Escherichia coli. However, little is known about the physiological implications of this regulatory disposition. In this study, we demonstrate that transcription of ompW is indeed mediated by a series of global regulators involved in the anaerobiosis of E. coli. We show that FNR can both activate and repress the expression of ompW through its direct binding to two distinctive sites, -81.5 and -126.5 bp respectively, on ompW promoter. ArcA also participates in repression of ompW under anaerobic condition, but in an FNR dependent manner. Additionally, ompW is also subject to the regulation by CRP and NarL which senses the availability and types of carbon sources and respiration electron acceptors in the environment respectively, implying a role of OmpW in the carbon and energy metabolism of E. coli during its anaerobic adaptation. Molecular docking reveals that OmpW can bind fumarate, an alternative electron acceptor in anaerobic respiration, with sufficient affinity. Moreover, supplement of fumarate or succinate which belongs to the C4-dicarboxylates family of metabolite, to E. coli culture rescues OmpW-mediated colicin S4 killing. Taken together, we propose that OmpW is involved in anaerobic carbon and energy metabolism to mediate the transition from aerobic to anaerobic lifestyle in E. coli.Understanding bacterial physiology relies on elucidating the regulatory mechanisms and cellular functions of those differentially expressed genes in response to environmental changes. A widespread Gram-negative bacterial outer membrane protein OmpW has been implicated in the adaptation to stresses in various species. It is recently found to be present in the regulon of the global anaerobic transcription factor FNR and ArcA in  Fumarate and Nitrate Reduction (FNR) and Aerobic respiratory control (ArcAB)  . While ttor ArcA . In additor ArcA . Of part. Although its functions remain obscure, it has been implicated in bacterial responses to various antibiotics stresses and environmental stimuli. For instance, OmpW expression was reported to be induced in the presence of minimal inhibitory concentration of tetracycline and ampicillin, in a nalidixic acid-resistant E. coli K-12 strain  and consequently the protein was suggested to mediate the eﬄux of MV  of Gram-negative bacteria provides as a permeability barrier that hinders the entry of both toxic molecules and nutrients. To facilitate the selective entry of nutrients and other molecules that are necessary for the growth and function of the cell, Gram-negative bacteria use protein channels called porins within their OM. In addition to the major porin proteins which form trimeric, hydrophilic barrels composed of 12-22 antiparallel β-strands, the OM also contains a considerable number of smaller, monomeric β-barrel proteins called minor porin proteins, which are composed of 8 or 10 β-strands . Althoug2 strain , and dur2 strain . Temperaded host . In Salmux of MV . However MV , Salmonella typhimurium, Enterobacter cloacae and Klebsiella pneumonia, suggesting potential roles of OmpW in bacterial adaptation or pathogenicity. Vibrio cholerae OmpW, which is present in all known Vibrio strains, has been found to be highly immunogenic and attracted interests for vaccine development  of carbon source , 0.2% casamino acids (w/v), 1 mM MgSO4, 1 mM CaCl2, 2.5 mg ml-1 ferric ammonium citrate, 2 mg ml-1 thiamine and 0.02% ammonium molybdate (w/v). For certain experiments, the medium was also supplemented with 0.4 mM potassium nitrate, 40 mM sodium fumarate, or 40 mM Trimethylamine N-oxide (TMAO). Appropriate antibiotics, when required, were supplemented to the media at the following concentrations: Ampicillin (100 μg ml-1), Chloramphenicol (20 μg ml-1), Kanamycin (25 μg ml-1), or Tetracycline (10 μg ml-1). Conditions for aerobic, micro-aerobic, and anaerobic culturing of bacteria are as followings. Aerobic: 3 mL of inoculum is cultured in a 15 mL volume culture tube with 220 rpm aeration; Micro-aerobic: 30 mL of inoculum is cultured in a 50 mL volume tube with 14 rpm aeration. The micro-aerobic growth condition in this system is achieved by limited air supply and aeration. The extent of oxygen limitation was recorded by the promoter-lacZ activity of the well studied reporter gene narG (5–107 cells are inoculated into a screw-capped Pyrex tube filled up with media and are incubated without shaking. Anaerobic growth condition in this system is generated through the following manners: (i) utilization of a screw-capped Pyrex culture tube filled with growth media; (ii) a small initial inoculums , which can rapidly consume the limited residual O2 in the media, and (iii) without aeration supply. The micro-aerobic culturing system described.pression . E. coliene narG , which aFastTaq (Roche) was used. And for molecular cloning and site-directed mutagenesis, iProof (BIO-RAD) was used for the amplification of DNA fragments. Strains containing PompW-lacZ or ompW-FLAG fusion on the chromosome of E. coli K-12 MG1655 were constructed following the method described by ompW-lacZ, promoter region of ompW (-215 to +56 relative to ompW transcription start site) flanked by XhoI and BamHI at its 5′- and 3′- respectively was amplified by PCR using E. coli MG1655 genomic DNA as the template. Following enzyme digestion and purification of the digestion product, the DNA fragment was ligated into pPK7035 (lacZ such that the expression of lacZ is driven by the ompW promoter (pPK7035-PompW). Subsequently, a linear DNA fragment containing 36 nt homologous region to lacI followed by kan-PompW-lacZ and ~900 nt homologous region to the downstream of lacZ was amplified by PCR using pPK7035-PompW as the template. Following Dpn I digestion to remove the template, the PCR product was purified using illustraTM GFX PCR DNA and Gel Band Purification Kit . The purified PCR product was then electroporated into the competent cells of MG1655 transformed with the plasmid pKD46 which contains λ-red recombinase. Desirable colonies were selected on LB plate containing kanamycin and were verified by DNA sequencing.Strains, plasmids and primer sequences described in this work are listed in Supplementary Tables  pPK7035  at the pPompW-lacZ fusions containing various mutations were constructed by site-directed mutagenesis of pPK7035-PompW followed by PCR amplification of the linear DNA fragment for the pKD46 mediated homologous recombination using the mutated pPK7035-PompW plasmid as the template. This resulted in the strains of AY0234, AY0284, AY0285, AY2010, AY2048, respectively. PompW-lacZ fusion was transduced from AY0210 via P1 vir into PK4811, PK8281, AY2203, AY2204, AY2206, AY2020 to study the effect of fnr, crp, narL, narP, deletion on the transcriptional activity of PompW-lacZ. Construction of ompW-FLAG was similar to that of PompW-lacZ except for the primers used to amplify the linear DNA fragment which contains 50 bp homologous region upstream of the ompW stop codon followed by the sequence of FLAG, kanamycin resistance gene, and 50 bp homologous to sequences downstream of ompW stop codon. Following the successful construction, the ompW-FLAG loci was also transduced from AY0264 to PK4811 via P1 vir to study the effect of deletion of fnr on the production of OmpW-FLAG. Other strains containing gene deletions were generally constructed by P1 vir-mediated transduction from the corresponding Keio collection strains [National BioResource Project : E. coli]. When necessary, pCP20, a plasmid which encodes FLP recombinase was used to remove the antibiotic resistance marker  served as the template. Reactions were as below: 5X iProof GC buffer, 2.0 μl; DMSO, 1 μl; 10 mM dNTP mix, 0.4 μl; 5 mM forward and reverse primers 2.0 μl each; template, 10–100 ng; iProof (2 units μl-1), 0.2 μl; add ddH2O to a total volume of 20 μl. The PCR was performed using a thermal cycler C1000 (BIO-RAD). The program was set as follows: 98°C * 3 min; 98°C * 10 s, 50°C * 1 min, 72°C * 5 min, for 18 cycles; 72°C * 10 min. PCR products were subsequently treated with 1 μl DpnI  at 37°C for 1 h to remove the remaining template. The resulting products were transformed into E. coli DH5α competent cells. Mutations were verified by DNA sequencing.To replace or delete nucleotides in the Escherichia coli strains containing promoter-lacZ fusions were inoculated in M9 minimal medium  containing appropriate concentration (w/v) of carbon sources , 0.2% CAA (w/v), 1 mM MgSO4, 1mM CaCl2, 2.5 mg ml-1 ferric ammonium citrate, 2 mg ml-1 thiamine and 0.02% ammonium molybdate (w/v). 0.4 mM potassium nitrate or 40 mM sodium fumarate was also added as indicated in certain experiments. Cells were grown aerobically by shaking at 250 r.p.m. to an OD600~0.3 or anaerobically in screw-capped tubes without aeration to an OD600 ~0.2 at 37°C. Chloramphenicol (50 μg ml-1) or Tetracycline (20 μg ml-1) was then added to terminate cell growth and any further protein synthesis. Cells were placed on ice until assayed as described previously  to a total volume of 1 ml and lysed by adding chloroform and SDS. Following thorough mixture and incubation at 28°C water bath for 5 min, 200 μl ortho-Nitrophenyl-β-galactoside  was added to initiate the reaction. Upon the development of yellow color, reactions were stopped by adding 500 μl freshly prepared Na2CO3 solution and vortex. A420 and A550 of the reaction solution, OD600 of cell culture, and time required for color development in each of the reactions were recorded to calculate the β-Galactosidase activity. The activity is expressed in Miller Units as the mean from three independent experiments. Error bars represent the standard deviation.eviously . Proper E. coli MG1655 was mixed with 1.25 ml ice-cold ethanol/phenol stop solution (5% water-saturated phenol pH4.5 in ethanol) and placed on ice for 10 min before being harvested by centrifugation at 4000 g for 9 min at 4°C. After removing supernatant the cell pellet was frozen in liquid nitrogen and stored at -80°C to aid lysis. Cells were lysed by resuspending in 800 μl TE buffer  containing 1.4 μl 36 kU μl-1 lysozyme (Epicentre), and then placed in 64°C water bath for 2 min. After incubation, 88 μl 3M NaOAc (pH 5.2) was added to adjust the pH and ion strength of the lysate solution. Subsequently, acid-phenol/chloroform extraction followed by ethanol precipitation was performed to obtain the total RNA following the manufacture’s instruction. To remove trace amount of genomic DNA contamination, the extracted RNA was subject to DNase I treatment using the turbo DNA Free Kit (Amibion). Absence of genomic DNA contamination was confirmed by PCR using the prepared RNA as template. The quantity of RNA was determined using NanoDrop 2000 (Thermo Scientific).Anaerobic or aerobic cell culture was obtained as described above in β-galactosidase assay. 