An analysis of histone acetylation at the Sry promoter in mutant gonads at 11.5 dpc shows a reduction in levels of the positive histone mark H3K27Ac. Our data suggest a role for CBP/p300 in testis determination mediated by control of histone acetylation at the Sry locus and reveal a novel element in the epigenetic control of Sry and mammalian sex determination. They also suggest possible novel causes of human disorders of sex development (DSD).CREB-binding protein  and its closely related paralogue p300 , together termed p300/CBP, are histone/lysine acetyl-transferases that control gene expression by modifying chromatin-associated proteins. Here, we report roles for both of these chromatin-modifying enzymes in mouse sex determination, the process by which the embryonic gonad develops into a testis or an ovary. By targeting gene ablation to embryonic gonadal somatic cells using an inducible Cre line, we show that gonads lacking either gene exhibit major abnormalities of XY gonad development at 14.5 dpc, including partial sex reversal. Embryos lacking three out of four functional copies of  In XY embryos, the transient expression of Sry (sex determination region on Y chromosome) in somatic cells of the undifferentiated gonad between 10.5 dpc and 12.0 dpc triggers testis differentiation (Sox9 (Sry-related HMG box 9)  ]. In XX G box 9) .Sry is crucial for activation of the testis-determining pathway of gene expression; a developmental window of approximately 6 h exists in which Sry expression is critical -binding protein, CBP , and p300 (EP300 or KAT3B) are two members of the KAT3 subfamily of histone acetyl-transferases (HATs). In addition to their ability to acetylate histones and transcription factors, they regulate gene transcription by recruiting transcription factors or by connecting them to the basal transcription machinery [reviewed in ]. CBP anequently . Conditiequently , photoreequently , lens inequently , spermatequently  and neurp300 and Cbp have been shown to be expressed in different cell lineages of the embryonic gonad in mice. Whilst there are no reports of in vivo roles for CBP/p300 in sex determination, a number of lines of evidence suggest that further investigation of this possibility is warranted, especially in the context of the function of SRY and SOX9: i) p300/CBP activity is positively implicated in functionality of the testis-determining gene SOX9 in other contexts, either by promoting SOX9 transcription  p30pression ,36; iii)ng-sites ; iv) p30ng-sites .p300 and/or Cbp in gonadal somatic cells prior to sex determination. Loss of either molecule individually results in partial XY gonadal sex reversal; loss of three out of four functional alleles, retaining either one functional p300 or one Cbp allele, causes complete XY gonadal sex reversal. Careful analysis of mutant gonads reveals that this sex reversal is associated with a reduction in expression of Sry and Sox9 at 11.5 dpc. Chromatin immunoprecipitation reveals a correlative reduction in levels of the positive histone acetylation mark H3K27Ac at the Sry promoter in mutant gonads at the same stage. Our data suggest a requirement for these HATs in establishing a normal profile of Sry expression mediated by histone acetylation at its promoter. They also indicate that CBP or p300 sequence variants identified in humans with disorders of sex development (DSD) warrant further investigation in respect of their possible pathogenic effects.These data suggest that CBP and p300 might play important roles in testis determination or differentiation. However, testing this hypothesis has been precluded by the death, at around mid-gestation, of embryos lacking these genes ,24. Hereflox/floxCbpor flox/floxp300mice with mice carrying floxCbpor floxp300 and tamoxifen-inducible Wt1CreERT2, in which Cre expression is driven by the endogenous Wt1 regulatory elements  gonads and the adjacent mesonephros at both stages, while gonads predicted to lack CBP  or p300  exhibited a strong reduction in CBP and p300 protein levels, respectively. These data suggest that one dose of tamoxifen at 9.5 dpc is sufficient to efficiently delete these two genes in the somatic cells as early as 11.5 dpc. However, as Wt1 is not expressed in germ cells, CBP and p300 were detected in these cells  for Sox9, a major gene controlling testis differentiation and a Sertoli cell marker, on gonads from ERT2Cre/+Wt1 embryos, with and without tamoxifen dosing of the dam . These results suggest that XY ERT2Cre/+Wt1gonads treated with tamoxifen can occasionally exhibit a short delay in testis determination, which explains the polar Stra8 expression; this is the case with other mutants that have a very minor effect on testis determination (ERT2CreWt1or tamoxifen was observed on XX gonad differentiation (data not shown). It is likely that these gonads are sensitized to any further disruptions to testis determination, rather like the situation in which B6 mice carry the domesticus Y chromosome from the AKR strain (B6.YAKR)  ,48. TherCbp and p300 on gonad differentiation, we analysed gonads at 14.5 dpc, a stage at which disruptions to sex determination are easily detectable. As expected, XY control gonads (ERT2Cre/+Wt1) exhibited well differentiated testis cords expressing Sox9 (Stra8 was observed (Cbp (Cbp cKO) or p300 (p300 cKO) ]  or a Leydig cell marker (Insl3), but which showed ectopic expression of female somatic and germ cell markers , similar to XX control gonads. XX Cbp, p300 dcKO gonads showed no overt abnormalities (data not shown). These data show that all testicular cell lineages examined have been affected in Cbp/p300 dcKO gonads, consistent with wholesale XY ovary development. In a similar fashion, XY embryos with conditional deletion of two copies of Cbp and one copy of p300  also exhibited complete gonadal sex reversal ] exhibited an intermediate phenotype with disruption of testis cord organization and significant expression of Stra8 and FOXL2 at the gonadal poles ] . Cbp/p30Sry and its principal target gene, Sox9, at 11.5 dpc, at the time testis determination is initiated. Sry mRNA and SRY protein levels were drastically reduced in Cbp/p300 dcKO gonads compared with XY controls (Sry in sub-dissected gonads (lacking the mesonephros) by qRT-PCR revealed a reduction of approximately 6-fold in Cbp/p300 dcKO gonads  from whole gonads (lacking the attached mesonephros) at 11.5 dpc   . In contcontrols . These dCbp and p300 knockout mice, we observed a partial male-to-female gonadal sex reversal in XY embryos with targeted ablation of either Cbp or p300 in somatic cells. Complete gonadal sex reversal occurred when only three out of four functional copies of Cbp/p300 were targeted for deletion. However, it should be remembered that the presence of ERT2CreWt1 means that embryos are haploinsufficient for Wt1, a gene with a known role in testis determination. Although tamoxifen-treated ERT2CreWt1 XY embryos develop testes as normal, it is likely that this haploinsufficiency makes some small contribution to the sex reversal phenotype. It is possible that in the presence of two wild-type copies of Wt1, it would be necessary to delete all four functional copies of Cbp/p300 in order to effect XY gonadal sex reversal.Here we demonstrate for the first time a role for CBP/p300 in mammalian testis determination. Using an inducible Cre recombinase system to circumvent the embryonic lethality seen in Stra8 and FOXL2, suggesting that the ovarian program of differentiation, in contrast to the testicular, can occur when there is a major functional deficit in these two proteins.Our observation that partial gonadal sex reversal is caused by deletion of either gene indicates that both CBP and p300 play a role during testis determination. Although we observed a strong decrease of CBP and p300 proteins in the somatic cells of the gonads at 11.5 dpc, we cannot formally exclude the possibility that deletion does not occur in all cells, or that this deletion is not early enough, and that, as a consequence, some residual p300 or CBP activity is responsible for the observed partial induction of testis differentiation in single gene mutants. However, previous studies have suggested that CBP and p300 can act redundantly. Both compound mutants exhibited complete gonadal sex reversal, with XY gonads developing as ovaries. These data suggest that both genes are involved in testis determination and can at least partially compensate for each other’s loss. Finally, it is noteworthy that sex-reversed mutant XY gonads robustly express p300/Cbp dcKO, Sry expression is dramatically decreased at 11.5 dpc. This is sufficient to explain the loss of Sox9 expression at the same stage (Sry expression. Lysine 27 of histone H3 (H3K27), a known target of CBP/p300 acetylation, shows higher levels of acetylation at the Sry promoter in XY gonads than in the mesonephros at the time of sex determination. Levels of pan-acetylated H3 and acetylated H3K9 are also higher in the gonad, suggesting a positive correlation between the level of H3 acetylation and Sry expression. These experiments were performed on whole gonads and, due to the heterogeneous collection of cell-types found in the gonad at this stage, may under-estimate the enrichment of acetylated H3 at the Sry promoter in gonadal somatic cells. In gonads depleted for both CBP and p300 using our conditional deletion strategy, the levels of H3K27 acetylation (H3K27ac) were significantly decreased, suggesting that the down-regulation of Sry in the absence of sufficient Cbp/p300 activity is caused by reduced histone acetylation at its promoter. Our data shed new light on a key developmental gene and its epigenetic control, which remains poorly understood  revealed no differences between CBP/p300-deficient gonads and controls , including partial and complete 46, XY gonadal dysgenesis , as a way of identifying the mutations causally responsible for the observed condition. It is noteworthy that two such studies report potentially disruptive mutations in human and p300 ,54. WhilMouse experimentation was approved by the Animal Welfare and Ethical Review Body at MRC Harwell. Breeding was performed by license under the Animals (Scientific Procedures) Act with approval from the U.K. Home Office (PPL 70/8898). Mice were housed in individually ventilated cages in a specific pathogen-free environment. Further details of micro- and macro-environmental conditions are available on request. Adult mice were humanely euthanized by dislocation of the neck and embryos were decapitated in ice-cold, phosphate-buffered saline solution.floxCbp and floxp300 lines have been previously described  ERT2Cre/+Wt1, flox/+p300x flox/floxp300; or iii) ERT2Cre/+Wt1, flox/+p300, flox/floxCbp x flox/floxp300. Deletion was induced with a single dose of tamoxifen (Sigma T5648), dissolved in corn oil, at 200mg/kg per mouse, administered by oral gavage at 9.5 days post coitum (dpc). Noon on the day of the copulatory plug was counted as 0.5 dpc.The escribed ,56 and wreallele , also maHprt1. Fold-change in expression was determined by the 2−ΔΔCT method. Statistical differences were determined by using a two-tailed Student’s t-test. Primers sequences are available on request.Total RNA was extracted from one pair of gonads, dissected away from the mesonephroi, using the RNeasy plus microkit (Qiagen). 300 ng of RNA was reverse-transcribed using the high capacity RNA to cDNA kit (Applied Biosystem). qPCR of cDNA (qRT-PCR) was performed on a 7500 Real-Time PCR system (Applied Biosystems) using Fast SYBR Green Master Mix (Applied Biosystems) according to manufacturer’s instructions At least 3 samples for each genotype were analysed. Data were normalized to Tissues were fixed overnight in 4% paraformaldehyde, embedded in paraffin and cut into 8μm sections. Analyses were performed on at least two independent samples per experiment. The following primary antibodies were used: AMH , SOX9 , FOXL2 (a kind gift from Dagmar Wilhelm), SRY (a kind gift from Makoto Tachibana), CBP  and p300 . Secondary antibodies were Alexafluor 594 and Alexafluor 488. Images were captured using a Zeiss 710 multiphoton microscope.in situ hybridization (WIMSH) protocol as well as the probes used have been previously described . A minimum of three samples per genotype were analysed for each probe.Tissues were fixed overnight in 4% paraformaldehyde before storing at −20 °C after serial methanol dehydration. The wholemount Gapdh. At least six measurements were made for each histone mark. Statistical differences were determined by using a two-tailed Student’s t-test. Primer sequences are available on request.Chromatin immunoprecipitation (ChIP) was performed by modifying published methods of low cell number ChIP ,58. BrieHMG online.Supplementary Fig S1Click here for additional data file.Supplementary Fig S2Click here for additional data file.Supplementary Fig S3Click here for additional data file.Supplementary Fig S4Click here for additional data file.


Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs.The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and  Lsd1, preventing the de-silencing and activation of additional OR genes  provides an extreme example of cellular diversity orchestrated by the seemingly stochastic, monogenic, and monoallelic expression of a single olfactory receptor (OR) gene. Each mature olfactory sensory neuron (mOSN) in the MOE expresses only one OR that is chosen from a pool of more than two thousand alleles . The basOR genes .In the context of this repressive chromatin environment, OR gene choice requires the action of intergenic enhancers that escape heterochromatic silencing and activate the transcription of their proximal ORs . These etrans enhancement in OR gene expression, and are consistent with the sufficiency of a multi-enhancer hub formation for OR gene choice.Here, we present a detailed molecular characterization of the Greek Islands, which revealed a common genetic signature and occupancy by shared sequence-specific transcription factors, allowing us, for the first time, to incapacitate them as a whole. ChIP-seq studies of FAC-sorted mOSNs revealed that most of the previously characterized Greek Islands, and several newly identified islands, are bound by two transcription factors: Lhx2 and Ebf. Computational analysis of the co-bound ChIP-seq peaks from Greek Islands revealed stereotypically positioned Lhx2 and Ebf binding sites that together constitute a ‘composite’ binding motif that affords cooperative binding in vivo. This motif is highly enriched in Greek Islands relative to OR promoters and Lhx2/Ebf co-bound sites genome-wide. Considering the prevalence and specificity of this composite motif in Greek Islands, we designed a synthetic ‘fusion’ protein that binds to this consensus sequence and not to individual Lhx2 or Ebf motifs in vitro. We found that overexpression of this fusion protein in mOSNs eliminated chromatin accessibility at most Greek Islands, and resulted in strong transcriptional downregulation of every OR, regardless of their genomic distance, or even their chromosomal linkage to a Greek Island. Finally, partial pre-assembly of a Greek Island hub in cis, by insertion of an array of 5 Greek Islands next to the Greek Island Rhodes, significantly increased the frequency of expression of Rhodes-linked OR genes. These manipulations provide genetic support for the requirement of + cells from the MOEs of Omp-IRES-GFP knock-in mice, as previously described . Notably, most Ebf and Lhx2 co-bound sites in OR clusters have much stronger ChIP signal than singly bound sites  signal from mOSNs, while Lhx2 protein levels are unaffected in progenitor and immature OSNs  at a proper distance, we could generate a DNA binding peptide that has high affinity for the composite but not for individual motifs. Because the DNA binding specificity of homeobox genes is low and is influenced by their partners , the LhxOmp-IRES-tTA knock-in mice  next to the endogenous Rhodes, a Greek Island from chromosome 1  in mOSNs, and by conditional deletion of transcriptional co-activator Bptf, either of which caused reduction of Greek Island interactions in trans and pervasive OR downregulation . HoweverLhx2 knock out and in fusion protein expressing mOSNs, clearly reveals the critical and ubiquitous role of the Greek Islands as key regulators of OR expression. The fact that these transcriptional effects extend to ORs that have neither a Greek Island in cis nor Lhx2/Ebf motifs on their promoters, is consistent with the role of Greek Islands as trans OR gene enhancers. However, it should be noted that we cannot exclude alternative interpretations of our data. For example, it is possible that there are additional intergenic OR enhancers with bona fide composite sites that are only utilized in neurons that express specific OR genes. Although our analysis in the 3 purified OSN populations expressing ORs Olfr1507, Olfr17 and Olfr171 does not support the existence of OR-specific Greek Islands, we cannot exclude the possibility that Greek Islands with such restricted activity exist in other OSN sub-populations. Furthermore, although our ATAC-seq analysis suggests that fusion protein overexpression affects predominantly Greek Islands, indirect effects on OR transcription cannot be excluded. Taking all these caveats into account, the fact that distinct genetic manipulations that target the Greek Islands, and their genomic interactions, cause widespread downregulation of OR expression, provides strong genetic support for the requirement of interchromosomal interactions in OR gene choice.To minimize indirect effects that may confound the interpretation of these manipulations, we targeted a common and highly specific genetic signature among Greek Islands, the composite motif. This DNA sequence constitutes a remarkable example of highly constrained and stereotypically distributed transcription factor binding motifs that is shared between most Greek Islands, and is highly enriched relative to OR promoters and co-bound sites genome-wide. Overexpression of a ‘synthetic’ fusion protein that specifically recognizes the composite motif eliminated ATAC-seq signal from Greek Islands in mOSNs, suggesting that it displaced the endogenous Lhx2 and Ebf proteins on most OR enhancers. Similar observations were made for the conditional Lhx2 deletion, which also reduced the chromatin accessibility of Greek Islands and abolished Ebf binding from these elements. The strong and specific downregulation of the OR transcriptome in both IFN beta enhanceosome , which exhibit steep concentration threshold requirements attributed to phase transition processesMice were treated in compliance with the rules and regulations of IACUC under protocol number AC-AAAI1108. All experiments were performed on dissected whole main olfactory epithelium (MOE) or on freshly isolated, FAC sorted primary cells collected from whole main olfactory epithelium.Omp-IRES-GFP mice, which were previously described (Olfr17-IRES-GFP mice (Olfr151-IRES-tauGFP mice (Olfr151tm26Mom)(Olfr1507-IRES-GFP mice (Olfr1507tm2Rax)(Mature olfactory sensory neurons (mOSNs) were sorted from escribed . Olfr17+GFP mice . Olfr1511tm26Mom). Olfr15007tm2Rax).Lhx2 conditional allele mice (Lhx2fl/fl: Lhx2tm1.1Monu) (Omp-IRES-Cre mice (Omptm1(cre)Jae) 26Sortm14(CAG-tdTomato)Hze/J )   with Ompcre)Jae) . In orde)Hze/J )  was alsoTetO-Fusion-2A-mCherry construct were generated at the Columbia University Transgenic Mouse facility at the Irving Cancer Research Center. Fusion protein expression in mOSNs was achieved by crossing these mice with Omp-IRES-tTA mice (Omptm1(tTA)Gogo)(Transgenic mice bearing the tTA)Gogo).Rhodes knock-in mice were generated by Biocytogen.For ATAC-seq and ChIP-seq experiments, cells were sorted from adult male and female mice ranging in age from 7 to 16 weeks of age. For RNA-seq, cells were sorted from male and female mice ranging in age from 6 to 10 weeks.Biological replicate samples were collected and processed separately from different mice.2 followed by cervical dislocation. The main olfactory epithelium (MOE) was dissected and transferred to ice-cold EBSS . The MOE was cut in to small pieces with a razor blade, and then dissociated with a papain dissociation system . Diced tissue was added to papain-EBSS, with at most 2 MOEs/mL, and incubated for 40 min at 37°C on a rocking platform. After 40 min, tissue was triturated 30 times, the supernatant containing dissociated cells was transferred to a new tube, and the cells were pelleted . Remaining papain was inhibited by resuspending the cell pellet with Ovomucoid protease inhibitor solution diluted 1:10 in EBSS, and the dissociated cells were pelleted .Mice were sacrificed using CO2, and 500 ng/mL DAPI (Invitrogen). These cells were passed through a 40 uM cell strainer, and then FAC sorted. Live cells were selected by gating out DAPI positive cells.For live cell sorts, dissociated cells were washed once with sort media , and then resuspended in sort media supplemented with 100 U/mL DNase I , 4 mM MgClFor formaldehyde-fixed cell sorts, dissociated cells were resuspended in PBS + 1% methanol-free formaldehyde (Pierce). Cells were fixed at room temperature for 5 min, and then fixation was quenched by adding 1/10th volume of 1.25M glycine. Fixed cells were pelleted , washed once with sort media, resuspended in sort media, passed through a 40 uM cell strainer, and then FAC sorted.Sorted fixed cells were pelleted . Cell pellets were resuspended in ChIP Lysis Buffer ) and incubated on ice for 10 min. Nuclei were collected by centrifugation . The nuclei pellet was resuspended in shearing buffer  and then sheared to a size range of 200–500 bp on a Covaris S220 Focused-ultrasonicator . Sheared chromatin was centrifuged  to remove insoluble material. The DNA concentration of the sheared chromatin was determined by fluorescent quantification . Shearing was assessed by agarose gel electrophoresis after DNA clean-up: chromatin was incubated for 30 min with RNase A, cross-links were reversed overnight at 65 C, and then DNA was column purified .AB_2045685) and Lhx2  in a thermomixer set to 900 rpm. This elution was repeated and the elution fractions were pooled. The eluted DNA was incubated overnight at 65°C. Input chromatin was brought up to 200 uL with elution buffer and also incubated at 65°C overnight. ChIP DNA and input DNA were column purified using Zymo ChIP DNA columns  and eluted in 20 uL of 10 mM Tris-HCl pH 8. Purified DNA was analyzed by quantitative PCR . Cells were lysed by adding an equal volume of Buffer 2 . After addition of Buffer 2, cells were incubated on ice for 10 min, and then nuclei were pelleted . Nuclei were resuspended in MNase buffer . Nuclei were digested for 1 min and 40 s with 0.2U of Micrococcal Nuclease (Sigma) per 1 million cells. Digestion was stopped by adding EDTA to a final concentration of 20 mM. Undigested material was pelleted , and the supernatant (S1 fraction) was retained and stored at 4°C. The pelleted material was resuspended in Dialysis Buffer , rotated overnight at 4°C. Following dialysis, the insoluble material was pelleted  and the supernatant (S2 fraction) was retained. MNase digestion was assessed by agarose gel electrophoresis. The MNase treatment was optimized to yield a nucleosomal ladder comprising mostly mono and di-nucleosome sized fragments in the S1 fraction and di-nucleosome and larger sized fragments in the S2 fraction. The concentration of nucleic acid in the S1 and S2 fractions was determined by fluorescent quantification . Prior to ChIP equal volumes of S1 and S2 fractions were combined, and the total quantity of nucleic acid in the pooled fractions calculated to normalize between experiments.Live sorted cells were pelleted  and then resuspended in Buffer 1 , H3K79me3 , and H3K27ac . Approximately 1 ug of MNase digested chromatin was used per IP, with approximately 100 ng reserved as an input control. Chromatin was diluted to 500 uL in Wash Buffer 1 , 1 ug of antibody was added, and the binding reaction was rotated overnight at 4°C. For each IP, 10 uL of Protein A Dynabeads (Life Technologies) and 10 uL of Protein G Dynabeads were blocked overnight with 2 mg/ml yeast tRNA and 2 mg/mL BSA in Wash Buffer 1. Blocked beads were added to antibody bound chromatin and rotated for 1–2 hr at 4°C. Bound beads were washed 4 times with Wash Buffer 1, 3 times with Wash Buffer 2 , and once with TE pH7.5. DNA was eluted from beads by incubating at 37°C for 15 min with 100 uL Native ChIP Elution Buffer  in a thermomixer set to 900 rpm. This elution was repeated and the elution fractions were pooled. Input chromatin was brought up to 200 uL with Native ChIP Elution Buffer. ChIP DNA and input DNA were column purified using Zymo ChIP DNA columns  and eluted in 20 uL of 10 mM Tris-HCl pH 8.MNase digested native chromatin was used for ChIP with antibodies for H3K9me3  added as a carrier. The RNA pellet was washed twice with 75% ethanol, dried, then resuspended in nuclease free water. 