle  rather than RNA polymerase II. Therefore, promoters specifically recognized by Pol III  are needed. Such promoters have been isolated from other eukaryotes for sgRNA transcription; but, as discussed earlier, they contain motifs  that Symbiodinium lack  then finally the plasma membrane  was developed  can have negative impacts on a number of coral fitness traits including growth and fecundity . Based on these discoveries, we have developed a tailored genetic engineering framework for Symbiodinium based on empirical data that may also be applicable to other dinoflagellate genera. In doing so, we have opened a new prospective avenue to decode Symbiodinium functional genomics that may ultimately allow for engineering increased stress tolerance of Symbiodinium to reduce coral bleaching.As we have discussed here, recent NGS breakthroughs have revealed natural genetic elements of RL conceived the manuscript concept, analyzed NGS data, and wrote the manuscript. CRV analyzed NGS data and critically edited the theory and writing of the manuscript. SA analyzed NGS data. PS, DS, and MvO critically edited the theory and writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AWL declared a shared affiliation, though no other collaboration, with one of the authors DS to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review.


In this study, Mueller et al. investigated whether changes in nucleosome occupancy occurred in the set of genes that is activated by the unfolded protein response (UPR). Their findings demonstrate no decrease in occupancy on most promoters, gene bodies, and enhancers, but instead there was an increase in the accessibility of nucleosomes, as measured by MNase digestion and ATAC-seq, that did not result from removal of the nucleosome. Activation of transcription requires alteration of chromatin by complexes that increase the accessibility of nucleosomal DNA. Removing nucleosomes from regulatory sequences has been proposed to play a significant role in activation. We tested whether changes in nucleosome occupancy occurred on the set of genes that is activated by the unfolded protein response (UPR). We observed no decrease in occupancy on most promoters, gene bodies, and enhancers. Instead, there was an increase in the accessibility of nucleosomes, as measured by micrococcal nuclease (MNase) digestion and ATAC-seq (assay for transposase-accessible chromatin [ATAC] using sequencing), that did not result from removal of the nucleosome. Thus, changes in nucleosome accessibility predominate over changes in nucleosome occupancy during rapid transcriptional induction during the UPR. Many sequence-specific DNA-binding regulatory factors and components of the general transcription machinery are inhibited by nucleosome formation . During Drosophila cells during the unfolded protein response (UPR), an evolutionarily conserved response to protein misfolding that changes the regulation of hundreds of genes  to map nucleosomes during a short (4-h) time course of activation of the UPR. The use of several MNase concentrations allowed us to capture nucleosomes that are preferentially released at both high and low levels of MNase, providing a more comprehensive occupancy map than is likely to be obtained at a single MNase amount . These eDrosophila S2 cells with 5 mM dithiothreitol (DTT), a reducing agent that prevents disulfide-linkages in proteins, thereby inducing misfolding. To identify time points at which we expected nucleosomes to change during the UPR, we looked at UPR initiation in S2 cells by measuring splicing of the key UPR TF XPB-1  and using RNA sequencing (RNA-seq) to examine genome-wide changes in regulation . We chose 1 h and 4 h to capture the UPR process at an early time point (1 h) after spliced XBP-1 had been fully generated and at a later time point after spliced XBP-1 had been present for a few hours . We observed hundreds of genes that were both significantly up-regulated and down-regulated at least twofold during that time course, indicating that treatment with DTT induces a rapid and general transcriptional response .We induced the UPR in Saccharomyces cerevisiae, we noted variations in measuring nucleosome occupancy from experiment to experiment that confounded interpretation. We attributed this to the fact that nucleosomes are variably released by different MNase concentrations, changes that are likely to be influenced by H1 association, histone variants, and compaction . MNase cleaves the linker DNA between nucleosomes, and thus MNase-seq allows an estimate of the portion of the genome that is occupied by nucleosomes . While umpaction . We thermpaction . We founmpaction . We concmpaction B.Supplemental Fig. 1D). We noted that, as we observed previously .We used a series of four increasing MNase concentrations (MNase titration) to profile nucleosomes, sequenced each individual titration point, and then combined (averaged) the data. We computed nucleosome occupancy throughout the genome before and during induction of the UPR. This measurement recapitulated known parameters for nucleosome occupancy, including decreased occupancy at TSSs and