Elucidation of interactions involving DNA and histone post-translational-modifications (PTMs) is essential for providing insights into complex biological functions. Reader assemblies connected by flexible linkages facilitate avidity and increase affinity; however, little is known about the contribution to the recognition process of multiple PTMs because of rigidity in the absence of conformational flexibility. Here, we resolve the crystal structure of the triple reader module (PHD-BRD-PWWP) of ZMYND8, which forms a stable unit capable of simultaneously recognizing multiple histone PTMs while presenting a charged platform for association with DNA. Single domain disruptions destroy the functional network of interactions initiated by ZMYND8, impairing recruitment to sites of DNA damage. Our data establish a proof of principle that rigidity can be compensated by concomitant DNA and histone PTM interactions, maintaining multivalent engagement of transient chromatin states. Thus, our findings demonstrate an important role for rigid multivalent reader modules in nucleosome binding and chromatin function.  •The ZMYND8 PHD/BRD/PWW cassette forms a stable structural reader ensemble•The reader ensemble binds chromatin via simultaneous engagement of histones and DNA•Each module of the ZMYND8 reader ensemble contributes to chromatin interactions•Genome-wide distribution of ZMYND8 at enhancers is mediated by K14ac interactions Savitsky et al. resolve the crystal structure of the PHD/BRD/PWWP cassette of ZMYND8 and demonstrate that tethering to K14ac-decorated chromatin is mediated through simultaneous engagement of histones and DNA by this triple reader ensemble. Such multivalent interactions control ZMYND8 participation in transcriptional complexes and recruitment to damaged DNA sites. Post-translational modifications (PTMs) on histones and DNA are critical regulators of chromatin stability, structure, and gene expression . Combina2) and histone 3 trimethyl-lysine 4 (H3K4me3) . For example, in the case of the transcription initiation factor TFIID subunit 1 (TAF1), two BRD modules exist in a rigidly confined topology, allowing for optimal recognition of multiple acetylated lysines on histone H4 with low micromolar affinity . Multi-dH3K4me3)  via the ylations . TherefoTo better understand how multivalent interactions involving more than two reader domains impact recognition of PTMs, affecting protein function, we studied the topology of the N-terminal triple reader domain architecture found in the zinc-finger MYND domain-containing protein 8 (ZMYND8), which contains, in addition to a PHD/BRD arrangement, a Pro-Trp-Trp-Pro (PWWP) domain within a PHD/BRD/PWWP cassette. ZMYND8 has been previously found to participate in transcriptional regulation complexes , is implGuided by sequence similarity to the recently structurally characterized BRD/PWWP tandem modules of ZMYND11  and by tx sequences found on histones; in the presence of a full aromatic cage, H3K4me3 inserts within the cage while a bulky residue separates the modified lysine from the adjacent arginine (R2), as exemplified in the case of the PHD finger of BPTF bound to a histone H3 K4me3 peptide  (3 and histone 3 lysine 18 acetyl (K18ac) on histone H3, the ZMYND8 topology orients the BRD module closer to histone 3 lysine 14 acetyl (H3K14ac). We therefore hypothesized that simultaneous engagement of histone peptides by the PHD and BRD modules may be possible and that histone 3 lysine 14 acetyl (K14ac) may be a target site of the ZMYND8 BRD module.Structural alignment of the ZMYND8 PHD domain with the TRIM33 PHD/BRD tandem module in complex with a methylated histone H3 peptide (histone 3 trimethyl-lysine 3 [H3K9me3])  revealed3]) F. In add3) (3) complex established that the C-terminal tail residue K396 of ZMYND8 mimics H3K36me3 insertion within the cage, suggesting that ZMYND8 may be able to bind to unmodified lysine residues   revealed3) G. In add3) A. Superiresidues A.Our structural analysis supports the notion that the triple reader assembly of ZMYND8 may be able to engage histone peptides. A schematic of such an possible interaction is shown in x , pS, and pT  and BRD modules was shown previously to bind to a phosphate ion in the ZMYND11 structure, in close proximity to T32 of histone H3 A. Threon, and pT . We obseTo assess peptide binding in solution, we used biolayer interferometry (BLI) and profiled commercial libraries containing histone H2A, H2B, H3, and H4 N-terminally biotinylated peptides and found strong binding primarily to H3 and H4 peptides. Although H3K14ac exhibited the strongest signal, we also observed binding to unmodified H3 as well as to all methylation states of K9 C. Interex, other than K4mex, systematically weakened binding , PWWP module (F288A/W291A), or all three reader modules  A. We tes binding C and 3D; binding E.Taken together, our data suggest that ZMYND8 relies on all three reader modules to engage histone H3 in cells, whereas binding to histone H4 mainly occurs through the BRD module.x binding . We found ZMYND8 signals enriched around transcriptional start sites (TSSs) together with H3K14ac A and 5B,WT, we observed fast recovery after bleaching, suggesting removal of ZMYND8 from chromatin, whereas the mutant BAZ2BNF masking did not have this effect  and proximity biotinylation (BioID) coupled to mass spectrometry (MS) . Using bFunctional enrichment analysis followed by annotation and clustering of the AP-MS network components confirmed a systematic loss of interactions with chromatin-associated functions upon mutation of the BRD domain for all statistically significant biological processes A and celIt has been proposed that multivalent binding results