3 ug of RNA was DNase treated using the TURBO DNA-free Kit (ThermoFisher) according to manufacturer’s instructions. cDNA was prepared from 800 ng of RNA using SuperScriptIII (ThermoFisher) and used for qPCR with gene specific primers . Fold chLive sorted cells were pelleted , the supernatant was aspirated until 250 uL of media remained, and then the cell pellet was resuspended in 750 uL Trizol LS (ThermoFisher). Total RNA was extracted by adding 200 uL chloroform, vortexing for 15 s, incubating at room temperature for 2 min, then centrifugation at 12,000 rcf for 15 min at 4°C. The aqueous phase was collected and RNA was precipitated with isopropyl alcohol with 10 ug/mL linear acrylamide (ThermoFisher) added as a carrier. The RNA pellet was washed twice with 75% ethanol, dried, then resuspended in nuclease free water. 1 ug of RNA was DNase treated using the TURBO DNA-free Kit (ThermoFisher) according to manufacturer’s instructions. RNA-seq libraries were prepared from DNase-treated RNA using a TruSeq Stranded Total RNA with Ribo-Zero Gold Set B kit (Illumina RS-1222302).Sequencing libraries were profiled on Bioanalyzer 2100 using a high sensitivity DNA kit (Agilent). Library concentration was determined by KAPA assay (KAPA Biosystems). Libraries were multiplexed and sequenced on an Illumina HiSeq with 50 bp single-end or paired-end reads. See Three versions of the fusion protein were designed with either 1, 2, or 4 repeats of a 5 amino acid linker sequence between the DNA binding domain of Ebf and the DNA binding domain of Lhx2 . Gene blThe 5-enhancer hub that was inserted into the Rhodes locus was generated using Gibson assembly of gene blocks synthesized by Integrated DNA Technologies .32P using T4 Polynucleotide kinase. Labeled probes were purified on a Sephadex G-50 column .Probe oligonucleotides  were annFusion protein, Lhx2, and Ebf were in vitro translated from pcDNA3 expression vectors bearing the T7 promoter . In vitro binding reactions were setup with 1 uL of in vitro translation product, 0.5 ug Poly(dI-dC), 1 ug BSA, and 1xProtease Inhibitor cocktail in EMSA Binding Buffer . For competition conditions, 2 pmol (20-fold excess) or 10 pmol (100-fold excess) of unlabeled, annealed oligonucleotides was added to the binding reactions. The binding reactions were incubated for 20 min on ice, and then 100 fmol of radiolabeled probe was added. Following probe addition, binding reactions were incubated at room temperature for 10 min. Binding reactions were loaded on a native TBE polyacrylamide gel , and electrophoresed at 180 V for approximately 3 hr. After running, gels were transferred to filter paper, dried, and exposed to a phosphoimager screen overnight. Phosphoimager screens were scanned on a Typhoon FLA7000 or FLA9500.Regions of Olfr12 and Olfr1410 were cloned and verified by Sanger sequencing . DNA forFor RNA Fluorescent In Situ Hybridization , MOE wasFor imaging GFP and mCherry, MOE was dissected and fixed in 4% PFA for 30 min on ice prior to being embedded in OCT. Coronal cryosections were taken at a thickness of 12 to 14 um and then air dried for 10 min. Slides were fixed with 4% PFA for 10 min. After fixation, slides were washed with PBST (PBS with 0.1% Triton X-100), blocked in PBST-DS (PBST +4% donkey serum), stained with DAPI (2.5 ug/mL) in PBST-DS, washed with PBST, and then mounted with Vectashield and imaged.For immunofluorescence , MOE wasConfocal images were collected with a Zeiss LSM 700. Image processing was carried out with ImageJ (NIH).SCR_011841). ChIP-seq and ATAC-seq reads were aligned to the mouse genome (mm10) using Bowtie2 (RRID:SCR_006646) (SCR_006525) and removed with Samtools (RRID:SCR_002105) (Adapter sequences were removed from raw sequencing data with CutAdapt (RRID:_006646) . Default_002105) . SamtoolSCR_010881)   was usedFor H3K9me3, H3K79me3, and H3K27ac ChIP-seq replicate experiments were merged, and HOMER was used to generate 1 bp resolution signal tracks normalized to a library size of 10,000,000 reads. Regions enriched for H3K9me3 were identified by running HOMER peak calling in region mode, with the following settings: -L 0 -F 2.5 -size 2000 -minDist 4000. A consensus set of H3K9me3 enriched regions was generated by selecting regions that were enriched in both biological replicates.For ATAC-seq, regions of open chromatin were identified by running HOMER peak calling in ‘region’ mode, with a fragment size of 150 bp and a peak size of 300 bp. For ATAC-seq signal tracks, replicate experiments were merged, and HOMER was used to generate 1 bp resolution signal tracks normalized to a library size of 10,000,000 reads. Reads were shifted 4 bp upstream in order to more accurately map the Tn5 insertion site. Reads were extended to the full fragment length, as determined by paired-end sequencing, except for signal tracks of ATAC-seq fragment ends , which wA small number of failed ChIP-seq and ATAC-seq experiments were excluded from analysis. Failed experiments were identified based upon the presence of very few enriched peaks and low fold enrichment of reads in the identified peaks.Omp-IRES-GFP sorted mOSNs. With the exception of P, all were bound by Ebf and Lhx2, and were present within a region enriched for H3K9me3. Using these criteria, we used Bedtools2 , was compared to the genome-wide rate (4729/8850) using a Binomial test, with the alternative hypothesis that the overlap rate is greater in OR clusters, yielding a p-value of p=2.557e-16. For Lhx2, 4729 peaks overlapped Ebf peaks, whereas 11468 did not. In H3K9me3 positive regions of OR clusters, 63 out of 114 Lhx2 peaks overlapped Ebf peaks. In R, this overlap rate (63 out of 114), was compared to the genome-wide rate (4729/16197) using a Binomial test, with the alternative hypothesis that the overlap rate is greater in OR clusters, yielding a p-value of p=5.702e-09.Heatmaps and signal plots were generated with Deeptools2. Unless GREAT  was usedMotif analysis was performed with HOMER. Motif searches were run on Ebf and Lhx2 consensus peak sets for the 200 bp region around the center of the peak, with repeat masking. The top de novo identified motif for Lhx2 and Ebf ChIP-seq was converted to TRANSFAC format, and plotted using Weblogo v3.5 , togetheHOMER was used to search Greek Island sequences for motifs with a variety of settings. A search for long motifs that allowed up to 4 mismatches  recovered a sequence motif that was highly enriched relative to random background sequences (p=1e-59). HOMER was then used to optimize this motif against a background set of all mOSN Ebf and Lhx2 co-bound sites. This optimized motif is reported as the composite motif in For To analyze specific instances of the composite motif for Composite motif multiple alignments  were genHOMER was used to identify all instances of the de novo, ChIP-seq derived Ebf and Lhx2 motifs genome-wide. Bedtools was then used to identify all instances of Ebf motifs that occur within Greek Islands with a composite motif score of 10 or above. Greek Islands without an Ebf motif were excluded from further analysis. For the remaining islands, Bedtools2 was used to identify the closest Lhx2 motif to each of Ebf motif. For Greek Islands with multiple Ebf motifs, only the closest pair was retained. Additional sets of sequences were analyzed in the same manner, and the distribution of motif distances was plotted in R. An identical analysis centered on Lhx2 motifs rather than Ebf motifs yielded similar results.SCR_012918)  . DiffbinSCR_005275)  using STAR . Samtool_005275) , either _005275) , or with_005275) . RNA-seq_005275) .Lhx2KO or Fusion protein expressing mOSNs. The base mean and log2fold change values from these analyses were used for plots. For MA-plots, significantly changed genes were identified with an adjusted p-value cutoff of 0.05.RNA-seq data analysis was performed in R with the DESeq2 package . Genes wFor density and scatter plots of log2 fold change in OR transcript levels , ORs witThe data reported in this paper will be available through GEO with accession number GSE93570. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Yin Shen (Reviewer #3).Thank you for submitting your article "Cooperative interactions enable singular olfactory receptor expression" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this study, Lomvardas and colleagues carried out a series of experiments to test the hypothesis that cooperative binding of the Lhx2 and Ebf transcription factors to OR enhancer elements is required for the singular OR gene expression in olfactory sensory neurons. The authors present compelling data demonstrating the enrichment of contiguous Lhx2 and Ebf binding motifs in the "Greek Island" elements linked to OR gene loci and their association with open chromatin as assayed by ATAC-seq; and likely cooperative binding of these two transcription factors to the combined motif as evidenced by the diminished binding of Ebf's in the Lhx2 conditional knockout. To demonstrate necessity of the proposed cooperative interaction, the authors then devised an ingenious experiment in which they first designed a linked Ebf-Lhx2 fusion protein that shows preferential binding to the combined motif and then use it to block OR expression through its transgenic expression in neurons in vivo. Sufficiency was inferred by observing an increase in expression of locus-specific ORs in a knock-in mouse in which a tandem repeat of Greek Island elements was inserted into the OR locus containing Rhodes.This work provides genetic insight for the stochastic mono-allelic OR gene expression program, and offer support to the model of inter-chromosomal interactions via the Greek Island elements in OR gene regulation. In addition, the results presented here suggest that cooperative binding of Lhx2 and Ebf transcription factors is involved in OR gene choice, an aspect of OR gene regulation that thus far has remained somewhat elusive by comparison to data supporting mechanisms of negative feedback regulation and stability of OR expression.Minor points:Reviewers raised the following points for further improvement of the manuscript:a) The Greek Island insertion experiment shown in b) The authors ought to be more open-minded when interpreting their results in the context of their model in which the Greek Islands mediate inter-chromosomal interactions required for high level, singular OR expression. It remains to be determined whether and how Lhx2-Ebf binding plays a role in such complexes. Indeed, if anything the data of c) In d) In  Minor points:Reviewers raised the following points for further improvement of the manuscript:a) The Greek Island insertion experiment shown in In our design of this knock-in experiment we did not introduce heterologous sequences that would allow us to distinguish between the endogenous Greek Islands from the ones inserted in the Rhodes cluster. Thus, we have no way to differentiate the state of the ectopic and endogenous enhancers in these mice. However, we reasoned that if the tandem insertion of 5 Greek Islands next to Rhodes increases occupancy by transcription factors we would see an in increase in the ChIP enrichment. ChIP-qPCR comparing Lhx2 occupancy on the 5 Greek Islands in wild type and homozygote knock-in mice does not show significant changes in Lhx2 enrichment on the 5 Islands between the two genotypes . These results, together with various normalization methods that take into account the existence of extra enhancer alleles, are shown in b) The authors ought to be more open-minded when interpreting their results in the context of their model in which the Greek Islands mediate inter-chromosomal interactions required for high level, singular OR expression. It remains to be determined whether and how Lhx2-Ebf binding plays a role in such complexes. Indeed, if anything the data of Changes are made in the Discussion and alternative explanations and possible caveats of our data are provided.c) In We added a new panel showing that the strongest effects in chromatin accessibility are observed on Greek Islands, consistent with the transcriptional changes following expression of the fusion protein .d) In As shown in the in situ hybridization experiments there are very few neurons in each section that express ORs from the Rhodes cluster, both in wild type and mutant mice. Thus, the main point of this experiment is that in each section of the olfactory epithelium the majority of the OSNs do not express the ORs from the Rhodes cluster. In other words, insertion of 5 strong transcriptional enhancers next to Rhodes does not result in the transcriptional dominance of the neighboring ORs. Determining the exact frequency of olfactory neurons expressing these ORs in an accurate fashion is beyond the point, since in both wild type and mutant mice they are expressed in less than 1% of the total population.Finally, we split the original


Escherichia coli kps capsule gene cluster. Transcription from promoter PR1-2 was dependent on the activity of the upstream promoter PR1-1, which activated PR1-2 via transcription coupled DNA supercoiling. During growth at 37 °C a temporal pattern of transcription from all three promoters was observed with maximum transcriptional activity evident during mid-exponential phase followed by a sharp decrease in activity as the cells enter stationary phase. The growth phase dependent transcription was regulated by Integration Host Factor (IHF), which bound within the PR1 region to repress transcription from PR1-2 and PR1-3. This pattern of transcription was mirrored by growth phase dependent expression of the K1 capsule. Overall these data reveal a complex pattern of transcriptional regulation for an important virulence factor with IHF playing a role in regulating growth phase expression.In this study we characterise three tandem promoters  within the PR1 regulatory region of the  Escherichia coli isolates from extraintestinal infections express a polysaccharide capsule or K antigen1. Capsule expression is an important virulence factor providing a selective advantage to the bacteria. It has been implicated in aiding transmission between hosts by preventing desiccation4 in adhesion5, resistance to host’s innate defences9, resistance to the host’s adaptive immune response12, and for intracellular survival, and crossing the blood brain barrier14. In addition, polysaccharide capsules also play signaling roles in mediating interactions between the pathogen and the host15. These include moderating induction of chemokines and cytokines, interacting with toll-like receptors (TLRs), and the perturbation of mucus clearance16.E. coli capsules have been classified into four groups (Groups 1–4) on the basis of a number of biochemical and genetic criteria17. The Group 2 capsule gene cluster (kps) is composed of three regions , located 225 bp upstream of kpsF, and Promoter Region 3 (PR3), 714 bp upstream of kpsM  that, in both cases, appears to be important in temperature regulation23 and in the case of PR3 important in attenuating the level of transcription that reaches kpsM22. In the case of PR1 two putative additional transcriptional start points have been located within the 225 bp UTR24. However whether these represent functional promoters and what role they may play in the regulation of transcription of region 1 is not known.s Groups –4 on thes Groups –4 on thekps expression and show that maximum PR1-2 activity is dependent on the transcription from the upstream PR1-1 probably through transcription-coupled supercoiling, while promoter PR1-3 can act independently. We show the contribution of each promoter to the temporal pattern of transcription during bacterial growth at 37 °C with a burst of transcription occurring in mid exponential phase which decreases as the cells enter stationary phase, a pattern mirrored by the cell surface expression of capsular polysaccharide. IHF binding in PR1 is critical to the growth phase regulation of transcription acting to repress transcription from PR1-2 and PR1-3 at the end of exponential phase. Taken as a whole these results add a new level of complexity to our understanding of regulation of transcription from the PR1 region, its regulation, and its role in the growth phase regulation of this capsule expression in pathogenic E. coli.In this paper we present a detailed analysis of the PR1 region. We demonstrate the presence of three functional promoters PR1-1, PR1-2 and PR1-3 that contribute to kpsF24, but to date, only the upstream start (PR1-1) has been studied in detail. We used a 5′ RACE (rapid amplification of cDNA ends) on mRNA extracted from strain UTI89 to confirm that these three start sites exist at +1, +132 and +181  was the most abundant mRNA  indicating that PR1-2 is functional on the chromosome of UTI89. However, the transcript from PR1-1 and PR1-2 contributes less than a quarter of the total transcript exiting PR1, indicating that transcription from PR1-3 is accounting for the majority of transcription that exits the PR1 region during exponential growth. In contrast, when the cells enter stationary phase both PR1-3 and PR1-1 appear less active, with the low levels of transcription at this point due to transcription from PR1-2  appears to contribute the least to the total mRNA exiting PR1 and this is true for all stages of growth tested Fig. . During 1-2 Fig. .Taken together these results clearly show that significant transcription is initiating from all three promoters on the chromosome of UTI89 and that transcription from PR1 is growth phase dependent with the three promoters contributing differently to the total transcript during the growth phase.600 = 1.0 and then appears to fall back which is in line with the decrease in transcription from the PR1 promoter region at late-exponential phase , using anti-K1 antibody, of UTI89 at various stages of growth was carried out Fig. . There iase Fig. . This islacZ transcriptional fusions were generated (using promoter-probe plasmid pRS415), as shown in Fig. lacZ, demonstrated significant β-galactosidase activity (1187 ± 174 and 1310 ± 191 Miller Units respectively). Likewise strain P90C(pJJ7) that contains PR1-1 and PR1-2 expressed comparable high level β-galactosidase activity (1432 ± 102 Miller Units). This indicates that when the PR1 region is cloned on a multi-copy plasmid PR1-1 is a powerful promoter  and P90C(pJJ5) was significantly lower (10 fold) than that of the full length PR1 construct (pJJ1) despite these constructs both containing the PR1-2 and PR1-3 transcriptional start sites  decreased β-galactosidase activity by approximately 99% compared to strain P90C(pJJ2)  we see a 79.1% reduction in copy number from pJTERM compared to pJJ1, which is consistent with transcription still initiating from PR1-3. These data confirm the dependence of PR1-2 on transcription coming from PR1-1 and that transcription from PR1-3 is independent of PR1-1. One mechanism by which transcription from PR1-1 could activate PR1-2 is by a transcription-coupled DNA supercoiling (TCDS)26. In this scenario RNAP creates local supercoiling during transcription with negative supercoils generated upstream and positive supercoils downstream of the translocating RNA26. In this case PR1-2 would be a supercoiling sensitive promoter such that TCDS from PR1-1 and PR1-3 might be the source of the local DNA supercoiling for promoter activation. To establish if PR1-2 is supercoiling sensitive, strain P90C(pJJ6) was grown in the presence of 5 μgml−1 of novobiocin, an inhibitor of the GyrB subunit of the DNA Gyrase enzyme27 and β-galactosidase activity measured. The presence of novobioicin caused an increase in β-galactosidase activity from 8 ± 1.3 Miller units to 19 ± 3 Miller units at mid log phase (p = 0.001) indicating that PR1-2 is sensitive to supercoiling. Both PR1-1 and PR1-3 were unaffected by the addition of novobiocin, indicating that these two promoters are not supercoiling sensitive (data not shown).To understand how the promoter activity at PR1-1 affects the activity of PR1-2, plasmid pJTERM was constructed  and P90CihfA::Tn10(pJJ5) lacking the IhfA subunit. As predicted the ihfA mutation decreased β-galactosidase activity from 1240 ± 110 Miller units to 947 ± 93 Miller units (p = 0.0176) in the case of pJJ1 and increased β-galactosidase activity from 54 ± 6 Miller units to 349 ± 29 Miller Units (p = 0.00006) in the case of pJJ5. As such we can be sure that heterodimeric IHF is acting