phasing of nucleosomes into the gene body . We show individual examples of genes and surrounding sequences in the following figures, demonstrating that, while there are some regions where occupancy changes, occupancy is surprisingly constant when examined either in total or by individual genes . As expected, chromatin accessibility and nucleosome occupancy did not change significantly in these genes between the UPR time points.In contrast, we found that accessibility on these up-regulated genes, as measured by MACC, increased significantly . When analyzing total up-regulated genes, unlike h-MACC, ATAC-seq does not show a clear increase in gene bodies during the UPR (We compared the accessibility metric h-MACC with the accessibility metric generated by ATAC-seq (see the figures below). ATAC-seq uses a transposase, Tn5, to add sequencing adapters into native chromatin in vitro. Only the “open” chromatin structure will allow the transposase to deposit the sequencing adapters, whose location is determined by high-throughput sequencing . We obseImpL3, ERO1L, HSC70-3/BiP, and Herp . We saw changes predominately in accessibility as measured by either h-MACC or ATAC-seq and some limited changes in occupancy . These data demonstrate that the occupancy measurements that we used are able to detect decreases during the UPR. We did not see significant decreases in occupancy in either the other highly regulated genes examined in Supplemental Fig. S2B), indicating that the occupancy decrease observed with BiP is the exception as opposed to the rule during the UPR . In contrast, h-MACC and ATAC-seq scores increased across these regions . Consistent with these observations, h-MACC showed positive correlation with gene expression levels at all three time points, while occupancy showed a negative correlation . In contrast, examination of promoters of genes whose expression was down-regulated during the UPR showed no significant changes in accessibility or occupancy .It is plausible that occupancy changes are found primarily in promoter regions of up-regulated genes. To test this, we focused on TSS-proximal regions (promoters) of up-regulated genes, which we defined as regions within 1 kb of TSSs. There was no general decrease in occupancy on promoters of up-regulated genes (ImpL3) . We conclude that increases in accessibility of nucleosomes are seen more generally than changes in occupancy.We examined individual promoters to look for evidence of a loss of nucleosomes at promoter regions. Four representative promoters all showed increased accessibility B. Only oImpL3) B. In addImpL3, sprt, and Mmp1) showed that h-MACC increased over the enhancer as acetylation levels increased, and transcription increased nearby; however, nucleosome occupancy was largely unchanged . We conclude that at up-regulated enhancers, the predominant change in nucleosomes was an increase in accessibility, not a decrease in occupancy.We examined this more broadly across a set of 422 up-regulated enhancers. Accessibility increased from 0 h to 1 h to 4 h . The nucleosome occupancy at these regions remained low throughout the UPR. In line with previous reports . The fraction of the genome that exhibited differences in accessibility was larger than the corresponding fraction estimated for nucleosome occupancy . If nucleosome occupancy changed during the UPR, then the values comparing 0 h with 1 h or 0 h with 4 h should show more variation in peak location than seen in the control; instead, these curves overlapped the control curve . Thus, nucleosome positions did not “move” more between the UPR time points than the fluctuations observed between repeat experiments at the same time point. These results confirm on the genome scale our initial conclusions that nucleosome occupancy does not change significantly during acute regulation.To further generalize our analysis, we identified all sites where either accessibility or titration occupancy changed significantly between the 0-h and 4-h time points , we note that MACC and ATAC-seq did not produce identical results in some gene regions  (ImpL3)   or promo (ImpL3) B, 4A, de (ImpL3) , 3, up-r/BiP  or  (ImpL3) , or a sa (ImpL3) .Drosophila . However, this decrease is compensated for by the increase of the occupancy of nucleosomes released at low MNase concentrations (accessible nucleosomes). This change in physical properties of the nucleosomes at the TSSs of the UPR-activated genes occurs without nucleosome repositioning and is reflected by the increases in the MACC or ATAC-seq signal measured in our study. We also note that the nucleosomes accessible under low MNase conditions are enriched in the “enhancer” histone mark H3K27ac , which is consistent with increased occupancy of accessible nucleosomes at active enhancers. Thus, our results indicate that regulatory regions do not need to become nucleosome-free during a rapid transcriptional induction, suggesting that nucleosomes are not a barrier to the interactions that activate and might even be a component of the activation process involving components such as pioneer factors  might alter accessibility. We infer from