8 ml anaerobically grown culture of -1 random primers (Promega), and incubated at 70°C for 5 min followed by an ice bath for 5 min. The following components were then added sequentially: 5 μl 5 × RT buffer (Promega), 5 μl 10 mM dNTP mix (Promega), 200 units of MMLV reverse transcriptase (Promega), and 20 units of RNase inhibitor (Roche). The mixture was incubated in 40°C water bath for 1 h and then the reaction was terminated by incubation at 70°C for 15 min. The quantity and purity of cDNA was determined using a NanoDrop 2000 (Thermo Scientific).~1 μg RNA prepared as described above was mixed with 1.5 μl 10 μM gene-specific reverse primer or 1 μl 200 μg μl1. rrsA gene of the 16S rRNA was chosen as the normalizing gene. RT-qPCR was performed with each specific primer pair  using SYBR Green Mastermix (ABI). The reactions were conducted on an ABI StepOnePlus real-time PCR detection system, and the fluorescence signal of SYBR green intercalation was monitored to quantify the double-stranded DNA product formed in each PCR cycle. Data was analyzed using ΔΔCt method according to the manufacturer’s instruction. Experiments were performed using the cDNA sample obtained from three independent isolates, and error bars represent the standard deviation.The specific primer pairs were designed using on-line toolE. coli MG1655 WT strain as mentioned above. The extracted RNA was first hydrolyzed by Tobacco Acid Pyrophosphatase (TAP) followed by ligation to the 5′ RACE adapter. The resulting RNA was then reverse transcribed to cDNA using reagents supplied in the FirstChoice RLM-RACE Kit (Ambion) following the manufacture’s instruction. The obtained cDNA was then utilized to perform outer (primer: ompW 5′race out-: 5′-GTTGGTGGCAGATGATGAACGGTT-3′) and inner (primer: ompW 5′race in-: 5′-CCGCTCGAGCGCTGCCAGTAATTCCACACCAATGT-3′) PCR using Fastart Taq (Roche) DNA polymerase. The PCR products were gel band purified and cloned into BamHI and XhoI sites of pPK7035 . The binding reaction was conducted in a 10 μl reaction system containing variable amount of protein and DNA fragment in the binding buffer . 0.2 mM cAMP was added to each reaction when the binding of His6-CRP to the ompW promoter was examined, and 5 μg of Bovine serum albumin (NEB) mixed with DNA served as a negative control. Following incubation at 37°C for 30 min, the reaction mixtures were loaded onto a 6% poly-acrylamide gel. Electrophoresis was conducted at 120V for 1.5–2 h in an ice-bath. The gel was stained with EB and photographed under UV illumination.Ethidium bromide (EB)-stained EMSA was performed according to a previous report  with sli6-NarL or phosphorylated His6-NarL (His6-NarL-P) to ompW promoter. Phosphorylated protein was obtained following a previous protocol with slight modification  as following: the DNA fragment was diluted to 10 ng μl-1 with ddH2O and mixed sequentially with labeling buffer, CoCl2-solution, DIG-ddUTP solution and terminal transferase; the mixture was incubated at 37°C for 15 min before the labeling reaction was stopped with 0.2 mM EDTA. The probe was then purified with illustraTM GFX PCR DNA and Gel Band Purification Kit  and eluted with ddH2O. Each EMSA reaction mixture contained 1 ng of DIG-labeled DNA and appropriate amount of phosphorylated protein as described in figure legends. Five microgram of Bovine serum albumin (NEB) mixed with DNA served as the negative control. The binding reaction, incubation and electrophoresis were performed as described in EB-staining EMSA. Following electrophoresis DNA probes were transferred to positive charged nylon membrane (Roche) in 10X SSC solution , followed by detection using Roche DIG Gel Shift Kit (second generation).Digoxigenin-labeled EMSA  was perffication : 1 μg ofg for 9 min at 4°C and subsequently resuspended in 30 μl BugBuster Protein Extraction Reagent (Novagen) containing 1 mg ml-1 lysozyme and 10 U ml-1 DNase I to lyse the cells. The mixture was incubated at 37°C for 30 min followed by centrifugation at 16000 g for 20 min at 4°C. The supernatant was transferred to another fresh tube. To avoid any insufficient lysis, the pellet was resuspended in 10 μl 4X SDS buffer followed by incubation at 55°C for 25 min. The supernatant was then combined with the 30 μl supernatant from lysis by the BugBuster Protein Extraction Reagent and incubated at 55°C for another 25 min. After centrifugation at 16000 g for 1 min, a portion of the protein extract was subject to total protein analysis using the Bio rad Bradford Protein Assay kit. ~ 40 μl supernatant  was loaded onto SDS-PAGE and then subjected to western blot analysis using 1:5000 mono-clonal anti-FLAG (Sigma) primary antibody and anti-mouse IgG secondary antibody (Sigma).Anaerobic and aerobic cultures of AY0264 and AY0265 were obtained as described in β-galactosidase assay. Eight mililiter culture (O.D. as 0.3) was pelleted by centrifugation at 4000 6-tagged FNRD154A, CRP and NarL, overnight cultures of AY2023, AY2030, AY0984 were diluted 1:50 to 50 ml LB containing 20 μg/ml kanamycin, and grown at 37°C with shaking for ~2.5 h (or ~5 h for AY0984) till OD600~0.6. 0.5 mM IPTG was then added to the culture to induce the expression of His6-tagged protein at 30°C for 4 h. For over-expression of His6-tagged Colicin S4, the plasmid p33-S4His containing the DNA sequence encoding Colicin S4 supplied by Prof. Dirk Linke’s group , 0.15 mM Phenylmethanesulfonyl fluoride (PMSF)]. Cell suspension was lysed by sonication for 10 min  using VWR SCIENTIFIC BRANSON SONIFIER 450. The resulting cell lysate was then subjected to ultracentrifuge at 45000 g for 30 min at 4°C. The supernatant was mixed with 2 ml Ni Sepharose 6 Fast Flow resin  and gently shaken for 1 h at 4°C to allow sufficient binding. The mixture was then poured into a 1.5 cm X 10 cm column and washed with 10 ml binding buffer . His6-tagged protein was eluted with 5 ml elution buffer . The elution was dialyzed for 3 h in 250 ml dialysis buffer  for three times at 4°C to remove imidazole. SDS-PAGE was used to monitor the purification process and analyze the purified protein. The concentration of purified protein was determined using NanoDrop 2000 (Thermo).For over-expression of His’s group  was tran2 (PDBID: 2F1T)  in 3D structure were completed as a loop using the SWISS-MODEL online homology modeling server. The structures of LDAO which was present in OmpW crystal, and the potential substrates fumarate and TMAO were generated by ChemBioDraw Ultra , which were subsequently optimized with the MM2 force filed shipped with ChemBio3D. Then, AutoDock 4.2.5.1 was used to study the binding conformations of LDAO, fumarate, or TMAO to 2F1TA, using the Lamarckian genetic algorithm (LGA) (The 3D structure of OmpW was obtained from RSCB Protein Data BankD: 2F1T) , which chm (LGA) . The defE. coli MG1655 strain was inoculated (cell density as ~105 cells ml-1) in M9 minimal medium containing 0.4% glycerol (w/v) and grown at 37°C with shaking till OD600~0.1. Colicin S4  and 0–10 mM of fumarate, succinate, or TMAO as indicated in Figure 8 was then added to the cell culture. Cells were continuously grown aerobically at 37°C and OD600 was recorded every 30 min using a SPECTRONIC 20D+ (Thermo Scientific) till the culture reached stationary phase. For the rescue assay under anaerobic conditions, E. coli MG1655 strain was inoculated (cell density as ~105 cells ml-1) in M9 minimal medium containing 0.4% glycerol (w/v) and 40mM TMAO and grown at 37°C anaerobically till OD600~0.1. Colicin S4  and 0–10 mM of fumarate as indicated in Figure 8 was then added to the cell culture. Cells were continuously grown anaerobically at 37°C and OD600 was recorded every hour till the culture reached stationary phase.For the rescuing assay under aerobic condition, ompW is indeed directly regulated by FNR as indicated in the genomewide studies by ompW-lacZ in E. coli MG1655 and its isogenic Δfnr strain and examined in vivo transcription of ompW gene under both aerobic and anaerobic conditions. β-galactosidase assay showed that transcription of PompW-lacZ under anaerobic condition was increased to ~3-fold of that under aerobic conditions, and deletion of fnr caused decrease of the transcription to the similar level as that of aerobic condition, suggesting that transcription of ompW was activated by FNR under anaerobic condition . To confirm this up-regulation at mRNA and protein level, we performed reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis using the chromosomal FLAG-tagged OmpW strain under identical conditions as in β-galactosidase assay. RT-qPCR and western blot showed that mRNA level and protein level of OmpW under anaerobic condition was also induced to ~3-fold compared with that under aerobic condition, and deletion of fnr caused reduction of ompW mRNA and production of OmpW-FLAG , confirming the anaerobic activation of ompW in an FNR-dependent manner.To investigate whether the expression of 4ATCAA” in the promoter of its regulated genes  using the FNRD154A variant which exists as a functional dimer even under aerobic conditions . Unexpectedly, an additional super-shifted band was observed in EMSA at higher concentration of FNRD154A protein implying the presence of a second FNR binding site on the promoter of ompW . To confirm this, we first performed bioinformatics analysis and found that indeed two putative FNR binding sites are present in PompW: in addition to the site centered at -126.5 bp (TTGATTTAAATCAC) upstream of ompW transcription start site which was identified by TTAATCCAGATCAA) which bears 9 out of 10 FNR consensus site was also identified . In order to verify whether FNR indeed binds to these two sites and regulates the expression of ompW, we mutated either or both of the sites and examined the effect of these mutations on the binding of FNR to ompW promoter. To disrupt the -81.5 site, we changed the last four nucleotides of the site from “TCAA” to “CTGG”. To disrupt the -126.5 site, the third nucleotide “G” in the first half of the binding site “TTGAT” was changed to “A” and the last four nucleotides “TCAC” were changed to “CTGG”. As shown in Figure 2C, two retarded bands corresponding to the DNA-protein complexes were observed in the EMSA assay of FNRD154A with the native promoter. However, only one retarded band was observed in the EMSA of FNR with either mutated -81.5 site or mutated -126.5 site . Furthermore, promoter DNA containing mutations on both sites failed to form any complex with FNRD154A . These results confirmed that FNR can directly bind to both -81.5 and at -126.5 sites in the promoter region of ompW.FNR regulation is mediated through its specific binding to the consensus DNA sequence of “TTGATNed genes . In mosted genes . To invenditions . EB staiompW in vivo, we mutated each of the two sites in the PompW-lacZ fusion and compared their transcription activity with that of the native promoter. As shown in Figure 1, mutation of the -81.5 site caused ~3 fold reduction of the transcription of ompW to the similar level as that of native promoter under aerobic condition or that in Δfnr strain under anaerobic condition, suggesting that binding of FNR to the -81.5 site was responsible for the FNR dependent activation of ompW gene. Surprisingly, mutation of the -126.5 site led to ~2-fold increase of the transcription of PompW-lacZ, suggesting that FNR binding of this site caused repression of ompW transcription under anaerobic condition , consistent with the finding from the CHIP-seq experiment by ompW-lacZ and found that transcriptional activity of the resulting Promoter-lacZ fusion was even further increased to the level of ~3-fold higher compared to that of native promoter , suggesting that binding of FNR to the -126.5 site indeed led to repression of ompW expression. The fact that deletion of the -126.5 site (Δ-126.5) led to a greater extent of derepression of ompW transcription than that of m-126.5 suggested that nucleotides change in m-126.5 was not sufficient to completely abolish the binding of FNR to this site. Furthermore, when mutation of the -81.5 site was combined with deletion of the -126.5 site, transcriptional activity of the resulting PompW-lacZ was similar as that of the -81.5 site mutation alone , suggesting that FNR binding of the -126.5 site was dependent on its primary occupancy of the -81.5 site. Taken together, these results suggested that FNR can bind to two distinctive sites on the promoter region of ompW and regulate its expression. While binding of the -81.5 site activated the expression of ompW, binding of the -126.5 site led to repression of ompW expression, and this repression was dependent on the initial occupancy of FNR on the -81.5 site.To investigate the role of FNR binding to each of the sites on the anaerobic regulation of ompW was not annotated properly previously. To elucidate this, we performed 5′ RLM-RACE to examine the transcription start site of ompW. 5′ RLM-RACE result showed a single amplified PCR product (data not shown) and sequencing of this fragment revealed that the first nucleotide being transcribed was “G” located 29 bp upstream of the ATG start codon , the identical site as annotated previously in a genome-wide study  and focA-pflB (encoding pyruvate formate lyase)  . The cooompW-lacZ at different time points following the transition from aerobic to anaerobic growth. As shown in Figure 3, transcription of ompW increased following the transition to anaerobic condition and achieved the maximal expression level after 10 min of the transition. Interestingly, transcription of ompW was then gradually decreased after 30 min time point. This pattern is consistent with our speculation that FNR binds to the two distinctive sites of ompW promoter and gene repression following the FNR binding of the second site (centered at -126.5) was dependent on its binding of the first site (centered at -81.5) which activated ompW expression . Consistent with this notion, transcription of PompW-lacZ which contains mutation of the -81.5 site was not activated after the transition to anaerobic growth and remained at a very low level during the time course tested; whereas transcription from PompW-lacZ which contained deletion of the -126.5 site was increased rapidly to the level that is even higher than that of native promoter and maintained at this level without obvious reduction for 4 h following the transition . These results confirmed the maximal expression of ompW under micro-aerobic condition during the transition from aerobic to anaerobic condition and the distinctive effect of the binding of FNR to the two sites, -81.5 and -126.5, on the expression of ompW. We also constructed ΔompW strain and performed competition assay to examine whether the regulated expression of ompW contributes to the competitive advantage or fitness of E. coli during the transition from aerobic to anaerobic growth. Growth competition index was calculated based on the colony forming units (CFU) as following: mutantanaerobic/parentanaerobic: mutantaerobic/parentaerobic; mutantmicroaerobic/parentmicroaerobic: mutantaerobic/parentaerobic. We found ΔompW cells displayed a significantly lower competition index (0.25) relative to the WT under microaerobic condition than under anaerobic condition (0.45) (data not shown), suggesting a role of OmpW in the microaerobic environment which occurred during the transition from aerobic to anaerobic lifestyle of the bacterium.To test this, we examined the transcription of PompW transcription that is even higher than that of the WT promoter at all time points following the transition to the anaerobic condition suggested that an additional repression factor may exist and its effect is dependent on the FNR binding to the -126.5 site. Since ompW transcription by CHIP-seq experiment, we then tested the effect of ΔarcA on the transcription of PompW-lacZ under anaerobic conditions. As shown in Figure 4, indeed, ΔarcA caused elevated transcription of ompW. However, ΔarcAΔfnr double deletion resulted in a similar level of ompW transcription as that of Δfnr alone, suggesting that ArcA repression of ompW transcription was dependent on the presence of FNR, presumably its sequential binding to the -81.5 and -126.5 sites as illustrated above. EMSA also confirmed the direct binding of ArcA to the ompW promoter (data not shown).The fact that deletion of the -126.5 site led to a level of ompW was controlled at low level under aerobic condition. ompW is also repressed by H-NS under aerobic condition. As shown in Figure 4, Δhns caused elevation of the transcription of PompW-lacZ under aerobic condition, suggesting that aerobic expression of ompW was indeed repressed by H-NS. Interestingly, Δhns did not lead to increased transcription of PompW-lacZ under anaerobic condition and ΔfnrΔhns double deletion caused increased transcription of ompW comparing with that of Δfnr alone, suggesting that FNR antagonized the H-NS mediated repression under anaerobic condition. Two different antagnization mechanisms of H-NS mediated gene repression has been described: the antagonist binds to a region previously bound by H-NS; or a region adjacent to the H-NS sites  which represents a well conserved CRP site  in M9 galactose medium. As shown in Figure 5A, disruption of this site caused significant decrease of transcription from the PompW-lacZ fusion in M9 galactose medium to the level that is similar as that of native promoter in M9 glucose medium, suggesting that the site “5′-TGTGATCTATGTAGGA” was responsible for the elevated transcription in M9 galactose medium. To confirm this in vitro, we performed EMSA using purified His-CRP protein and PompW DNA fragments. As shown in Figure 5B, EMSA demonstrated that native PompW formed a retarded complex with CRP and mutation of the -42.5 site was sufficient to abolish the binding of CRP to the promoter DNA . It is noteworthy that binding of CRP to the native ompW promoter in EMSA was dependent on the presence of cAMP , indicating that the binding of CRP to ompW promoter was specific and functionally relevant. Together these results confirmed that CRP directly binds to ompW promoter and activates its expression in the absence of the preferred carbon source glucose.Bioinformatics search revealed a putative CRP binding site, “5′-CRP site . To examE. coli, nitrate is preferred to all other compounds owing to its significantly positive standard redox potential and consequently the highest energy yield , suggesting that nitrate repressed the transcription of ompW. It is known that nitrate regulation is mediated by the NarX-NarL and NarQ-NarP two-component systems , suggesting NarL was responsible for the transcriptional repression of ompW in the presence of nitrate. Furthermore, NarL mediated repression was found to be dependent on FNR, since fnr narL double deletion resulted in a similar transcription activity of PompW-lacZ as that of Δfnr alone and ΔnarL no longer had de-repression effect in the Δfnr strain background. On the other hand, transcription of PompW-lacZ was not affected by nitrate or narL deletion under aerobic growth condition (data not shown). Although the exceptionally high β-galactosidase activity of PompW-lacZ resulted from ΔnarL was ambiguous, nonetheless, these results suggested that expression of ompW was subject to nitrate repression in a NarXL dependent manner under anaerobic condition.Depending on the presence or absence of alternative electron acceptors, facultative bacteria either respire or ferment under anaerobic conditions. Since anaerobic respiration conserves energy with higher efficiency than fermentation, this mode of metabolism is preferred under anaerobic condition when alternative electron acceptors are present. Among the various electron acceptors which can be utilized by gy yield . Thus, pcceptors . Hence,  systems  in which systems , 1994. TompW, we analyzed the sequence of its promoter region and identified a potential NarL binding site centered at -18.5 bp (TACTCCAATGTAGGTA) upstream of the transcription start site . Mutation of the first three nucleotides of the site from “TAC” to “CAT” relieved the nitrate repression of PompW-lacZ . We next performed EMSA to detect the direct binding of NarL to the PompW DNA fragment. As shown in Figure 6B, a retarded band corresponding to the protein-DNA complex formed by phosphorylated NarL (His6-NarL-P) and native PompW was observed and His6-NarL-P displayed a significantly higher binding efficiency than that of unphohphorylated His6-NarL, suggesting the specific binding of the phosphorylated NarL to the ompW promoter. However, DNA fragment containing mutation of the -18.5 site failed to form a complex with His6-NarL-P , indicating that NarL bound to the site centered at -18.5 on PompW and repressed its expression, presumably by interfering with the binding of RNAP to the promoter given the close proximity of this site to the -10 and -35 elements of the promoter.To further