at the +140 site.There is an IHF consensus binding site in the UTR centred at +140 that has been shown to bind IHFyed Fig. . In straity Fig. . This coity Fig. . However140 Fig. . These d140 Fig.  indicatecis-activity of IHF in regulating transcription at PR1, an IHF binding site mutant was constructed in strain UTI89 (UTI89IHF) as described in experimental procedures. In this strain the consensus IHF binding sequence 5′-TTACAACCCATTG30 was replaced by 5′-TTATGTGACGTTG – the mutated nucleotides are underlined  between the total transcript exiting the PR1 region (detected using primers kpsF-F and kpsF-R) at any of the time points  does not decrease as the cells move into stationary phase . As such at this stage we cannot preclude that these promoters may function to differentially control capsule expression in different niches within the host. The observation that PR1-1 had significantly more activity when cloned independently on a multi-copy plasmid suggests that additional regulation is occurring when the PR1-1 promoter is located on the chromosome and in single copy. One possibility is that on multi-copy PR1-1 is titrating out a repressor and we are currently investigating this.In this study we have demonstrated the presence of three functional tandem promoters PR1-1, 1-2 and 1-3 in the PR1 promoter region of the 26, in which RNAP creates local supercoiling during transcription with negative supercoils generated upstream and positive supercoils downstream of the translocating RNA36. In this scenario, PR1-2 would be a supercoiling sensitive promoter such that transcription-induced DNA positive supercoiling from PR1-1 could activate its expression. The observation that novobiocin, which inhibits GyrB and increases positive supercoiling, leads to increased transcription from PR1-2 indicates that PR1-2 is a supercoiling sensitive promoter activated by increased positive supercoils. This is in keeping with our hypothesis of TCDS in activating expression from PR1-2. Previous studies have demonstrated that promoter-promoter interaction via TCDS is normally short-range37, the separation between PR1-1 and PR1-2 is around 132 bp, so the notion that transcription from PR1-1 can influence the activity of adjacent promoter PR1-2 on the same DNA chain though local supercoiling is therefore reasonable. In the fis promoter there are three tandem RNAP binding sites and it has been proposed that idling of RNAP at an upstream promoter acts as an accessory factor to stimulate transcription from the downstream promoter38. The observation that the mutation at position 2 of the -10 of PR1-1, prevented transcription from PR1-2, would argue against RNAP idling at PR1-1 being the mechanism by which PR1-2 is activated, since mutations at this site in the -10 region will not prevent RNAP binding and idling at the PR1-1 promoter39.The three promoters PR1-1, PR1-2 and PR1-3 contribute differentially to the total transcript that exits the PR1 promoter region. By both site directed mutagenesis to disrupt the -10 of PR1-1 and by introduction a Rho independent terminator between the two promoters we demonstrate that transcription initiation from PR1-2 is dependent on transcription initiation from PR1-1. Our interpretation of this is that transcription from PR1-1 could activate PR1-2 by TCDSkps gene cluster and group 2 capsule expression in pathogenic E. coli. Implicit in this is that PR3 is also growth phase dependent since transcription from PR3 will control expression of region 2 and expression of the polysaccharide biosynthesis genes  to which 2.7 µl of 2-Mercaptoethanol (0.05 M) was added and the cell lysed by the addition of 40 µl choloroform, 20 µl 0.01% (w/v) SDS. After lysis 35 µl O-nitrophenyl-β-D-galactoside (ONPG: 4 mg/ml in Z-Buffer) was added to 176 µl of sample and the reaction proceeded in the dark at 28 °C until a yellow color developed when the reaction was terminated by the addition of 88 µl 1 M Na2CO3 to all wells. The plates were then read by measuring the OD420nm in plate reader (Synergy™ HT Multi-Mode Microplate Reader) for each reaction and the β-galactosidase activity in Miller units determined using the equation:Assays were carried out essentially as described47 was used to construct strain UTI89IHF. A 801 bp fragment containing the IHF binding site was amplified by PCR from UTI89 (using primers kpsF-F and kpsF-R) and was cloned in pBluescript SK+. The IHF binding site mutated by site directed mutagenesis (described below) and the mutated fragment was then subcloned into pDOC-C downstream of a Kan cassette. A 588 bp upstream fragment amplified by PCR from the UTI89 chromosome (using primers Fup-F and Fup-R) was then cloned upstream of the Kan cassette to provide the left flank for homologous recombination. The resulting plasmid was transformed into UTI89 alongside pACBSce1. Transformants were patched on 5% sucrose before induction of recombination onto the chromosome as described previously47. The introduction of the IHF binding site mutation on the chromosome of UTI89 was confirmed by PCR and sequencing.The gene doctoring methodE. coli cultures were inoculated into fresh LB medium (1:100) and grown at 37 °C to the appropriate OD600. 1 ml of culture was extracted and immediately mixed with 2 ml RNA Protect Bacteria Reagent (Qiagen). RNA was subsequently extracted using an RNAEasy Mini Kit according to the manufacturers instructions (Qiagen). RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop), where an A260 of 1.0 equals 40 μg ml−1.For RNA extraction, overnight −1 to 95 °C for 15 s).Reverse transcription of RNA was performed using QuantiTect Reverse Transcription Kit for fast cDNA synthesis enabling sensitive real-time two step RT-PCR for gene expression analysis (Qiagen) with 1 µg total RNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on an ABI Prism sequence detector (Applied Biosystems) using FAST SYBR Green Master Mix (Applied Biosystems). The thermal cycling conditions were as follows: 20 s holding step at 95 °C was followed by 40 cycles of a 1 s denaturation step at 95 °C and a 30 s annealing/polymerisation step at 60 °C. For each primer set a standard curve was obtained for absolute quantification by plotting the threshold cycle against the logarithm of a known amount of copy numbers and the quantities of target copies contained in an unknown sample were determined by extrapolation from the linear regression of the standard curve. A negative control (just RNA) that had not undergone the reverse transcription step was also included as a negative control in each run. In addition, each set of primers were also checked every time to make sure no primer-dimers occurred and that there was no other DNA contamination in the reaction by carrying out a melting curve stage after the total amplification cycle , 2 µl of 25 ng µl−1 template plasmid DNA, 12.5 µl of 10 ng µl−1 of each primer, 2.5 µl of 2.5 mM dNTP, 1 µl Strategy Pfu Ultra polymerase (Stratagene) and 14.5 µl ddH20. PCR was carried out under the following cycles: 95 °C × 5 min (1 cycle) 95 °C × 50 sec, 60 °C × 50 sec, 68 °C × 1 min + 1 min per 1 kb template (30 cycles), 7 min × 68 °C (1 cycle). The PCR product was immediately digested with Dpn I before transforming into E. coli DH5α and then plated out on the LB plate with appropriate antibiotics. Successful mutated plasmids was purified from cultured single colony and verified by sequencing.This protocol was performed as described previouslyE. coli strain K5746 containing plasmid pPLhiphimA-5 and purified using a heparin column as described49.Briefly, IHF was overexpressed in Competitor DNA and the promoter regions fragment were amplified from pBluescript or the promoter constructs with primers designed to anneal to the plasmid backbone either side of the cloning site. For each construct, PCR amplification products were checked by agarose gel electrophoresis and purified by MiniElute PCR purification kit (Qiagen). Equal quantities (25ng) of the target and competitor DNA were mixed and added to binding reactions containing reaction buffer  glycerol; 25 µM spermidine) and varying concentrations of purified IHF which had been pre-equilibrated for 10 min. Polyacrylamide gels underwent preliminary electrophoresis in 1 × TBE at 90 V for 90 min before samples loading. Samples were run into the gel at 250 V for 3 min before being separated at 120 V for 60 min. One lane containing DNA loading buffer was used as an indicator for migration of DNA fragments through the gel. Following electrophoretic fractionation of samples, gels were stained with 0.5 µg/ml Ethidium Bromide for 30 min and visualized under UV light.8 cells were washed three times in PBS, blocked for 1 h in 1% (w/v) BSA/PBS and treated with anti-K1 Ab (1/400 dilution) in buffer A  for 1 h at RT. After one wash with buffer A bacteria were treated with secondary Ab (1/500 dilution donkey anti-mouse Alexa Fluor 594) in buffer A for 1 h at RT and then washed three times in buffer A. 100,000 cells were analyzed for fluorescence per sample using a BDFortessa flow cytometer.Bacteria were grown to the OD indicated. Around 10All data generated or analysed during this study are included in the published article (and its Supplementary Information files).The gel images for Fig. Supplementary data