our analyses that characterizing mechanisms for regulating accessibility that do not involve nucleosome movement or removal will be important for understanding how regulation occurs during the UPR and perhaps during many other types of activation.Drosophila cells were grown at 28°C in Schneider's medium with 10% heat-inactivated fetal bovine serum (FBS). Cells were treated with 5 mM DTT, and 1 × 107 cells were harvested at 0 h (before treatment), 1 h, and 4 h and immediately put on ice. Cells were then cross-linked and stored as described previously  buffer conditions  with 1 mL of ChIP buffer. After tumbling for 10 min at 4°C, the digests were spun at high speed for 10 min at 4°C, and the supernatant was incubated with anti-H3 antibodies  as described .DNA was analyzed on the BioAnalyzer and then used directly for library preparation as described previously . Librari7 cells was performed as described previously  in a ratio of 100 µL of cold sonication buffer to 1 × 107 cells. Cells were lysed for 10 min on ice. Cell lysates were sonicated in a Qsonica at 4°C. After a hard spin for 10 min at 4°C, the supernatants corresponding to 2.5 million cells were removed and either flash-frozen and kept at −80°C (as input) or diluted into 1 mL of ChIP buffer  to be used for ChIP after a hard spin for 10 min at 4°C. The immunoprecipitation solution was incubated with the respective immunoprecipitation antibody overnight at 4°C with tumbling. The next day, protein A Dynabeads (Life Technologies) were added, and, after a 2-h incubation while tumbling at 4°C, the immunoprecipitation samples were washed once with low-salt wash buffer , three times with high-salt wash buffer (same as the previous buffer but with 500 mM NaCl), and once with LiCl wash buffer . The beads were rinsed with TE buffer, and the proteins were eluted with 500 µL of elution buffer  for 30 min at room temperature. At this point, the input samples were also taken up in elution buffer.From each treatment group, 3 × 10Downstream experiments were performed as described previously .The antibodies used were anti-Pol II , anti-H3K27ac , and anti-H3 .4 cells were collected and washed in cold PBS. ATAC-seq libraries were prepared as described previously . The sequenced paired-end reads were mapped to the Drosophila melanogaster genome (dm3) using Bowtie aligner version 0.12.9 . We also compared the H3 tag frequency profiles averaged over a set of ∼7000 randomly selected sites, which corresponded to the number of the expressed genes identified in this study , and confirmed that H3 levels were similar for all titration and time points as expected . Input correction was not used in most analyses in this study; however, similar results were obtained when either H3 enrichment (ChIP/input) or input-subtracted H3 frequency was used . Traditional nucleosome occupancy was computed in a similar way using the data generated for the samples where different digests were pooled before sequencing. For consistency, ATAC-seq signal was summarized in the same bins as MNase-seq data. The ATAC-seq, H3 occupancy, and traditional nucleosome occupancy values in bins were further scaled to facilitate the comparison with h-MACC. Specifically, the values of each metric were scaled so that median values of the first and 20th quantiles were scaled between 0 and 1. To confirm that our findings did not depend on the scaling or binning procedures, the main results were reproduced with the unscaled metrics. In particular, tag coverage was computed for nucleosome and H3 occupancy data as well as ATAC-seq data as the library size-normalized number of paired-end fragments spanning over each genomic location.H3 occupancy was estimated as tag frequency averaged over all four titration points either in bins of the same size as the bins used for MACC estimation (200 bp) for direct comparison of MACC and H3 occupancy e.g., E or withP < 0.05) changes in their expression levels more than twofold at the time intervals 0–1 h and 0–4 h were called as differentially expressed.RNA-seq tag frequencies were normalized for GC content using Bioconductor package EDASeq, and the expression estimates for each gene were obtained using Bioconductor package DESeq . DiffereZ-threshold of 10 were used to select the H3K27ac-enriched regions. The selected enriched regions closer to each other than 150 bp were joined. Only regions enriched in both replicates within the same time point were preserved and identified as enhancers. Enhancers located closer than 1 kb upstream of TSSs or 500 bp downstream from TSSs were excluded from the analysis of TSS-distal enhancers. The enhancers were classified as “up-regulated” if the corresponding H3K27ac enrichment significantly increased twofold over the time intervals 0–1 h and 0–4 h. The enhancers that did not exhibit significant twofold changes in the H3K27ac levels but had the H3K27ac enrichment (ChIP/input) above fourfold at all time points were identified as those having higher levels of H3K27ac at all time points.The Spp package  was usedAll sequencing data are available under Gene Expression Omnibus accession numbers number GSE95689.