confirm NarL-dependent repression of ompW is subject to tight controls by the availability of O2, carbon sources, and the anaerobically preferable electron acceptor nitrate suggested that OmpW may be involved in the carbon and energy metabolism of the bacterium during anaerobiosis. Interestingly, a previous proteomic study suggested that OmpW directly interacts with fumarate reductase Frd  using the SWISS-MODEL online homology modeling server. Since the detergent molecule LDAO was present and bond to OmpW in the original 3D structure of OmpW, we first conducted molecular docking of LDAO to OmpW using the in-house LGA algorithm developed by us. LDAO was predicted to bind OmpW with an estimated free energy of binding as -6.17 kcal/mol (Table 1) by the algorithm, consistent with the high affinity of LDAO to OmpW pore as indicated in the original 3D structure. Notably, LDAO was also predicted to be present in its native position as in the original 3D structure by our algorithm. Its predicted binding pocket  was also consistent with its native position  as present in the original 3D crystal structure except a slight shift of about 2-carbon toward the inner pore of OmpW, which probably was caused by the presence of solvent molecules in the crystal structure and the absence of solvent in the docking prediction. After confirming the feasibility of the docking and the algorithm, binding of fumarate to OmpW was analyzed and it was shown to bind to a side pocket of OmpW that differs from that of LDAO  with an estimated free energy of -3.91 kcal/mol (Table 1). Binding of fumarate to this pocket was predicted to be stabilized by L28, Q171, and Y165 . To test whether this binding is specific, we examined the binding of another alternative electron acceptor of anaerobic respiration, trimethylamine N-oxide (TMAO), with OmpW and found that TMAO was not predicted to bind to OmpW, since only -1.9 kcal/mol of free energy of binding is predicted by the algorithm. These results suggested that OmpW might be able to bind fumarate and serve as a specific receptor of fumarate.In bacteria, there are often strong correlations between how transcription of the gene is regulated and the physiological roles of the encoded proteins . The factase Frd  which isE. coli cells  significantly inhibited the growth of E. coli under both aerobic and anaerobic conditions, and addition of fumarate with various concentrations indeed can rescue the Colicin S4 mediated growth inhibition of E. coli. Interestingly, it requires higher concentration of fumarate to rescue the Colicin S4 mediated killing under anaerobic than under aerobic conditions, consistent with the higher expression level of OmpW under anaerobic conditions. Since fumarate belongs to a class of metabolites called C4-dicarboxylates, we also tested the effect of another C4-dicarboxylate, succinate, on the growth of E. coli in the presence of Colicin S4. As shown in Figure 8C, succinate can partially rescue the growth inhibition of E. coli by Colicin S4. However, another alternative electron acceptor TMAO which showed poor or no binding to OmpW in molecular docking is not able to rescue the growth inhibition caused by Colicin S4. These results together suggested that OmpW might be involved in the binding and/or utilization of C4-dicarboxylates in E. coli during the transition from aerobic to anaerobic lifestyle of E. coli.To test our speculation of the physiologically relevant function of OmpW to bind fumarate experimentally, we performed growth rescue assay. It has been known that OmpW serves as the receptor of Colicin S4 during its mediated killing of li cells . If OmpWtor FepA . In all tor FepA . Thus, tOmpW remain obscure. Expression of Salmonella ompW has been reported to be up-regulated by MarA and SoxS in response to menadione and down-regulated by ArcA in response to hypochlorous acid and hydrogen peroxide  which is responsible for its activation. We also revealed the regulation of ompW by CRP and NarL which responds to the availability of carbon sources and electron acceptors in the growth environment respectively, and the repression of ompW expression by the nucleoid-associate protein H-NS under aerobic conditions. On the basis of these regulatory mechanisms, molecular docking and the growth rescue assay of Colicin S4 mediated killing of E. coli suggested a role of OmpW in the binding of fumarate and other C4-dicarboxylates in responses to the availability of O2, carbon sources, and electron acceptors.In recent decades, genome-wide analyses such as microarray, CHIP-chip, and CHIP-seq have allowed identification of global gene expression changes in response to specific physiological stresses and/or the activation of specific transcription regulators. However, a full understanding of bacterial physiology relies on the elucidation of the functions of each of the genes which expression is altered in response to the stimuli. OmpW has been identified as a core regulon of the anaerobic global transcription regulator FNR  and its peroxide . HoweverompW was first activated by FNR through its binding to the -81.5 site and subsequently repressed through binding of the second FNR molecule to the -126.5 site, and further repressed by ArcA which functions primarily to repress TCA cycle enzymes. Unlike fum and frd, which are located in different operons and consequently are controlled independently by different regulators, OmpW as a membrane protein that is capable of binding fumarate and potentially involved in its metabolism is located in a separate location and its expression is driven by a single promoter. This perhaps explains the fact that expression of ompW is subject to both activation and repression by a series of the global regulators involved in anaerobic carbon and energy metabolism. Among them, since FNR directly senses the absence of O2 molecule and regulates global gene expression involved in the anaerobic lifestyle of E. coli, it acts as the primary regulator of ompW. Other transcription factors, such as ArcA , CRP , and NarL (senses the availability of respiratory electron acceptor nitrate) are the secondary regulators that modulate ompW expression in response to the availability and status of energy, carbon, and electron acceptors in the anaerobiosis of the bacterium. The relationship and roles of these regulators on ompW expression are supported by our genetic studies. A model to explain this regulation is summarized in Figure 9.Fumarate is a key metabolic intermediate in the TCA cycle and can serve as an alternative electron acceptor during anaerobic respiration. As an intermediate in the TCA cycle, fumarate is oxidized to malate under the catalysis of fumarase Fum. When serving as an alternative electron acceptor of anaerobic respiration, fumarate is reduced by the fumarate reductase Frd to yield succinate. Notably, fumarate is the only intermediate in the central metabolic pathway that can also serve as an electron acceptor in anaerobic respiration. During the transition from aerobic to the anaerobic metabolism, TCA cycle enzymes which function primarily in carbon oxidation are generally repressed, whereas the enzymes that are involved in anaerobic respiration of alternative electron acceptors in the absence of the preferred electron acceptor nitrate are activated. That is, the flux of TCA cycle intermediates is expected to be reduced while the flux of the alternative electron acceptors needs to be increased during the transition to the anaerobic lifestyle. The requirement of this metabolic adaptation probably explains why the transcription of cydAB operon encoding cytochrome d oxidase which also displays maximal expression under microaerobic condition during the transition of bacteria from aerobic to anaerobic growth. Interestingly, FNR binding sites on cyd promoter, as well as other 15 similar promoters as identified by E. coli.Notably, this type of coordinated regulation by the global regulators FNR, ArcA and H-NS is also observed in the case of 4-dicarboxylates such as L-malate and succinate, are common products of plant and bacterial metabolism. They serve as the major carbon sources of rhizobia which form symbiosis with plants  and CRP sites in several clinically significant species, such as S. typhimurium, Y. pestis, K. pneumoniae, and E. cloacae . All these strongly support a conserved role of OmpW in the cellular fumarate metabolism in Gram-negative bacteria.The OM porin proteins play an important role in the permeability of Gram-negative bacteria which dictate the entry of both nutrients and toxic compounds, such as antibiotics. This is because the OM of Gram-negative bacteria is generally non-permeable to both hydrophilic and hydrophobic molecules owing to the presence of lipopolysaccharide within the outer leaflet of the OM. To facilitate the entry of nutrients into bacteria, bacteria express OM porins (OMP) which form hydrophilic pores and channels in the hydrophobic lipid bilayer of the Gram-negative bacteria OM. The type and expression levels of these porins play a critical role in the selective acquisition of nutrients or toxic compounds in the environment . Regulath plants . FumaratAn open question is how OmpW participates in the cellular utilization of fumarate. We have attempted to answer this question by extending the grid box in molecular docking to cover the entire trans-membrane part of OmpW (data not shown). However, the docking results showed no further evidence for the transport or flux process. The X-ray crystal structure of OmpW does not show a continuous channel spanning the entire membrane either. However, this could be due to the lack of amino acid residue dynamics in the static X-ray crystallographic structure and the fact that OmpW proein was treated rigid in the docking study. Future work will focus on identifying the proteins interact with OmpW and characterization of OmpW homologues in those species which utilize fumarate as their main carbon source to understand how OmpW facilitate the physiological process of fumarate utilization during the transition of bacteria to their anaerobic lifestyle.AY, MX, JS, and GC designed experiments; MX, YL, and JS performed experiments; MX, YL, and AY prepared 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.


Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/β-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/β-catenin signaling and allosteric regulation of a transcription factor by DNA.The Wnt/β-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in  Drosophila as a model system. This pathway is known to activate gene expression in cells receiving the Wnt signal, working through a transcription factor known as TCF. But sometimes Wnt signaling also instructs TCF to repress target gene expression. What determines whether TCF will positively or negatively regulate Wnt targets? We demonstrate that activated and repressed targets have distinct DNA sequences that dock TCF on their regulatory DNA. The type of site determines the output, i.e., activation or repression. We find that TCF adopts different conformations when bound to either DNA sequence, which most likely influences its regulatory activity. In addition, we demonstrate that Wnt-dependent repression occurs robustly in the fly larval lymph gland, the tissue responsible for generating macrophage-like cells known as hemocytes.During development and in adult tissues, cells communicate with each other through biochemical cascades known as signaling pathways. In this report, we study the Wnt signaling pathway, using the fruit fly  It is a common theme in gene regulation that the same transcription factor (TF) can directly activate or repress target gene expression, increasing the transcriptional complexity these TFs can achieve Drosophila NF-κB family member Dorsal, mutation of TF sites flanking Dorsal binding sites converts CRM reporters that are repressed by Dorsal into ones that are activated The sequence specificity that determines the activation/repression choice of TFs can occur in the TF binding sites themselves, or the surrounding sequences. Several TFs that appear to be intrinsic transcriptional activators can also repress transcription when bound to CRMs in conjunction with other TFs Dual regulation of transcription has also been seen in Wnt/β-cat  signaling, an important cell-cell communication pathway that plays various roles throughout animal development, stem cell biology and disease Drosophila TCF/Pangolin (TCF/Pan) mediate both sides of the transcriptional switch The best-characterized TFs that recruit β-cat to W-CRMs are members of the T-cell factor (TCF) family All TCFs contain a sequence-specific DNA binding domain called the HMG domain, whose high affinity consensus is SSTTTGWW,  While TCF/β-cat complexes are commonly associated with transcriptional activation, there are a few cases where they appear to directly repress target gene expression Ugt36Bc locus through HMG sites with a consensus that is distinct (WGAWAW) from classic ones Ugt36Bc in the absence of signaling Ugt36Bc compared to the switch seen in activated targets. Instead of TCF/Pan default repression and Wnt-dependent activation, the reverse switch consists of TCF/Pan basal activation and Wnt-dependent repression.In contrast to the aforementioned examples, we previously showed that TCF/Pan mediated Wnt-dependent repression of a W-CRM from the Tiggrin (Tig) gene, which contains functional WGAWAW sites bound by TCF/Pan. Regulation of the Ugt36Bc and Tig W-CRMs by TCF/Pan requires the C-clamp, which binds to Helper-like (r-Helper) sites adjacent to the WGAWAW sites. Swapping these sites in the Tig W-CRM to classic HMG and Helper sites converts the W-CRM into one that is activated by Wnt signaling. Conversely, an activated W-CRM from the naked cuticle (nkd) locus was converted to a repressed W-CRM by replacing its classic HMG-Helper pairs with pairs from the Tig W-CRM. Partial protease digestion indicates that TCF/Pan adopts a different conformation when bound to classic or repressive sites, supporting allosteric regulation of TCF/Pan by its binding sites. In addition, we have extended this work from cell culture to the fly, showing that WGAWAW and r-Helper sites mediate basal activation and Wnt-induced repression in the larval lymph gland (LG). Wnt signaling is known to play an important role in regulating hematopoiesis in the LG In this report, we have explored the mechanism of this reverse switch/direct repression mechanism by TCF/Pan and Wnt signaling in more detail. We identified another repressed W-CRM from the Ugt36Bc was originally identified as a candidate for repression by Wnt signaling from a microarray screen performed in Kc167 (Kc) cells Drosophila cell line likely of hemocytic origin TigTig expression was repressed by DisArmed, a mutated version of Armadillo  which is defective in gene activation but is still competent for repression Tig being directly repressed by Wnt signaling, the cis-regulatory information responsible for Wnt regulation of Tig expression had not been identified.Tig locus is compact, with a small (∼1 kb) intergenic region and six introns, only the first of which is larger than 500 bp (Fic domain-containing protein (Fic), a gene involved in fly vision Fic was expressed in Kc cells, but was not regulated by Wnt signaling , sequences were cloned upstream of the hsp70 core promoter, which is unregulated by Wnt signaling Tig promoter. These reporters (Tig2–Tig5) all had basal expression higher than the hsp70 promoter control  was also examined . As prevTig contained several sequences that were similar to sites in the Ugt36Bc W-CRM that were footprinted by the HMG domain of TCF/Pan Tig intron containing these putative sites  was constructed  and WGAWAW-r-Helper pair (WH) were distinct, with several proteolytic fragments observed with TH that were not detectable with WH . AnalyziThe HMG domain of LEF1 (a vertebrate TCF) is known to induce a sharp bend in DNA when bound to a classic HMG site Tig promoter (Tig1) or a heterologous one from hsp70 (minR). Transgenic lines were established and analyzed for LacZ expression in embryos and larva. Both reporters were active in embryonic hemocytes, as indicated by co-localization with MDP-1, a hemocyte marker  of the LG, an irregularly shaped region containing mature hemocytes enriched in the periphery of the LG . This paTig and minR reporters in the CZ without detectably altering cell fate. In addition, the finding that DisArmed can mediate this regulation suggests that the transcriptional activation activity of Arm is not required for this regulation.To test whether the Tig1 and minR reporters were repressed by Wnt signaling in the LG, the Gal4 misexpression system rd instar larvae (∼88–96 AEL). This is prior to release of LG hemocytes, so all circulating hemocytes are of embryonic lineage at this developmental stage To test whether the Tig1 and minR reporters were repressed by Wnt signaling in embryonic hemocytes, we expressed Arm* or DisArmed under the control of two embryonic hemocyte drivers, Srp-Gal4 or Croquemort-Gal4 . No detectable repression was observed (data not shown). To examine whether the negative results were due to perdurance of LacZ, we assayed circulating hemocytes from mid 3DN and Fz2DN) Our working model is that TCF/Pan activates Tig1 and minR expression in the CZ of the LG, while Wnt signaling represses these reporters in the MZ. To test this, we examined reporter expression when dominant-negative versions of Frizzled and Frizzled2  with low basal expression and a high degree of Wnt activation completely inverted the regulation: the altered W-CRM had high basal expression and was repressed by Wnt signaling  promoter and a small amount of Cu2+ was added hsp70 promoter was used, the altered Ugt36Bc W-CRM was not active, similar to the HMG site only swap in the Tig1 W-CRM  or synthetic (minR), are active in embryonic and larval hematopoietic systems  and 10, Tig and minR W-CRMs in the LG is likely direct, based on site-directed mutagenesis of the WGAWAW and r-Helper sites , which provide basal activity in the imaginal discs 6 cells/ml in growth media supplemented with 10 µg/ml dsRNA for 4 days, diluted to 1×106 cells/ml without additional dsRNA, and grown for 3 more days for luciferase assay using Tropix Chemiluminescent Kits (Applied Biosystems) or 2 more days for mRNA preparation using Trizol Reagent (Invitrogen). dsRNAs targeting the 3′UTR of TCF/PanAxin or a control gene (β-lactamase) were used tubulin56D levels.Kc cells were cultured and transient transfections were carried out as previously described 5 cells. For standard reporter assays, 50 ng luciferase reporter and 6.25 ng LacZ per well were transfected with Axin RNAi or control RNAi. For TCF/Pan rescue assays, same amount of reporter and LacZ plus 50 ng TCF/Pan-expressing plasmid and 250 ng Arm* per well were transfected with TCF/Pan RNAi. pAc5.1-V5/His-A vector was used to equalize DNA content between samples and as a negative control for expression vectors. Luciferase activity was normalized to β-galactosidase activity from pArm-LacZ to control for differences in transfection efficiency among samples. In the figures, each bar represents the mean of biological triplicates and the data shown are representative of three independent experiments. All RLA units are arbitrary units unless otherwise specified.Each treatment in reporter assays was done in triplicate wells, each containing 2.5×10nkd-IntE and all site mutants and swaps based on these W-CRMs were cloned into pGL3-basic containing an Hsp70Bb minimal promoter. Vector with a Hsp70Bb promoter but containing no W-CRM was used to control for basal promoter activity. A MluI site was introduced into Tig1 upstream of the TCF sites for the ease of cloning of the swap constructs. Sequence changes were done using site-directed mutagenesis  or recursive PCR All luciferase reporter vectors are derivatives of pGL2 or pGL3 (Promega). pHsp-178, Tig1, Tig5 and all site mutants and swaps based on these W-CRMs were cloned into pGL2-basic. Tig2–4, minR, For expression plasmids, pAc-TCF (WT/C-mut), pAc-Arm*, pGEX-GST, pGEX-GST-HMG and pGEX-GST-HMG-C-clamp (WT/C-mut) have been described elsewhere E. coli. 4 nM biotinylated probe  and 7–20 µM protein were used in each reaction. The conditions in the DNA bending assays were similar to the EMSA assays except for the following modifications: 4 nM biotinylated probe was incubated with 20 µM (for WH and WS), 200 nM (for TH) or 500 nM (for TS) protein before separating on 5% native PAGE gel.EMSAs were performed as previously described EcoRI, whose sites were introduced by the primers, and biotinylated through Klenow reaction using Biotin-16-dUTP (Roche). Probes containing a SS site (with both HMG site and Helper site mutated from TH) were generated to confirm that TCF/Pan has no detectable affinity to the surrounding sequences on the probes (The probes for the DNA bending assays were generated according to a previously described strategy e probes . The seqTig intronic W-CRM and the full length Ugt36Bc W-CRM (178 bp) were footprinted  or not (for reverse EMSA) at a final concentration of 5–50 ng/µl for chymotrypsin (Roche) or 50–150 ng/µl for endoproteinase Glu-C (New England Biolabs). The mixture was incubated at 25°C for 2.5–3 hours. Then the digested product was loaded onto 16% tricine SDS-PAGE gel Tig (Tig1) and minR fly reporters were generated by cloning the corresponding sequences into pPelican and pHPelican vectors, respectively DN and UAS-Fz2DNDrosophila RNAi Screen Center.All the Gal4 and UAS lines used in this study have been previously described: Srp-Gal4 ts. Crosses were set up at 18°C, and the larvae were transferred to 25°C for 2 days  Kc cells were treated with control (Wnt Off) or Axin (Wnt On) dsRNA for six days and processed for transcript analysis as described in Tig expression is repressed by Wnt signaling (A), which Fic expression is unaffected (B).  