Thermus thermophilus HB27 is a metal tolerant thermophile containing a set of genes involved in arsenic resistance which, differently from other microbes, are not organized into a single operon. They encode the proteins: arsenate reductase, TtArsC, arsenic efflux membrane transporter, TtArsX, and transcriptional repressor, TtSmtB.The characterization of the molecular determinants of metal resistance has potential biotechnological application in biosensing and bioremediation. In this context, the bacterium TtArsX and the arsenic responsive transcriptional repressor TtSmtB are required to provide resistance to cadmium. We analyzed the sensitivity to Cd(II) of mutants lacking TtArsX, finding that they are more sensitive to this metal than the wild type strain. In addition, using promoter probe reporter plasmids, we show that the transcription of TtarsX is also stimulated by the presence of Cd(II) in a TtSmtB-dependent way. Actually, a regulatory circuit composed of TtSmtB and a reporter gene expressed from the TtarsX promoter responds to variation in Cd(II), As(III) and As(V) concentrations.In this work we show that the arsenic efflux protein TtSmtB and TtArsX is responsible for both the arsenic and cadmium resistance in T. thermophilus. The data also support the use of T. thermophilus as a suitable chassis for the design and development of As-Cd biosensors.Our results demonstrate that the system composed by The online version of this article (10.1186/s12934-018-0918-7) contains supplementary material, which is available to authorized users. Facilitated efflux transporters fall into two wide, functionally and evolutionary distinct membrane protein families, the P-type ATPases and the Major Facilitator Superfamily (MFS) of transporters , after an energy minimization step.The docking calculations were obtained using Hex Protein Docking server  with TtATtarsX (TtarsXp) previously named TTC0354p, was amplified by PCR using the primer pairs new 0354pr fwEcoRI and R0354NdeI, respectively , treated with different concentrations of NaAsO2, KH2AsO4 and CdCl2 (Sigma) as source of As(III), As(V) and Cd(II) respectively, and grown at 60 °C for 16 h. β-galactosidase activity assays were carried out on permeabilized cells in 96-well microplates at 70 °C with a Synergy H4 microplate reader (BioTeK) as described by Miller [For measuring reporter β-galactosidase activity, the growing transformants were diluted to 0.1 ODy Miller .420 = OD of the chromogenic product, OD550 = OD of the cellular debris, t = time of reaction, V = volume of used cells and OD600 = OD of the cell culture. β-galactosidase activity of T. thermophilus negative control (transformed with pMHPnorbgaA vector) was subtracted from that of the samples. The activity reported is the average of two or three independent experiments each made in triplicate. The error bars indicate the standard deviation of the average values. Miller Units expressed as a percentage, were calculated assuming that the Miller Units value of not treated cells (control) was 100%. Statistical analysis was performed using a Student’s t test; significant differences are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Miller units (U) were calculated by the equation:TtSmtB was purified to homogeneity using the procedure already described, consisting of thermo-precipitation of the E. coli BL21-Codon Plus (DE3) RIL/TtSmtB cell extract followed by HiTrap Heparin chromatography. The histidine tag was removed from purified TtSmtB by thrombin digestion (Sigma). The purified protein was stored in aliquots at − 20 °C [Recombinant  − 20 °C .TtSmtB, electrophoretic mobility shift assays (EMSA) were performed. The TtarsX promoter region was amplified by PCR using specific primer pair: 0354footprint fw and 0354footprint rv, .To determine if cadmium was ligand of TtArsX, is a membrane metal-transporter involved in arsenic detoxification; in fact, a mutant strain in which this gene had been knocked out, was about 15-fold more sensitive to both As(III) and As(V) treatment than the wild type [TtArsX is composed of six TM helices containing all the conserved signatures of the characterized P1B-2-subclass members which generally display a dual role in Zn2+ transport and toxic metal ion detoxification, especially Cd(II) [5QN(X)7K motif in TM5 and a DXG(X)7N in TM6  , 35. The in vivo . The oveTtArsX to the well characterized P1B-2-subfamily, show the presence of a soluble heavy metal associated domain (Pfam: PF 00403) and suggest a wider metal ion specificity than that previously known.Taken together, the results of in silico analysis assign TtArsX and the transcriptional repressor TtSmtB [To analyse the role of r TtSmtB  in resisT. thermophilus HB27 and two mutants defective in TtArsX or TtSmtB  in the presence of different Cd(II) concentrations, and determined the MIC values which are reported in Table T. thermophilus TTC0354::pK18 revealed a 15-fold increase in cadmium sensitivity in comparison to the wild type, thus showing a role of TtArsX in Cd(II) resistance; on the other hand, T. thermophilus ΔsmtB::kat, showed a 3-fold increase in cadmium sensitivity as compared to wild type HB27. One possible explanation for this unexpected result is a polar effect of the TtsmtB::kat mutation: in the mutant strain the kanamycin resistance gene is in counter sense; therefore, the basal levels of TtArsX could be lower than in the wild type, making the cells more sensitive.First, we compared the growth of T. thermophilus HB27 is almost 14-fold more sensitive to Cd(II) than to arsenic  and As(III) showed that TtarsX promoter had Cd(II) responsive activity, the regulatory region was cloned in a promoter probe vector upstream of the bgaA gene, encoding a thermostable β-galactosidase [TtarsXpbgaA  treatment at 10, 20 and 100 µM (see “TtarsX promoter in the reporter system is activated by Cd(II) by twofold at 20 µM  in comparison to values in untreated cells (393 ± 40 MU). At 100 µM Cd(II) transcription is also increased in comparison to the control, but at lower levels; the reduced activity could be the consequence of partial toxicity, since reduced growth rates were observed under these conditions , we performed in vivo and in vitro experiments. The comparison of β-gal activities in T. thermophilus HB27-pMHTtarsXpbgaA and T. thermophilusΔsmtB::kat-pMHTtarsXpbgaA grown in the absence of metals , is consistent with the in vivo role of TtSmtB as a repressor. Moreover, EMSA assays were carried out with recombinant TtSmtB on the TtarsX promoter after preincubation with or without Cd(II). As shown in Fig. TtarsXp and indicate that, upon binding with Cd(II), binding of TtSmtB to the promoter is hampered. TtSmtB DNA-binding behavior upon interaction with Cd(II) is similar to that already observed with As(III) and As(V) [TtSmtB in Cd(II) mediated TtarsX transcription where it works as repressor.To check whether nd As(V) , suggestTtSmtB regulates cadmium tolerance by controlling at transcriptional level the metal efflux gene, adopting a derepression mechanism similar to that employed for arsenic detoxification [Altogether, these results indicate that fication .TtSmtB and TtarsX responsive promoter as components of a bioreporter system for toxic metal detection, we aimed at characterizing their cadmium and arsenic sensing potential in engineered T. thermophilus cells. Hence, we generated a new reporter plasmid, pMHTtarsXpbgaA-nqoTtSmtB, to measure the transcription of the reporter gene from the TtarsX promoter in a context in which the TtsmtB gene was expressed constitutively from the nqo promoter , we assayed the bioreporter in T. thermophilusTTC0354::pK18 defective mutant. As expected  used to cope with arsenic; to the best of our knowledge this is the first functional characterization in a bacterium of a common/promiscuous mechanism to defend from both arsenic and cadmium, giving new venues for the understanding of the metal response evolution and the adaptation of environmental thermophilic microorganisms to deal with high concentrations of metals under enhanced solubilization conditions [ars operon, cells may have evolved arsenic resistance from preexisting metal detoxification systems. It is also plausible that promiscuous detoxification systems have developed according to the hypothesis that in the genomes of thermophilic microorganisms the genetic information is condensed. Moving to biotechnologically relevant applications in the improvement of biosensor field, this study outlines the importance of a detailed characterization of the molecular components  binding affinities) and points to T. thermophilus as suitable chassis cell for design and development of robust metal biosensors. In this context, advantageous modifications can be programmed to increase biosensor sensitivity, selectivity and/or ability to detect metal mixtures.This work reports for the first time the identification of a molecular mechanism responsible for cadmium tolerance of the thermophilic bacterium nditions . NotablyT. thermophilus HB27 transformed with the vector pMHTtarsXpbgaA in the absence (circle) and presence of 100 μM Cd(II) (triangle).Additional file 1: Table S1. Strains used in this work classified according to their genotype. Table S2. Oligonucleotides used in this work. Table S3. Plasmids used in this work classified according to their features. Figure S1. Growth curves of


GDNF) gene. This hyperacetylation significantly enhanced Egr-1 binding and increased the recruitment of RNA polymerase II (RNA POL II) to that region (P < 0.05). Egr-1 expression was abnormally increased in C6 glioma cells. Further overexpression of Egr-1 significantly increased Egr-1 binding to GDNF promoter II, while increasing RNA POL II recruitment, thus increasing GDNF transcription (P < 0.01). When the acetylation of H3K9 in the Egr-1 binding site was significantly reduced by the histone acetyltransferase (HAT) inhibitor curcumin, binding of Egr-1 to GDNF promoter II, RNA POL II recruitment, and GDNF mRNA expression were significantly downregulated (P < 0.01). Moreover, curcumin attenuated the effects of Egr-1 overexpression on Egr-1 binding, RNA POL II recruitment, and GDNF transcription (P < 0.01). Egr-1 and RNA POL II co-existed in the nucleus of C6 glioma cells, with overlapping regions, but they were not bound to each other. In conclusion, highly expressed Egr-1 may be involved in the recruitment of RNA POL II in GDNF promoter II in a non-binding manner, and thereby involved in regulating GDNF transcription in high-grade glioma cells. This regulation is dependent on histone hyperacetylation in GDNF promoter II.The specific mechanisms for epigenetic regulation of gene transcription remain to be elucidated. We previously demonstrated that hyperacetylation of histone H3K9 in promoter II of glioma cells promotes high transcription of the glial cell line-derived neurotrophic factor ( GDNF is a single-copy gene with two promoters and six exons located on chromosome 5 at p12-p13.1 [GDNF transcription in glioma cells was related to histone hyperacetylation in its promoter II rather than gene mutation [GDNF promoter II [GDNF transcription in C6 glioma cells [Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor (TGF-β) superfamily and an important neurotrophic factor. It was initially cloned from the rat B49 glial cell line . In huma12-p13.1 . Its tra12-p13.1 . We prevmutation . A subseGDNF transcription [GDNF transcription. In addition, bioinformatics analysis revealed that the upstream sequence of the GDNF gene transcription initiation site was rich in CG bases. Moreover, no typical TATA-box motif was found from −25 to −30 bp, which is to say GDNF promoter II region is TATA-less. CG-rich TATA-less promoters are thought to recruit RNA polymerase II (RNA POL II) by increasing their binding to gene-specific transcription factors, thereby initiating or promoting gene transcription [GDNF promoter II [GDNF transcription in high-grade glioma cells by increasing RNA POL II recruitment to the Egr-1 binding sites of GDNF promoter II.The transcription factor Egr-1 has been shown to regulate cell growth, differentiation, and tumor progression –16. It icription , 7. We tcription . Three cmoter II . TherefoGDNF promoter II, as well as their binding to Egr-1 and RNA POL II, in normal and diseased human tissue by chromatin immunoprecipitation (ChIP). Subsequently, the expression of Egr-1 in human high-grade and low-grade glioma tissues, normal brain tissue, and rat C6 astroglioma cells and normal astrocytes were measured by real-time polymerase chain reaction (PCR) and western blot. Next, the effects of Egr-1 overexpression on Egr-1 binding to GDNF promoter II, RNA POL II recruitment, and GDNF transcription in C6 glioma cells were determined by gene overexpression, ChIP, and real-time PCR. The effects of Egr-1 overexpression on these outcomes were elucidated using a C6 glioma cell model with decreased histone acetylation in GDNF promoter II following treatment with the histone acetyltransferase (HAT) inhibitor curcumin [GDNF in glioma cells.We examined acetylation of histone H3 Lys9 (H3K9) in the Egr-1 binding sites of curcumin –24. FinaGDNF promoter II  and the off-target region (P < 0.05) in high-grade glioma tissue compared with normal brain tissue. Moreover, the degree of acetylation was significantly higher in the Egr-1 on-target region than in the off-target region (P < 0.01). This is consistent with our previous report of a difference in H3K9 acetylation in GDNF promoter II of rat C6 glioma cells [P > 0.05, Figure Three consecutive and conserved Egr-1 binding sites were present −32 bp upstream of the transcription initiation site in human I Figure . H3K9 acs Figure . Acetylama cells . By contGDNF promoter II was measured by ChIP-PCR. The results showed that Egr-1 could bind between −98 and +56 of GDNF promoter II in all three types of tissues but not to the off-target region (between −864 and −793). This demonstrated the specific binding of Egr-1 to the Egr-1 binding sites of GDNF promoter II . This is consistent with the difference in H3K9 acetylation in GDNF promoter II of rat C6 glioma cells we previously reported [P > 0.05, Figure Egr-1 binding to I Figure . Comparereported . There wGDNF promoter II was measured by ChIP-PCR to assess the recruitment of RNA POL II in the Egr-1 binding sites of GDNF promoter II in different grades of glioma. The results showed that RNA POL II could bind to different extents in all three types of tissues . In contrast, there was no significant difference between low-grade glioma tissue and normal brain tissue . In contrast, there was no significant difference between low-grade glioma and normal brain tissues  after infection, indicating the successful establishment of a C6 glioma cell model with overexpressed Egr-1. Furthermore, Egr-1 overexpression in C6 astroglioma cells significantly increased both binding of Egr-1 to the Egr-1 binding sites of GDNF promoter II .To investigate the relationship of Egr-1 overexpression with RNA POL II recruitment to n Figure  and 5B. n Figure  and protn Figure  expressiGDNF promoter II is necessary for Egr-1 to regulate enhanced GDNF transcription in glioma cells, a C6 glioma cell model with histone hypoacetylation in the Egr-1 binding sites of GDNF promoter II was established by treatment with curcumin . Moreover, Egr-1 overexpression failed to significantly increase RNA POL II recruitment to the Egr-1 binding sites of GDNF promoter II or GDNF expression (P > 0.05). Conversely, we observed significantly increased GDNF transcription in the DMSO treatment group , the Egr-1 binding to GDNF promoter II (P = 0.061), and RNA POL II recruitment in that region  acquired from the affiliated hospitals of Suzhou University were utilized for ChIP and Western blot assays. Biopsied glioma tissues derived from patients who had not yet undergone cancer therapy were randomly sampled. From these, the specimens with pathological grades I–II were assigned to the low-grade glioma group, while those with pathological grades III–IV were assigned to the high-grade glioma group.2 humidified atmosphere. Healthy cells derived after two or three passages were chosen for the following experiments.Cryopreserved rat C6 astroglioma cell line and normal astrocytes were used for the experiments. Cell culture was performed as previously described , using m−ΔΔCt method, and the results were displayed as % Input = 2 (CTInput-CTChIP) × Input dilution factor × 100. The PCR primer was synthesized by Kangchen Bio-tech Inc. , and the sequence is shown in Table Using the acetyl-histone H3 (Lys9) polyclonal antibody , Egr-1 polyclonal antibody , RNA POL II monoclonal antibody  and normal IgG , ChIP was performed according to the the EZ-chip chromatin immunoprecipitation kit manufacturer's instructions . Routine polymerase chain reaction (PCR) and real-time PCR were used to detect DNA from immunoprecipitated target antibody. After conventional PCR, the PCR products were detected by agarose gel electrophoresis. After real-time PCR, data quantification was analyzed relative to gene expression data using the 2Egr-1 cDNA fragment (NM_012551.2) was amplified by PCR technology. The PCR primers were synthesized as follows : Egr-1-AgeI-F 5′-ATGGACAACTACCCCAAACT-3′, and Egr-1-AgeI-R 5′-TTAGCAAATTTCAATTGTCCTAGG-3′. The lentiviral vector Ubi-MCS-GFP (Genechem) was digested by the restriction enzyme AgeI. Then the Egr-1 cDNA fragments were ligated into the lentiviral vector Ubi-MCS-GFP. The primer (5′-AAGCGCTGCTGCCGCTGTTGCT-3′) located in the coding sequence of the Egr-1 cDNA was used in PCR to identify positive transformants. Positive clones, as confirmed by PCR, were chosen for sequencing. Recombinant lentiviruses, which coexpress enhanced green fluorescent protein (EGFP) and Egr-1 sequence, were produced by 293T cells following the cotransfection of Ubi-Egr1-EGFP and the packaging plasmids pHelper1.0 and pHelper2.0 (Genechem). The virus titer was detected by real-time PCR after concentrating and harvesting the viral supernatant. C6 astroglioma cells were plated at a density of 5 × 104 cells/well in a 6-well plate . After 24 h, cells were infected by 100 MOI concentrated lentivirus in the presence of polybrene (8 μg/ml). 72 h after infection, cells were harvested for the following experiments.The full-length rat −ΔΔCT). The forward and reverse primer sequences for the target gene amplification as well as internal reference gene are shown in Table RNA extraction and real-time PCR were performed as previously described . GlyceraTotal protein was extracted from each sample with NP-40 lysis buffer. Total extracts were separated on a 10% SDS-PAGE gel and transferred onto NC membrane . Blots were blocked using TBST solution containing 5% non-fat milk, and incubated in rabbit anti-mouse Egr-1 polyclonal antibody  or a mouse anti-avian β-Actin monoclonal antibody . After series of washings with TBST, the NC membrane was incubated in IR-Dye 800cw or 680rd labeled secondary antibodies , and then scanned using a LI-COR Odyssey imaging system . β-actin was used as the internal reference protein.Equal amounts of nuclear protein extracts prepared from C6 cells were incubated with the mouse monoclonal RNA polymerase II antibody , rabbit polyclonal Egr-1 antibody  or normal IgG  overnight at 4°C. Then, the agarose-conjugated protein-A/G beads (SCBT) were added into the immunocomplex and the mixture was incubated at 4°C for another 12 h. After extensive washing with ice-cold WB/IP lysis buffer with protease inhibitors, the beads were mixed with SDS loading buffer and boiled. Immunoblotting were performed as Western Blot described above.The C6 cells were grown on coverslips in a 24-well plate, fixed with 4% paraformaldehyde (w/v) for 40 min, and permeabilized with 0.5% (w/v) Triton X-100 in PBS for 15 min. The cells were then blocked for 30 min in PBS containing 10% fetal bovine serum (FBS) followed by overnight incubation with the primary antibodies against Egr-1  and RNA polymerase II . After series of washings with PBS, cells were incubated for 2 h with secondary antibodies  conjugated with fluorescein isothiocyanate or tetra methyl rhodamine isothiocyanate (TRITC) in dark moist environment. The coverslips were mounted with hydromount containing DAPI to stain the nuclei . The localization of Egr-1 and RNA polymerase II protein was examined using fluorescence confocal microscope (Leica Microsystems).t test was used to test the mean value in 2 groups. One-way analysis of variance (ANOVA) was used to determine the significance of any differences between experimental groups. A value of P < 0.05 was considered as significantly different.Statistical analyses were carried out using the SPSS 16.0 software package  and expressed as mean ± standard deviation (mean ± SD). Independent sample


Caudal-related homeobox transcription factor 2 (CDX2) plays a key role in the homeobox regulatory network and is essential in regulating the expression of several homeobox (HOX) genes during embryonic development, particularly in the gut. Genome-wide CDX2 chromatin immunoprecipitation analysis and expression data from Caco2 cells also suggests a role for CDX2 in the regulation of HOXB4 gene expression in the intestinal epithelium. Thus, the aim of this study was to investigate whether HOXB4 gene expression is regulated by CDX2 in the intestinal epithelium. We demonstrated binding of CDX2 to four different CDX2 binding sites in an enhancer region located upstream of the HOXB4 transcription start site. Mutations in the CDX2 binding sites reduced HOXB4 gene activity, and knock down of endogenous CDX2 expression by shRNA reduced HOXB4 gene expression. This is the first report demonstrating the CDX2 regulation of HOXB4 gene expression in the developed intestinal epithelium, indicating a possible role for HOXB4 in intestinal homeostasis.The mammalian  HOX genes in humans can be divided into four different clusters, HOXA, HOXB, HOXC and HOXD, which are located on different chromosomes[HOX genes. HOX genes are transcription factors belonging to the HOX protein family that share a DNA-binding domain, termed the HOX domain. The HOX domain is comprised of 60 amino acids that bind DNA in a sequence-dependent manner[HOX gene expression is organ-specific, and the deregulation of specific HOX genes has been associated with cancer development[HOXA9, which has been implicated in the development of acute myeloid leukemia[HOXD3, which increases invasiveness and metastasis in lung cancer[HOXA4 and HOXD10, which are overexpressed in colorectal cancer[Homeobox (HOX) proteins are important regulators during development and the formation of the anterior and posterior body plane in vertebrates. The 39 different romosomes. Each clnt manner. In adul leukemia, HOXD3, ng cancer, and botHOXB4 is still poorly understood.Another member of the HOX family is HOXB4, which is expressed in different tissues such as colon and liver and is aCaudal-related homeobox 2 (CDX2) gene is specifically expressed early after fertilization in the trophectoderm and is important for the correct formation of the blastocyst and subsequent implantation in the uterus[CDX2 gene