Ketamine was introduced into clinical practice in the 1960s and continues to be both clinically useful and scientifically fascinating. With considerably diverse molecular targets and neurophysiological properties, ketamine’s effects on the central nervous system remain incompletely understood. Investigators have leveraged the unique characteristics of ketamine to explore the invariant, fundamental mechanisms of anesthetic action. Emerging evidence indicates that ketamine-mediated anesthesia may occur via disruption of corticocortical information transfer in a frontal-to-parietal (“top down”) distribution. This proposed mechanism of general anesthesia has since been demonstrated with anesthetics in other pharmacological classes as well. Ketamine remains invaluable to the fields of anesthesiology and critical care medicine, in large part due to its ability to maintain cardiorespiratory stability while providing effective sedation and analgesia. Furthermore, there may be an emerging role for ketamine in treatment of refractory depression and Post-Traumatic Stress Disorder. In this article, we review the history of ketamine, its pharmacology, putative mechanisms of action and current clinical applications. Fifty years ago, Corssen and Domino  publisheAlthough initially developed as an anesthetic, over the past several decades ketamine has been revealed to have greater potential in the field of medicine. A growing body of literature has demonstrated the clinical value of ketamine across diverse settings, with emerging roles in pain medicine and treatment-resistant depression. Concurrently, efforts to uncover the mechanisms underlying ketamine’s actions are providing researchers with new insights into the relationship between consciousness and anesthesia.Since the first clinical report in 1966, ketamine has become arguably the most unique anesthetic agent used today and also one of the most promising and exciting in terms of its potential. The purpose of this article is to provide an overview of ketamine’s history, pharmacology, putative mechanisms and clinical applications.Ketamine’s history begins with phencyclidine, which was first synthesized in 1956 by chemists at Parke Davis Company  and R(−) isomers and is commonly marketed as a racemic mixture of the two Figure . IsolateSoluble in both water and lipids, ketamine can be safely administered through multiple routes: intravenous (IV), intramuscular (IM), oral, nasal, rectal, subcutaneous and epidural. IV administration is 100% bioavailable and considered the ideal route of administration. However, in certain settings such as emergencies or with uncooperative patients, IM ketamine is commonly used and has only a slightly lower bioavailability of 93%  and has been used cautiously in patients with space-occupying cerebral lesions brain injury  have also been reported in long-term ketamine users  transmission. Early work on the mechanisms of ketamine found that it reduced neuronal excitation by NMDA, the agonist for which the glutamatergic receptor type is named  preferentially suppresses spinal microglia hyperactivation after nerve injury and may explain its potent effects on neuropathic pain  and other intracellular signals, which could then induce altered protein phosphorylation or signaling. In rodent models, systemic ketamine administration suppresses immediate early gene expression  in the cortex and hippocampal dentate gyrus after mechanical brain injury . In septic patients requiring emergency intubation, ketamine may also be a safer alternative to etomidate, which may cause adrenal insufficiency and is associated with higher in-hospital morbidity in such patient populations , although studies examining this remain limited (Feder et al., Since its discovery, ketamine has been observed to produce symptoms similar to those of schizophrenia. As a result, researchers have used these drugs extensively as models to study schizophrenia. While it now appears that overlaps in symptoms and even receptor effects are insufficient to explain the complex neuropathology of schizophrenia, ketamine and has undoubtedly facilitated and stimulated research efforts into understanding schizophrenia (Domino and Luby, When the first clinical use of ketamine was reported in 1966 in Anesthesia and Analgesia, it quickly became a popular induction agent among anesthesiologists. Now, 50 years later, ketamine is increasingly being used