Mutations in r-Helper sites (H) or WGAWAW sites (W) greatly decrease the basal activity and repression of the Tig and Ugt36Bc W-CRM reporters in Kc cells by Arm* expression. *p<0.05; n.s., not significant (Student's T-test).Expression of (TIF)Click here for additional data file.Figure S2Tig intron. (A) The 200 bp stretch of the Tig probe containing all footprinted regions is shown, with the HMG domain and C-clamp protected regions indicated. Two WGAWAW sites (red) are bound by the HMG domain, as well as several other sites (green). r-Helper sites bound by the C-clamp are shown in blue. The sequences that were mutated for the reporter assays shown in B) Tig1 reporters containing mutations in the TG-rich regions footprinted by the HMG domain were similar to the wild-type control.Sequences protected by GST-HMG and/or GST-HMG-C-clamp in the (TIF)Click here for additional data file.Figure S3Tig W-CRM. Regions where the blue signals are higher than the green signals were protected by GST-HMG-C-clamp and not by GST-HMG. Note that the arbitrary colors are switched compared to those shown in Footprinting chromatographs showing the C-clamp-specific protection of the r-Helper sites in the (TIF)Click here for additional data file.Figure S4nkd-IntE and Tig1 “swapped site” reporters. For all constructs, classic HMG binding and WGAWAW sites shown in red, while Helper and r-Helper sites shown in blue. (A) minR W-CRM and variations with the WGAWAW or r-Helper sites mutated . (B) The entire 255 bp nkd-IntE W-CRM, with sites to be swapped underlined and the sequence of the W-CRM with classic sites converted into WGAWAW and r-Helper sites. (C) Portion of the Tig first intron containing the two functional WGAWAW and r-Helper sites, plus the sequences where these motifs are swapped into sites typical of activated W-CRMs. The altered nucleotides in the swapped reporters are shown in lowercase.Sequence information of minR and (TIF)Click here for additional data file.Figure S5A) Similar to Tig1, the minR reporter is repressed by Arm* expression. (B) When either r-Helper or WGAWAW sites were mutated, the basal activity of minR reporter and its response to Wnt signaling (Axin RNAi) were both strongly decreased. *p<0.05; ***p<0.001; n.s.: not significant (Student's T-test).The activity of minR is dependent on r-Helper and WGAWAW sites. ((TIF)Click here for additional data file.Figure S6A) Cartoon showing a series of seven probes, each with a bipartite TCF binding site (red/blue boxes) located along the 139 bp oligonucleotide. These TCF sites could consist of a classic HMG or WGAWAW site (TS or WS) or HMG-Helper or WGAWAW-r-Helper pair (TH or WH). If DNA bending occurs upon protein binding, the complex will run slower in an EMSA when the binding site is in the middle of the probe B) GST-HMG protein bends TS slightly more than WS. (C) The presence of a Helper site does not increase the bending observed when GST-HMG-C-clamp binds to a HMG site. (D) GST-HMG-C-clamp bends TH slightly more than WH. (E) Probes with the identical spacer sequences as the TS, WS, TH and WH probes but lacking HMG and Helper sites (SS) were not bound by GST-HMG-C-clamp. Two SS probes, corresponding to the fourth and seventh probes in the series of seven probes (A), were tested. Each experiment was performed at least three times with similar results.DNA bending by the HMG domain of TCF/Pan. ((TIF)Click here for additional data file.Figure S7A–H) Larval LGs from late 3rd instar larvae containing p[Lz-Gal4] and p[UAS-mCD8::GFP] and the minR (A–D) or Tig1 (E–H) lacZ reporters, with LacZ immunodetection (red). Both fluorescent signals are cytosolic. Panels B–D and F–H are higher magnification of the boxed regions in A and E, respectively. The expression patterns of the reporters are largely exclusive with Lz≫GFP, a marker of crystal cells which often express Wg The Tig and minR reporters are not active in crystal cells. ((TIF)Click here for additional data file.Figure S8A–F) and Hml≫GFP (green) (G–I), two established CZ markers Cut immunostaining marks the CZ of the larval LG. Cut (red) colocalizes with Cg≫GFP ((TIF)Click here for additional data file.Figure S9A–F) Micrographs of older 3rd instar larval LGs from strains containing the minR reporter and P[Srp-Gal4], without (A–C) or with P[UAS-Arm*] (D–F). Activation of Wnt signaling by Arm* expression greatly reduces the size of CZ, indicated by Cut (red), and expression of the lacZ reporter (green) is greatly reduced.Activation of Wnt signaling in the larval LG is able to affect cell fate and reduce the size of the CZ. ((TIF)Click here for additional data file.Figure S10DN and Fz2DN in the MZ derepresses minR signal but also reduces the size of MZ. (A–F) Micrographs of younger late 3rd instar larval LGs (∼94–98 hr AEL) from strains containing the minR reporter, P[UAS-mCD8::GFP] and P[Dome-Gal4] without (A–C) or with P (D–F). Dome≫GFP indicates MZ cells, while lacZ positive cells are in the CZ. Inhibition of Wnt signaling by FzDN and Fz2DN expression increases lacZ reporter signal (red) in the CZ, but also reduces GFP expression, indicating an decrease in the size of the MZ.Inhibition of Wnt signaling by Fz(TIF)Click here for additional data file.Figure S11A–F) Micrographs of older late 3rd instar larval LGs from strains containing the minR reporter, P[UAS-mCD8::GFP] and P[Hml-Gal4], without (A–C) or with P[UAS-TCF/Pan-RNAi] (D–F). Depletion of TCF reduces minR reporter expression (red), but also reduces the GFP signal indicating a reduction in the size of the CZ.TCF/Pan knockdown in the CZ reduces minR expression but also reduces the size of the CZ. ((TIF)Click here for additional data file.Figure S12A) A cartoon showing the structure of 3×GRH-W-CRM reporters, containing three Grainyhead (GRH) binding sites which provides basal activity in the tissues being tested and a W-CRM followed by a EGFP reporter gene. (B–P) Micrographs of wing (B–F), leg (G–K) and eye-antenna (L–P) discs from 3rd instar larvae carrying indicated 3×GRH-W-CRMs. 3×GRH-4TH contains four classic HMG-Helper site pairs, and displays high expression in regions where Wg is known to be expressed. 3×GRH-SS contains randon sequences and has the low level, ubiquitous pattern previously described Q) Sequence information for the 3×GRH-W-CRM reporters.WGAWAW, r-Helper site pairs do not affect transcription in several other tissues outside the hematopoietic system. ((TIF)Click here for additional data file.Figure S13A–E) Comparison between minR and Wnt-activated reporters stained with X-gal. The minR reporter (B) shows strong staining in the CZ, while all the Wnt-activated W-CRMs tested (C–E), as well as the negative control w1118 (A), have no detectable staining. The minR reporter was stained for the same amoun of time as the other reporter lines, resulting in over-staining. (F) Micrograph of an older 3rd instar larval LG stained with X-gal, taken with DIC optics, highlighting the larger, less densely packed cells of the CZ. (G) Brightfield image of the same LG where the LacZ staining is more pronounced. (H) Brightfield image of the same LG where the DIC image was used to draw a broken white line separating the CZ and MZ.Several Wnt-activated W-CRMs have no detectable activity in the LG. ((TIF)Click here for additional data file.Table S1Preference of WGAWAW binding by the HMG domains of vertebrate TCFs, taken from Badis et al. (2009). HMG binding sites from five repressed and eight activated W-CRMs were analyzed (XLSX)Click here for additional data file.Table S2Oligonucleotide sequences used in this paper. Note that the sequences for DNA bending assay were presented in a longer probe (see (XLSX)Click here for additional data file.


The spatial distribution of chromatin domains in interphase nuclei changes dramatically during development in multicellular organisms. A crucial question is whether nuclear organization is a cause or a result of differentiation. Genetic perturbation of lamina–heterochromatin interactions is helping to reveal the cross-talk between chromatin states and nuclear organization. Since the earliest days of microscopy, there have been studies indicating that chromatin and chromosomes are not randomly distributed in interphase nuclei . We now One of the most pronounced and conserved features of genome organization — particularly in the nuclei of differentiated cells — is the close proximity of heterochromatin to the lamina or nuclear envelope (NE) , 5. NoneLMNA, which encodes both lamin A and lamin C  had almost no effect on gene expression [me3 levels fluctuate a great deal at specific promoters during ESC differentiation — hundreds of promoters gain this mark, whereas as many others lose it, during the transition from ESC to NPCs, and from NPCs to differentiated neurons [me3, they generally become activated in later differentiated states, suggesting that polycomb keeps different sets of genes poised for appropriate expression at later stages of differentiation [Naturally, histone modifications correlate with ESC differentiation , 23. In pression . Nonethe neurons , 19. Whentiation , 25–28.me2 during ESC-to-neuron differentiation, although localized changes were found at specific genes [me2 coverage in large chromatin domains increased from a range of 17.5–24 % in pluripotent human stem cells to a range of 39.3–44.8 % in differentiated cell lines [me2/me3 is associated with repetitive DNA . It is a matter of debate whether the overall amount of the heterochromatic histone H3K9 di- and tri-methylation increases during differentiation of ESCs , 30. Lieic genes . By contll lines . The bion genome ), the qun genome . Finallyme2/me3 ligand, heterochromatin protein 1α (HP1α) [me3 and HP1β act as barriers to the reprogramming of differentiated cells [Drosophila embryos [The imaging of condensed heterochromatin and chromocenters by microscopy confirms that there are major changes in heterochromatin during differentiation: dense-staining foci of heterochromatin are less obvious in undifferentiated than in differentiated ESCs and are less frequently perinuclear –36. Simiα (HP1α) , 42. Twoα (HP1α) . Surprisα (HP1α) . At the α (HP1α) , 42. Thied cells –47. None embryos .In summary, the modulation of chromatin states during differentiation provides a basis for changes in nuclear morphology, as well as for changes in gene expression. In general, pluripotent genomes are less rigidly organized than differentiated states, as demonstrated both by biochemical and fluorescence recovery after photobleaching (FRAP) methods , 49, 50 Drosophila tissue culture cells were used to define chromatin classes by principle component analysis [+ T cells [In order to classify the chromatin states that exist in differentiated cells, several laboratories have used principal component analysis and/or hidden Markov models (HMMs) to analyze histone modifications and non-histone protein binding patterns. Genome-wide mapping data from analysis , 54, andanalysis . TranscrThe nuclear lamina is a meshwork of proteins that lies adjacent and anchored to the nuclear membrane. Its main structural components are the type V intermediate filament proteins lamin type A and lamin type B  has been used to map sequences genome-wide that interact with the nuclear envelope in multiple cell types. Initially, the group of Bas van Steensel identified approximately 500 genes in these lamin-associated domains (LADs) in the ell line . They weell line . The aveell line , 70. Usienriched . Interesenriched , 72 (revenriched ).Several studies have monitored the progressive association of repressed pluripotency genes and silent tissue-specific genes with the nuclear lamina during differentiation , 70, 71.pha-4, a marker of endoderm differentiation, occurred before its activation [C. elegans embryos and adults showed significant concordance, tissue differentiation in adults was associated with an increased separation between NE-bound and NE-excluded regions [In the other direction, the correlation was more robust: many of the genes that were released from the lamina upon differentiation were shown to be ‘unlocked’ or ‘open’ for lineage-specific transcription, even though active transcription occurred only much later. This is consistent with a recent study that showed that it is sufficient to unfold chromatin to provoke the shift of a promoter away from the nuclear lamina in mouse ESCs . The auttivation . Finally regions . CollectC. elegans [met-2 set-25 double-mutants lack all H3K9 methylation in embryos and during somatic cell differentiation, which not only de-represses a heterochromatic reporter but releases both it and endogenous H3K9me-enriched chromatin from the nuclear periphery, as mapped by lamin-DamID [To go beyond a simple correlation of H3K9 methylation and heterochromatin anchoring, genetic approaches are needed. The most extensive screen for factors involved in sequestering chromatin at the NE was a genome-wide RNA interference (RNAi) screen in  elegans . Using a elegans . MET-2 ain-DamID , 74 .In worms, the single  on H3K9 . Intrigume2/me3 [me2 HMT, G9a, reduced but did not ablate lamin interaction, particularly of constitutively bound LADs [. While this suggests a positive role for H3K9me2 in perinuclear anchoring of chromatin in mammalian cells, another study based on fluorescence in situ hybridization (FISH) showed that mutation of G9a did not affect the localization of most tested lamin-bound loci in mouse ESCs, even though H3K9me2 levels were strongly reduced [me1 or H3K9me2 has a conserved role in perinuclear heterochromatin anchoring, even if it is unlikely to be a sufficient signal in mammalian cells. Histone H3K9 methylation is not the only perinuclear targeting signal in worms either, since a second, H3K9me-independent, anchoring system has been shown to be induced in differentiated larval and adult tissues, to anchor heterochromatin . Thus, in both worms and mammals, anchoring pathways depend upon the differentiation state of a cell.When mammalian LADs were mapped in vivo, 80 % were enriched for H3K9me2/me3 , 70. Morund LADs . While t reduced . Harr an reduced . The soume in heterochromatin anchoring in mammals is that this modification accumulates on centromeric satellite repeats and serves an essential role in kinetochore function [me depletion leads to chromosomal missegregation in mitotically dividing mammalian cells. In contrast, holocentric worms (where the entire length of the chromosome acts as the centromere) have no mitotic defects in the absence of H3K9me . Moreover, HMT functions appear to be more redundant in the mammalian systems: not only can HMTs G9a and both Suv39H1 and Suv39H2 deposit H3K9me2/me3, but, in cells lacking both isozymes of Suv39H, centromeres lose H3K9me3 yet remain clustered owing to a compensatory function of H3K9me1, which accumulates at the centromere [me3 and G9a-mediated H3K9me2 [me2 and H3K9me3 [me3 was also implicated in reporter association with the lamina [Part of the difficulty in defining the role of H3K9function . Becausentromere , 81. In  H3K9me2 . Consist H3K9me3 . Intrigue lamina .me3-marked heterochromatin, presumably through its interaction with the H3K9me reader HP1 [C. elegans embryos, the double deletion of genes encoding HP1 homologs (hpl-1 and hpl-2), even in combination with loss of a third H3K9me reader, LIN-61, left heterochromatic reporters anchored [me-recognizing anchors exist.Assuming that histone H3K9 methylation, either alone or together with other modifications, targets sequences to the NE, it remains unresolved what factor(s) recognize the anchoring signal. It is unlikely that lamins bind specific lysine-methylated residues directly — rather, this is a job for specific ‘reader’ proteins that contain structurally defined chromo, PHD, MBT or tudor domains . In HeLaader HP1 . Interesader HP1 , yet no ader HP1 ; moreoveanchored . It is tme3-marked chromatin [me2 in vitro [me2 is distributed broadly across the genome, without significant enrichment in LADs [me3 is enriched in centromeric satellite DNA, which does not always bind the nuclear envelope [uv420h2 knockout mice, which have strongly compromised levels of H4K20me3, peripheral heterochromatin appears normal [me2 and HP1 in mammalian heterochromatin anchoring.In mammals, one of these anchors could be the lamin B receptor LBR, which co-immunoprecipitates with H3K9hromatin  and appehromatin . Unlike in vitro . Unfortu in LADs , and H4Kenvelope . Moreoves normal . Nonethes normal , it willC. elegans, targeted RNAi and mutagenesis screens aimed at identifying factors that compromise heterochromatin anchoring in either embryos or differentiated tissues have been performed. In embryos, a novel H3K9me reader appears to mediate anchoring in embryos , whereas in differentiated tissues methylation marks other than H3K9 contribute to heterochromatin anchoring. These differentiation-induced alternative pathways, along with a lack of centromeric heterochromatin, are the likely explanation for the near-normal development of H3K9-deficient worms [In nt worms .me-independent, pathways for chromatin anchoring. The polycomb-deposited mark H3K27me3 is a plausible candidate as it marks facultative heterochromatin, particularly at developmentally regulated promoters [me3 contributes to the peripheral relocation of a sequence located at the edge of a LAD [mes-3 and mes-6 leads to de-repression of a heterochromatic reporter in embryos and differentiated tissues, but no release from the NE [me3-positive foci in differentiating cells are not perinuclear [me3 with H3K4me3, a mark that is actively excluded from the NE.Preliminary data address the nature of these alternative, differentiation-driven and H3K9romoters , and is romoters . Recent of a LAD , whereasm the NE . Moreoveinuclear . In uncoFurther evidence for redundant, accumulative, but also alternative, pathways of heterochromatin tethering came from an elegant study that examined nuclei in differentiated tissues of wild-type mice and mice deficient for LBR and/or lamin A/C . In the IgH LAD in mice [C. elegans showed that, despite an overlap of 89 % between DamID profiles, these two NE proteins also were bound by different sets of tissue-specific genes [cis-acting elements, proximity to developmentally regulated promoters, and possibly on cell-type variations in the composition of the NE [Finally, sequence-specific binding sites might also play a role in locus-specific lamin attachment, as evidenced in a study of the  in mice . In this in mice , 96. Howic genes . The domf the NE , 97.Schizosaccharomyces pombe Sad1 and C. elegans Unc-84 proteins), a class of NETs that are anchored both by lamins (in vertebrates) or by interaction with chromatin (in yeast). In the intermembrane space, their C-terminal SUN domain interacts with nesprins, which extend through the outer nuclear membrane to the cytoskeleton . Thiewed in –102. Intiewed in .me3 by PRC2 [me3 in fission yeast by Clr4 [me3 at centromeric repeats in mammals by Suv39 [me2 by G9a [C. elegans, SET-25 becomes enriched in foci that colocalize with the mark it deposits, H3K9me3, in a manner that is independent of the HP1 homologs, even though the worm Hpl-1 and Set-25 proteins colocalize in heterochromatic foci.During cell differentiation, uncommitted cells with identical genetic information acquire epigenetic changes that need to be passed on through mitotic cell division in order to maintain lineage specification. Current models for the epigenetic inheritance of histone methylation propose that HMTs are recruited to chromatin by the marks they deposit, thus ensuring both the modification of neighboring nucleosomes and the propagation of the mark onto newly deposited nucleosomes at the replication fork. Good support for this mechanism exists for the propagation of H3K27 by PRC2 , for the by Clr4 , for theby Suv39 , 107 and2 by G9a , 109. SiThe fact that H3K9 mono- and di-methylation is a trigger for perinuclear chromatin anchoring suggests that the pathway towards heterochromatin can itself drive its spatial segregation from active chromatin domains. Furthermore, the finding that the HMT that deposits the terminal, repressive H3K9 methylation mark remains bound to perinuclear heterochromatin explains how the nuclear periphery is favorable for both the establishment and propagation of repression. This