is specifically expressed posteriorly in the tailbud. The gene encoding the transcription factor CDX2 is also a member of the parahox gene family. DNA interactions mediated by the homeodomain of CDX2 can activate gene expression in a manner similar to the HOX proteins. CDX2 also plays an important role during development and establishment of the gastro-intestinal tract, and loss of CDX2 during development is fatal[HOXB4.The he uterus. During  is fatal. Later i is fatal. Loss of is fatal. Moreove is fatal. We cond2.SW480 (ATCC: CCL-228), Caco2 (ATCC: HTB-37) and HEK293 (ATCC: CRL-1573) cells were cultured as monolayers in Dulbecco's Modified Eagle’s Medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (FCS), 100 μg/ml streptomycin and 100 U/ml penicillin. All cell lines were cultured in a humidified atmosphere under 5% COCDX2 knock down was created using the lentiviral pLKO.1 puro shRNA expression vector. The shRNA expression vector (pLKO.1 puro) was a kind gift from Dr. Bob Weinberg [AgeI and EcoRI, and the DNA oligos CDX2 shRNA 735 Forward and CDX2 shRNA 735 Reverse were hybridized and inserted into pLKO.1. A lentiviral control vector containing scrambled non-target shRNA (pLKO.1 scramble) was used as a negative control and was a kind gift from Dr. David Sabatini [6 HEK293 cells were plated in 6-well plates one day prior to transfection. HEK293 were co-transfected with either scrambled shRNA or CDX2 shRNA expressing lentivirus, psPAX, which was a gift from Malin Parmar , and pMD2.G, which was a gift from Didier Trono . The transfected HEK293 cells were maintained for 48 hours in DMEM supplemented with heat inactivated 10% FCS. The supernatant was then sterile filtered using a 0.45-μM filter and added to 5x105 SW480 cells, followed by transduction for 24 hours. After 24 hours, the medium was replaced with DMEM supplemented with 10% heat inactivated FCS and 10 μg/ml puromycin , and the cells were reseeded twice per week for 3 weeks. Thereafter, cells were reseeded twice per week and cultured in DMEM supplemented with 10% heat inactivated FCS. CDX2 knock down was verified by RT-qPCR and western blotting.An SW480 cell line with stable  MA, USA). pLKO.1  Addgene). Next, 6HOXB4 enhancer was identified using previously published chromatin immunoprecipitation-sequencing (ChIP-seq) data [A potential CDX2 binding region in the eq) data , which wHOXB4 promoter and enhancer region. The HOXB4 promoter and enhancer region was cloned using an In-Fusiontm cloning kit . A 4997-bp fragment was amplified using Phusion HOTSTART II HD polymerase , the Infusion HOXB4 Enhancer/promoter forward primer and the Infusion HOXB4 reverse primer (HindIII according to the manufacturer’s protocol (Thermo Fisher Scientific), and the promoter and enhancer were inserted using the In-Fusiontm HD cloning kit (Clontech) according to the manufacturer’s protocol (Clontech). A 2850-bp fragment excluding the up-stream CDX2 binding sites was amplified using Phusion HOTSTART II HD polymerase (Thermo Fisher Scientific), the Infusion HOXB4 promoter forward primer and the Infusion HOXB4 promoter reverse primer (HindIII according to the manufacturer’s protocol (Fisher Scientific), and the HOXB4 promoter was inserted using the In-Fusiontm HD cloning kit (Clontech) according to the manufacturer’s protocol (Clontech)The BAC clone RP11-111C6, which covers chromosome 17, positions ‒48583316 to ‒ 48578320 (GENBANK ID: NC_000017.11), was obtained from the BACPAC Resource Centre  and used as a template to amplify the e primer . The pGLe primer . The pGLHOXB4 promoter/enhancer region (4997-bp fragment) and in which each of the CDX2 binding sites, sequence a/cATAAAa/t[EcoRI cleavage sequence, GAATTCG, were synthesized by Eurofins . The HOXB4 pGL4.10 reporter plasmid was digested with NdeI (Thermo Fisher Scientific) and PsyI (Thermo Scientific) according to the manufacturer’s protocol. Each of the four CDX2 mutated constructs was used as template, and a 1724-bp fragment containing the mutated CDX2 binding site was amplified using Phusion HotStart II polymerase (Thermo Fisher Scientific), following the manufacturer’s protocol, and the Infusion HOXB4 Move forward and reverse primers (NdeI- and PsyI-digested HOXB4 pGL4.10 reporter plasmid using the In-Fusiontm HD cloning kit (Clontech) according to the manufacturer’s instructions, resulting in four different plasmids with each of the CDX2 binding sites mutated.Four different constructs containing the cATAAAa/t, were re primers . The fra4 cells/well and transiently transfected the following day. The cells were transfected using polyethylenimine at a final concentration of 2 μM and 0.3 μg DNA/well. Twenty-five nanograms of luciferase reporter gene plasmid and 50 ng of β-galactosidase expression plasmid were used per well, and the DNA concentration was adjusted to 0.3 μg with pBluescript SK+ . Forty-eight hours post-transfection, cells were harvested and lysed; luciferase and β-galactosidase activities were determined using the Dual Lighttm luminescent reporter gene assay , and luminescence was determined using a GloMax® 96 Microplate Luminometer (Promega) according to the manufacturer’s instructions.One day prior to transfection, SW480 cells were seeded in 24-well plates at a density of 5x10CDX2 shRNA) using Trizol (Life Technologies) as described by the manufacturer. Ten micrograms of total protein were analyzed by SDS-PAGE using a NuPAGE 4–12% Bis-Tris protein gel (Life Technologies) followed by transfer to a PVDF membrane via electroblotting using the iBlot Western blotting system (Life Technologies). The membrane was incubated with primary antibodies against human CDX2  or glyceraldehyde 3-phosphate dehydrogenase .Total proteins were extracted from the lentiviral-modified shRNA-expressing SW480 cells  on the minus strand, position ‒2737 to ‒2747 (site-3) on the plus strand, position ‒2836 to ‒2846 (site-2) on the minus strand, and position ‒3030 to ‒3040 (site-1) on the plus strand. The electrophoretic mobility shift assay (EMSA) was performed as previously described[The amplified region of the g of 80% . Four didescribed. BrieflyT method, and ribosomal protein large P0 (RPLP0) was used as a reference gene[Total RNA was extracted from SW480 cells using an E.Z.N.A. Total RNA KIT I . RNA extraction was performed according to the manufacturer’s instructions. cDNA was synthesized according to the manufacturer’s instructions, using qSCRIPT cDNA Supermix . Relative quantification was calculated using the ΔΔCence gene.HOXB4 are listed in Confluent Caco2 cells were cross-linked, sonicated, and immunoprecipitated with an antibody specific for either human CDX2 (BioGenex) or an antibody specific for the influenza hemagglutinin (HA) epitope  as a negative control, as previously described. Purifiet-tests, and values are shown as the mean and standard deviation (SD). P-values of less than 0.05 were considered significant.Statistical analysis was performed using unpaired Student’s HOXB4 enhancer, located from 2640 to 3050 bp upstream of the HOXB4 transcription start site  compared to the negative control , indicating that CDX2 is involved in maintaining the endogenous expression of HOXB4 in SW480 cells.To determine whether endogenous er groups. In SW48HOXB4 expression, a luciferase reporter construct containing the human HOXB4 enhancer and promoter as well as another construct containing only the HOXB4 promoter were created. The activity of the reporter constructs containing the HOXB4 promoter or HOXB4 promoter and enhancer were measured both in SW480CDX2 and SW480NEG cells. The HOXB4 promoter exhibited activity on its own when comparing the expression levels in SW480CDX2 and SW480NEG cells (HOXB4 enhancer and promoter was significantly reduced (P<0.01) when examining the expression levels in SW480CDX2 compared to SW480NEG cells  relative to cells without co-transfection of the CDX2 expression vector . The enhancer activity of each construct was measured using transient transfection in SW480 cells, and mutation of all four sites individually significantly reduced HOXB4 enhancer and promoter luciferase activity  compared to the wild type reporter construct We used computational DNA sequence analysis to identify potential CDX2 binding sites in the r region . To furtonstruct . When coHOXB4 enhancer/promoter constructs containing mutations within the CDX2 binding sites , we performed EMSA with oligonucleotides covering each of the four potential CDX2 binding sites and nuclear extracts from Caco2 cells , or an unspecific oligonucleotide were utilized. Unlabeled HOXB4 site-2 and site-4 probes completely out-competed CDX2/DNA complexes  antibodies. The addition of an antibody against CDX2 effectively supershifted the CDX2/DNA complex in site-2, -3, and -4 , lane 5 HOXB4 expression is poorly understood in the intestinal epithelium and, until now, no other transcription factors in the intestinal epithelium have been shown to directly induce HOXB4 expression. Here, we demonstrate that CDX2 regulates HOXB4 gene expression in the intestinal epithelium-derived cell line SW480. Induction of HOXB4 expression has previously been studied during fetal development, and expression is induced in the hindbrain by retinoic acid. The induction of HOXB4 expression is mediated by the retinoic acid response element located within the HOXB4 enhancer[HOXB4 is also expressed during hematopoiesis and regulated by the transcription factor GATA-2 in hematopoietic stem cells and progenitor cells[The regulation of  enhancer. HOXB4 itor cells.HOXB4 expression levels, achieved using siRNA-mediated CDX2 knock down in the COLO320 cell line, has previously been demonstrated[A reduction in onstrated. In the in vitro binding of CDX1, CDX2 and CDX4 has been described, and binding has been demonstrated by all three CDX transcription factors in vivo[Previous studies of CDX1, 2 and 4 have shown that they bind to the promoter of HOXA5, and it has been suggested that CDX4 modulates HOXA5 expression during embryonic development. CDX2 exs in vivo.HOXB4 binding sites have been investigated in hematopoietic stem cells and progenitor cells, and HOXB4 has been shown to both up- and down-regulate the expression of target genes. HoweverHOX genes are transcription factors, their downstream targets must be identified to clarify the function of HOXB4 expression in the intestinal epithelium. It is possible that HOXB4 induction plays a role in cellular differentiation or intestinal homeostasis.The function of the CDX2-mediated induction of HOXB4 in intestinal differentiation and homeostasis is not known. As We show that CDX2 binds to the HOXB4 enhancer, regulates HOXB4 expression in intestinal-derived epithelial cell lines, and has a possible role in the regulation of HOXB4 expression in the intestinal epithelium and intestinal homeostasis.