beyond the operating room, demonstrating clinical utility in the fields of emergency medicine, critical care medicine, pain medicine and psychiatry.The growing interest of ketamine in a range of settings and patient populations may reflect an impressive benefit-to-risk ratio. Unique compared to other anesthetic agents, ketamine produces potent anesthesia, sedation and analgesia while maintaining cardiopulmonary stability and airway patency. Ketamine offers flexible options for administration, and the adverse psychological effects are often transient and either prevented by or alleviated with premedication or combined use with other agents. Nonetheless, the psychoactive effects often remain the limiting factor for its expanded clinical use, and just as ketamine was originally developed to reduce the emergence delirium that prevented phencyclidine’s use in humans, researchers have now moved towards developing shorter acting analogs of ketamine to further reduce the psychogenic effects (Harvey et al., Scientifically, investigators have taken advantage of ketamine’s complex molecular and neurophysiological mechanisms of action to gain further insights into the neural correlates of consciousness. Ketamine appears to inhibit information transfer in cortical networks (Bonhomme et al., Over the past 50 years, countless patients have benefited greatly from ketamine. We anticipate many more exciting discoveries in the next 50 years of investigating its clinical applications and mechanisms of modulating the mind.Both authors contributed to the writing of the manuscript, approved the final version to be published, and are accountable for manuscript accuracy and integrity. LL: conception, design and drafting of the initial manuscript. PEV: critical manuscript review and revision of all final content.PEV is supported by the National Institutes of Health, Bethesda, MD, USA, Grant T32GM103730. Funding also provided by the Department of Anesthesiology, University of Michigan Medical School.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Computational evaluation of variability across DNA or RNA sequencing datasets is a crucial step in genomic science, as it allows both to evaluate reproducibility of biological or technical replicates, and to compare different datasets to identify their potential correlations. Here we present fCCAC, an application of functional canonical correlation analysis to assess covariance of nucleic acid sequencing datasets such as chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). We show how this method differs from other measures of correlation, and exemplify how it can reveal shared covariance between histone modifications and DNA binding proteins, such as the relationship between the H3K4me3 chromatin mark and its epigenetic writers and readers.http://bioconductor.org/packages/fCCAC/.An R/Bioconductor package is available at Bioinformatics online. Overall, fCCAC greatly facilitates the assessment of covariance in genomic applications.Computational assessment of reproducibility across nucleic acid sequencing data is a pivotal component in genomic studies. Moreover, the ever-growing list of available datasets demands robust methods to quickly mine such resources to identify novel potential functional correlations between various genetic and epigenetic regulations. Chromatin immunoprecipitation followed by sequencing, or ChIP-seq, is a widely used method to profile histone modifications (HMs) and transcription factor (TF) binding at genome-wide scale. For each dataset, a set of peaks  can be obtained . Here, wN genomic regions (provided in BED format) we have two sets of curves, , ix and iy in terms of probe weight functions ξ and η can be estimated  for the H3K4me3 ChIP-seq triplicates, as expected  and KDM4A (JMJD2C) showed higher F value than DPY30 . We took advantage of recently published H3K4me3 ChIP-seq data  to evaluate reproducibility, and flag datasets showing low canonical correlations; (ii) or to investigate covariation between genetic and epigenetic regulations, in order to infer their potential functional correlations. Overall, this method will facilitate the development of new hypothesis regarding how TFs, chromatin remodelling enzymes, histone marks, RNA binding proteins, and epitranscriptome changes can cooperatively dictate the specification of cell function and identity.Supplementary DataClick here for additional data file.