circularity could act as a self-reinforcing mechanism that ensures the robust separation of active and inactive chromatin domains.C. elegans, where H3K9me1/me2-containing chromatin binds to the NE before establishment of the repressed state. Thus, peripheral sequestration of chromatin both nucleates and propagates repression. Given that peripheral attachment also favors late replication [We note that a similar mechanism has been demonstrated for SIR-mediated silencing in budding yeast, where peripheral anchoring is mediated by the chromatin-bound Sir4 protein (reviewed in ). Sir4 ilication , 51, theIt is clear that the NE cannot be considered exclusively as a repressive compartment, nor is the nuclear interior uniformly active. Nonetheless, elegant gain-of-function targeting assays show that subnuclear compartments can influence gene expression. In particular, the tethering of genes to repressive zones of the NE, notably to NE-bound telomere clusters in yeast or to emerin/lamin zones in mammals, can facilitate gene repression (reviewed in ). In mouC. elegans [Nonetheless, by now a large number of examples show the relocation of a transcriptionally active, developmentally regulated gene from the NE to the interior lumen of the nucleus in a tissue-specific or cell type-specific manner (reviewed in ). In sev elegans , develop elegans .HSP70 in human. In C. elegans, this locus (hsp16.2) is found juxtaposed to nuclear pores, independent of its expression status, and the gene becomes even more tightly associated with nuclear pores upon induction of heat shock [Drosophila [An exception to this trend of shifting inwards during activation is the major heat-shock gene, a conserved gene homologous to at shock . In flieat shock . This miosophila , 119. WhC. elegans following depletion of the lamin homolog LMN-1, in Drosophila testis, and finally in mammalian cells lacking lamins or associated components [In general, the integrity of the inner nuclear envelope is important for stable gene expression. This was shown for a heterochromatic array in mponents , 120–122mponents   or mechanotransduction signaling is compromised, thereby perturbing cell differentiation . ClOne frequent pathology arising from lamin A/C mutations is the autosomal-dominant Emery–Dreifuss muscular dystrophy (AD-EDMD) , which cC. elegans, by contrast, has suggested that hyper-sequestration of genes at the nuclear lamina leads to a dominant, striated-muscle defect [C. elegans LMN-1 (Y59C) led to an inability to release muscle-specific genes from the NE in muscle tissue at a stage when these promoters should normally be induced. Although the muscles could still develop, the tissue was misorganized, and there was a noticeable loss of muscle function in adult worms [A genetic study of a specific AD-EDMD mutation in e defect . The ectlt worms . If muscLMNA allele that has been studied in detail in both mouse and C. elegans encodes a protein lacking lysine 32 (ΔK32). Mice homozygous for the ΔK32 LMNA mutation show a delay in maturation of striated muscle and have metabolic defects that include reduced adipose tissue and hypoglycemia, which leads in turn to premature death. The transcription factor SREBP-1, which was previously shown to interact directly with lamin A protein [C. elegans, the equivalent mutation (ΔK46) caused alterations in the in vitro lateral assembly of dimeric head-to-tail lamin polymers, which is a prerequisite step for the formation of filaments. This led to an abnormal organization of lamin protofilaments and a decreased affinity for emerin in vitro [C. elegans, the ΔK46 mutation caused lamin aggregation with LEM-2 in vivo, and emerin displacement to the cytoplasm, and provoked motility defects and abnormalities of muscle structure [A further laminopathic  protein , showed  protein . In C. ein vitro . Remarkatructure .Drosophila has provided another genetic model for lamin deficiencies. Drosophila larval cells lacking the A-type lamin C have NE defects, including changes in nuclear morphology and the clustering of nuclear pore complexes, much like those observed in human laminopathies [opathies . Ectopicopathies . The smaopathies . In bothA wide range of mutations in lamin A/C have been correlated with changes in higher-order chromatin organization, and particularly severe effects accompany the C-terminal deletion that provokes systemic progeria, or Hutchinson Gilford progeria syndrome. It is unclear whether its chromatin effects cause or result from the premature-aging phenotypes as the progeria mutation also affects cell metabolism and WNT and NOTCH signaling , 135. InLess-dramatic phenotypes arise from lamin point mutations that appear to cause a loss or gain of interaction with specific transcription factors. One well-studied case is that of SREBP1, a transcription factor that binds to the sterol regulatory element on DNA and regulates the genes required for de novo lipogenesis. SREBP1 is a lamin A binding partner in mouse adipocytes, and lipodystrophy-linked mutations map to the SREBP1-binding domain in lamin A/C. Inappropriate sequestration or improper release of SREBP1 might thus be responsible for the fat loss seen in patients carrying these mutations. In a further exciting study, lamin A/C and emerin were shown to regulate the nuclear localization of the mechanosensitive transcription factor myocardin-like protein 1 , possibly by modulating the balance between G-actin and F-actin . Indeed,Other transcription factors that associate with lamin or lamina-associated proteins include germ-cell-less (GCL), which binds to the INM protein LAP2β in mouse, and the DP3 subunit of the E2F-DP3 heterodimer, which influences the regulation of E2F-dependent genes . The traClearly, there is much left to discover about how nuclear lamins and nuclear positioning affect tissue-specific gene expression, yet in all cases it is necessary to demonstrate causality and not simply correlation. Future research must focus on the crucial link between chromatin states and NE partners, while dealing with the redundancies that we know exist among factors that anchor chromatin in the interphase nucleus. Clever screens in organisms that are partially compromised for aspects of nuclear organization should provide the means to identify essential components of other redundant pathways. Forward-genetic screens for dominant, gain-of-function phenotypes will also be needed to verify new components. Deciphering the mechanisms that determine the spatial organization of the genome in differentiated tissues requires that one monitors tissue-specific spatial distributions, which presents a challenge for high-throughput genetic approaches, yet clues can be gained from human diseases that affect nuclear organization. Fortunately, chromatin modifications and NE proteins — with the exception of the absence of lamin in plants and yeast — appear to be some of the most highly conserved proteins in our genomes. Thus, it is likely that we will be able to discover and test new molecules involved in the organization of the interphase genome through development and tissue-specific differentiation by capitalizing on trans-species studies of nuclear organization.Imaging approachesFluorescence in situ hybridization (FISH)FISH is based on the hybridization of fluorescent probes with specific DNA, RNA or whole-chromosome sequences. Drawbacks include artifacts that might arise during the fixation steps of cells and/or tissues and the denaturation of DNA that is required for hybridization.LacO/LacI–GFP or TetO/TetR–GFPLacO together with the labeled lac repressor LacI–GFP, or the tetracycline (tet) operator TetO together with the labeled Tet repressor TetR–GFP [In order to analyze the position and dynamics of chromatin loci in living cells, arrays of bacterial operators can be integrated at a site of interest and the corresponding bacterial ligand, fluorescently labeled with green fluorescent protein (GFP), is expressed constitutively at low levels  operator TetR–GFP ). DrawbaTetR–GFP .Other fluorescence-based applicationsThe fusion of fluorescent proteins to specific nuclear proteins can also be used to monitor chromatin dynamics and nuclear organization. However, one must always test for genetic complementation by the fusion protein. Photoactivation of labeled histones at specific nuclear compartments allows the determination of subnuclear localization of the perinuclear chromatin after cell division . ImaginThe use of microscopy has the advantage of revealing the spatiotemporal localization of a defined genetic locus in the nucleus in relation to other landmarks at a single-cell level.3C/4C/5C/HiC methodsThe chromosome conformation capture (3C) technique and various derivative methodologies  enable low-resolution analysis of DNA–DNA interaction probabilities, over approximately 10 kb to roughly 1 Mb. This technology uses crosslinking, enzymatic digestion, ligation, amplification and determination of the interactive sequences by PCR or deep sequencing . 3/4/5C DNA adenine methyltransferase-fusion identification (DamID)Escherichia coli DNA adenine methyltransferase (dam), which leads to preferential methylation of GATC motifs that are in the vicinity of the fusion protein. The sequences become differentially sensitive to restriction enzymes, allowing their selective amplification for detection by microarrays or deep-sequencing [The DamID technique is an alternative method for detecting protein–DNA contacts based on fusing a chromatin or nuclear protein of interest to quencing , 148. VaChromatin immunoprecipitation (ChIP) variants: ChIP-chip/ChIP-seq/ChIA-PETThese approaches are used to investigate interactions between proteins or specifically modified proteins and DNA in vivo and at a genome-wide level. The ChIP-chip, ChIP-seq and ChIA-PET methods are based on the recovery of DNA that is crosslinked to a specific antigen of interest, followed by microarray, high-throughput sequencing or 3C technology . The ChIGenetic approaches: gain of function, loss of function and spatially targeted functionTo test for correlations between position and function revealed by the above-mentioned methods, one needs to perturb normal function. Classically, truncations, frameshifts or deletions of genes provide loss-of-function data, whereas gain-of-function mutations or fusion proteins help confirm that the effects are not indirect. One commonly used gain-of-function example is the targeting of a specific protein or DNA locus to a nuclear subcompartment, accompanied by monitoring the resulting changes in function , 150.