Pausing during the earliest stage of transcript elongation by RNA polymerase II (Pol II) is a nearly universal control point in metazoan gene expression. The substoichiometric Pol II subunit Gdown1 facilitates promoter proximal pausing in vitro in extract-based transcription reactions, out-competes the initiation/elongation factor TFIIF for binding to free Pol II and co-localizes with paused Pol II in vivo. However, we have shown that Gdown1 cannot functionally associate with the Pol II preinitiation complex (PIC), which contains TFIIF. In the present study, we determined at what point after initiation Gdown1 can associate with Pol II and how rapidly this competition with TFIIF occurs. We show that, as with the PIC, Gdown1 cannot functionally load into open complexes or complexes engaged in abortive synthesis of very short RNAs. Gdown1 can load into early elongation complexes (EECs) with 5–9 nt RNAs, but efficient association with EECs does not take place until the point at which the upstream segment of the long initial transcription bubble reanneals. Tests of EECs assembled on a series of promoter variants confirm that this bubble collapse transition, and not transcript length, modulates Gdown1 functional affinity. Gdown1 displaces TFIIF effectively from all complexes downstream of the collapse transition, but this displacement is surprisingly slow: complete loss of TFIIF stimulation of elongation requires 5 min of incubation with Gdown1. The relatively slow functional loading of Gdown1 in the presence of TFIIF suggests that Gdown1 works in promoter-proximal pausing by locking in the paused state after elongation is already antagonized by other factors, including DSIF, NELF and possibly the first downstream nucleosome. The promoter proximal pause, in which Pol II typically accumulates at about 50 bp downstream of transcription start, is a universal feature of transcription by RNA polymerase II in metazoans. Promoter proximal pausing plays a critical regulatory role in transcription and disruption of pausing can have widespread deleterious effects on gene expression –3. In a th subunit of Pol II, present in 30–50% of polymerases isolated from tissue sources CTP from PerkinElmer Life Science and MP Biomedicals.Reagents were sourced from the following: DNA primers from Integrated DNA Technologies, NTPs from New England Biolabs, CpA from TriLink, RNasin Plus from Promega, Streptavidin-coated M280 Dyna Beads from Invitrogen, and 800 Ci/mmol CTP and 0.5 units/μl RNasin plus; for 32P]CTP and 0.5 units/μl RNasin plus. In all cases reactions were incubated for 2 minutes at 30°C followed by addition of 50 μM CTP for 30 seconds at 30°C to guarantee advancement of the nascent RNAs to the first downstream G stop. To test for the ability of Gdown1 to functionally associate with these complexes, 240 fmole of Gdown1, or buffer as a control, were incubated with each reaction for 0–10 minutes at room temperature prior to chase with nonlabeled NTPs. Some reactions were stopped prior to chase to assess the lengths of the pulse labeled RNAs; these transcripts were purified by extraction with phenol and CHCl3 and resolved by denaturing PAGE.Our various AdML-based templates have non-template strands in which the first G residue downstream of transcription start is located from +6 to +31, as noted in the figure legends. To generate EECs, PICs were incubated in 10 μl reactions with the following: for Figs 32P]CTP, and 0.5 units/μl RNasin plus for 2 minutes at 30°C followed by addition of 50 μM CTP for 30 seconds at 30°C to guarantee advancement of the nascent RNAs to the first downstream G stop.To assess the ability of Gdown1 to functionally associate with open complexes, PICs assembled on bead-attached AdML 31g(M) templates were incubated with 100 μM ATP and 240 fmol of Gdown1 or buffer for 5 minutes at room temperature. The supernates with ATP and Gdown1 were removed and replaced with buffer M5. To prevent loss of activity during open complex formation, 13 fmol of TFIIE were added to each reaction before pulse labeling . Early eACT (underlined A residue is +1). 1 mM CpA, 25 μM dATP, and 1 μM CTP were added to PICs for 5 minutes with either buffer or 240 fmol of Gdown1 to generate complexes undergoing continuous abortive initiation CTP, and 0.5 units/μl RNasin plus for 2 minutes at 30°C followed by incubation with 50 μM CTP for 30 seconds at 30°C to guarantee advancement of the nascent RNAs to the first downstream G stop.To assess the ability of Gdown1 to functionally associate with complexes at the point of initiation, we incubated PICs assembled on bead-attached AdML 31g(M) templates with substrates allowing the formation of only the first bond, The sequence of the nontemplate strand of the AdML templates near the transcription start site is 5’Citiation . To end 3 and resolved by denaturing PAGE.EECs prepared as described above were chased with 200 μM NTPs  at 30°C for 30–60 seconds, as indicated in the figure legends. Reactions in which Gdown1 was incubated with PICs, open complexes, or abortively initiating complexes also had 95 fmol of TFIIF added with the chase. When Gdown1 was added at the zero time point, Gdown1 was combined with the chase NTPs just prior to addition to the reaction. Reactions were stopped by the addition of buffer containing 10 mM Tris-HCl (pH 7.9), 100 mM NaCl, 10 mM EDTA, 1% sarkosyl, and 0.2 mg/ml tRNA. Transcripts were purified by extraction with phenol and CHClAll gels were imaged with a Typhoon Trio and gel quantitation was performed with ImageQuant TL software. The fractions of TFIIF-dependent transcription  were calculated in Microsoft Excel from the total transcription, and then normalized to the fraction of TFIIF-dependent transcription in the buffer control reactions. The percent of TFIIF-dependent transcription in the presence of Gdown1 relative to the buffer control reactions was then plotted. Each plotted value represents at least three independent replicates.


Transcriptional control of gene expression is a widely utilized regulatory mechanism in synthetic biology, biotechnology and recombinant protein production. It is achieved by utilization of naturally occurring promoters responding to nutrients or chemicals. Despite their regulatory properties, these promoters often possess features which diminish their utility for biotechnology. High basal expression level and low induction ratio can be removed using genetic engineering techniques, although this process is often laborious and time-consuming.Bacillus subtilis synthesizes levans which cause Eschericha coli cell lysis. However, in the absence of sucrose the enzyme does not affect the growth of the host. We utilized this feature to develop a two-step protocol allowing for efficient selection of inducible promoter variants. Using TECS we were able to modify the well-described pBAD promoter to decrease its leakage while maintaining high activity upon induction. Furthermore, we used the method to test transcriptional interference of lambda phage-derived sequence and optimize it for higher induction levels through random mutagenesis.In order to facilitate optimization process for inducible promoters, we developed a simple method based on a conditional toxin expression which we abbreviate as toxin expression control strategy (TECS). In the presence of sucrose, SacB enzyme from E. coli. Our strategy is very effective in the selection of promoter variants with improved properties. Its simplicity and short hands-on time make it an attractive method to optimize existing promoters and to construct novel, engineered genetic elements which improve properties of an inducible promoter system.We show that TECS is an efficient tool for optimization and development of inducible promoter systems in The online version of this article (10.1186/s12934-018-0891-1) contains supplementary material, which is available to authorized users. Biotech markets are among the fastest developing markets worldwide . One of tac and l-arabinose-inducible pBAD are the most frequently used [Escherichia coli in which they regulate lactose operon and arabinose operon expression respectively [tac promoter is a flagship example of an inducible promoter engineering since the modern version is a highly modified version of plac promoter of E. coli. The original plac promoter, in spite of being regulated by lactose, was dependent on cAMP levels and had low intrinsic activity [lacUV5 mutant was selected which had decreased dependency on cAMP [trp promoter was created. This resulted in over tenfold increased induction ratio with respect to the wild-type promoter [tac promoter is rarely used directly for recombinant protein production. Instead, it is used to control T7 polymerase expression which drives the expression of a target gene under the control of the T7 promoter in pET series of expression vectors [There are several, well-studied models of inducible promoters among which lactose-inducible ptly used . Both prectively , 6. The activity . In the  on cAMP . Next, ipromoter . However vectors .tac, the pBAD promoter used in biotechnology is the unmodified version of E. coli promoter. In general, it has similar induction level as ptac promoter and all-or-none induction profile what does not allow for gradual induction level [BAD promoter offers remarkably robust gene expression regulation, efforts were made to eliminate all-or-none induction profile and increase the strength of the promoter. However, it was shown that interference with promoter sequence or flanking sequences results in alleviation of AraC-mediated regulation of expression or decreased activity of the pBAD promoter [araE encoding arabinose transporter [araC-pBAD sequence.In contrast to the pon level , 11. Nonon level . Since ppromoter . Therefonsporter  or enginnsporter , 16. Nevtac promoter. The second approach is to generate a randomized or synthetic promoter library (SPL), either through oligonucleotide introducing random sequences between core promoter elements, or through mutagenic PCR [There are two main approaches used in the promoter engineering process. One of them is the rational design, which relies on the available knowledge about output provided by particular changes to the promoter sequence . Mutatioenic PCR . Furtherenic PCR , 22, 23.enic PCR , 25. AltThe major obstacle in engineering inducible promoters is the need to preserve regulatory properties of the promoter namely low leakage level and activation by the inducer. These requirements complicate the process of selection hindering the use of high throughput technologies (FACS) which leads to the laborious testing of individual clones. We found that using a conditionally toxic gene as a reporter can be successfully utilized for selection of inducible promoters. In this work we show that our approach is efficient in modification of an inducible promoter as well as in testing effects of exogenous regulatory sequence addition and its modification. We show that TECS allows for efficient selection of inducible promoter variants with various expression levels form relatively low number of clones. In addition to these features, the protocol presented in this work requires only basic microbiological equipment and simple procedures, making it an attractive choice for generation of custom, inducible promoter libraries.araC-pBAD sequence, pBR322 ori and ampicillin resistance [bla) was replaced with chloramphenicol acetyltransferase (cat) sequence. Remaining plasmids are derivatives of pBAD24 cm and were constructed through overlap extension PCR [cat gene was amplified from pACYC184 plasmid (ATCC 37033) with primers cat_overlap_fw and cat_overlap_rev. The sacB gene was amplified from the pKOV plasmid (Addgene 25769) with primers sac2bad_fw and sac2bad_rev. GFP sequence was amplified from pEGFP plasmid with primers gfp2bad_fw and gfp2bad rev. Sequence of relevant regions was confirmed by Sanger sequencing with pBAD_seq2 primer (Macrogen Europe). All experiments were performed in E. coli DH5α . For standard growth LB Miller liquid and solid medium was used . For plasmid isolation cultures were grown in terrific broth . Minimal medium was composed of M9 salts, 0.2% glucose, 0.2% casamino acids and 1 mM thiamine.Plasmids and primers used in this study are listed in Additional file sistance . pBAD24 sion PCR . The catAll cloning procedures as well as phenotype analyses were performed in DH5α strain. Competent cells were prepared with Inoue method . RoutineO-oopRNA fragment was assembled from five oligonucleotides  at 488 nm excitation and 510 nm emission wavelengths. Cell density was measured by absorbance at 595 nm wavelength. Results of GFP intensity were represented as a ratio of GFP fluorescence to the culture density. All experiments were performed in at least three biological replicates. Significance of differences in GFP relative fluorescence between mutants and the wild type pConceptual backbone of TECS is the utilization of toxic protein as an in vivo, self-selection factor. The strategy is based on three, simple assumptions: (i) basal expression or promoter leakage will result in the toxin production and growth impairment during the first selection step; (ii) mutations enhancing regulatory properties will decrease the leakage thus prevent toxin production and allowing normal growth; (iii) enhanced regulatory properties are valid only if leakage repression can be bypassed by the induction of the promoter, which would lead to toxin production and growth inhibition.E. coli [E. coli and plated on media with 5% sucrose and resistance marker compatible with the vector. Fast growing, putative SacB-negative colonies are inoculated into a 96-well plate and replica-plated on selective media with sucrose and inducer  mutation in the BamHI site which decreased basal expression  and high-leakage variants are counter-selected in the first step where they are unable to grow on sucrose. The second factor is the induction, since inducible promoter should be activated by specific metabolite or conditions. Thus the second step of selection tests promoter induction and ability to produce toxin by selected mutants. This simple strategy allows TECS to select potentially interesting mutants from randomized libraries. Cells harbouring a leaky promoter will be unable to grow on sucrose-containing medium and the use of solid medium ensures the stringent selection for plasmids . In the In our opinion TECS fills a gap in available methods for promoter libraries generation. Computational methods which allow for in silico design of promoters with different strength, despite their increasing accuracy often yield unpredictable results . It is iBAD promoter, which was not changed since its introduction into expression vectors [BamHI site by mutagenesis, despite previous reports that insertions and deletions in this affect the leakage and activity of the promoter [BAD promoter. However, TECS could be used to optimize any inducible promoter. In this work we present two convenient methods for randomization of particular DNA sequence. The first method utilizes a well described mutagenesis with primer containing a random sequence patch (Additional file O promoter sequence. However, we are aware that in our experiments we tested only a minor subset of possible combinations. In theory, 35 nucleotides long random sequence generates over 1021 combinations. In order to achieve onefold coverage it would be necessary to generate over 1021 vector molecules and corresponding amount of primers would be 1.66 mmol of each, that is over 109 times more molecules that are used in a standard PCR. Nevertheless, despite the fact that we covered merely a pinch of possible combinations, TECS allowed for selection of mutants which were better than the initial vector.One of the best arguments supporting the usefulness of TECS is improving the p vectors . We werepromoter . InteresE. coli. We show that TECS allows for convenient, in vivo selection of mutants with desired properties. Although the method was tested on the well-studied pBAD promoter, it can be directly applied to modify any system. Optimization with TECS can be performed in any microbiological laboratory, using the basic equipment. The versatility and simplicity of the method make it an attractive choice for the generation of in-house-optimized vectors for controlled gene expression.In this work, we described TECS as a novel tool for optimization of inducible expression systems in Additional file 1. Additional Tables S1, S2; Figures S1–S3.