in higher specificity accompanied by affinity enhancement and loss of translational/rotation freedom to gain favorable entropy . InteracHere we report the structure of the triple N-terminal reader modules found in ZMYND8, which form a stable ensemble capable of simultaneously engaging histones and DNA. Binding is mainly driven by H3K14ac in vitro, and we find ZMYND8 to be associated with this mark in cells, whereas other histone modifications seem to be tolerated, with the exception of higher lysine methylation states. Two recent studies suggested that H4K16ac is a main point of histone H4 contact based on antibody evidence ; howeverInteraction with the entire N-terminal histone tail may result in “shielding,” preventing remodeling, a topology that we find by molecular modeling to be possible see ; we alsohttp://www.cbioportal.org/) revealed that 25% of ZMYND8 mutations occur within the reader ensemble, with enriched sites on the PHD and PWWP modules. Mapping of the mutated sites onto the crystal structure suggests that most, if not all, affect binding to histones either by disrupting the interaction interface or by destabilizing the domain topology  consortium  WT, or mutant was cloned into pDONR221, pDEST 5′ Triple-FLAG-pcDNA5-FRT-TO, or pDEST 5′ BirA∗-FLAG-pcDNA5-FRT-TO vectors; the PHD/BRD/PWWP cassette was cloned into the pNIC-ZB vector (GenBank: GU452710). Proteins of interest were stably expressed in T-REx Flp-In HEK293 cells as described previously  or peptides synthesized as described previously .Experiments were carried out on an ITC200 titration microcalorimeter, and data analysis was performed using the MicroCal Origin software as described previously ; data arPurified recombinant protein was crystallized at 4°C. Zinc single wavelength anomalous dispersion phasing (Zn-SAD) data were collected at Diamond Lightsource. Identification of three putative Zn sites in the SAD dataset allowed phasing, followed by manual model building and refinement as explained in the Chromatin fractionation assays were performed with HEK293T cells transfected with ZMYND8 WT or mutant plasmids as described previously .Cells were lysed, chromatin was digested, and co-immunoprecipitation was performed using antibodies described in the FRAP experiments were performed using a protocol modified from previous studies  as descrOligonucleotide probes encoding AT-rich or GC-rich sequences were used together with recombinant ZMYND8 constructs (WT or mutant) to determine protein binding in electrophoresis experiments. For details, see the Samples were analyzed by mass spectrometry in liquid chromatography-tandem MS experiments on a TripleTOF 5600 instrument. AP-MS as well as BioID-MS were performed as described previously .Small RNAi was used to knock down ZMYND8 or HATs in HEK293 cells expressing inducible 3×FLAG-ZMYND8. For details, see the Details regarding chromatin preparation, ChIP, and ChIP-PCR can be found in the Reads were mapped to hg19 (NCBI build 37) using BWA-MEM, allowing up to two mismatches. Genome ChIP-seq profiles were generated using HOMER , and enrP values were calculated using two-tailed Student’s t test or one-way ANOVA followed by Dunnett’s multiple comparison test. ChIP-qPCR P values were determined using one-way ANOVA followed by Dunnett’s multiple comparison test on biological replicates (n = 3). In AP-MS and BioID-MS experiments, data were analyzed with SAINTexpress to calculate the probability value of each potential protein-protein interaction from background contaminants using default parameters. Controls were compressed to six samples, and a false discovery rate (FDR) of 1% or lower was required for proteins to be classified as significant interaction partners of ZMYND8.For FRAP experiments, mean values and SEM were determined from 15 cells per condition tested over multiple experiments, and P.S. prepared recombinant proteins, designed and cloned full-length ZMYND8 constructs and mutants, and performed crystallization trials. P.S. and S.P. designed, carried out, and analyzed biophysical experiments. T.K. performed crystallographic studies. T.F. carried out FRAP studies and immunoblotting. J.P.L established stable cell lines. J.P.L. and A.C.G. designed and performed proteomic experiments and analyzed data. E.S., B.S., and K.K. prepared recombinant peptides and performed chromatin fractionation assays. T.F., H.F., and C.G. designed and performed EMSA assays. C.Y.W. performed ChIP-seq assays. C.Y.W. and T.F. performed ChIP-qPCR assays. A.K., A.S., and P.F. analyzed ChIP-seq data. P.F. designed and supervised the study and prepared the manuscript. All authors read and commented on the manuscript.


Streptomyces antibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM. A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimRSARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding.The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A IMPORTANCE Here, we have shown that a modulation of the production of the antifungal pimaricin in Streptomyces natalensis can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator pimM. The expression of this gene is controlled by the Streptomyces antibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator PimR, which binds a series of heptameric direct repeats in its promoter region. The structure and importance of such repeats in protein binding, transcriptional activation, and polyene production have been investigated. These findings should provide important clues to understand the regulatory machinery that modulates antibiotic biosynthesis in Streptomyces and open new possibilities for the manipulation of metabolite production. The presence of PimR orthologues encoded by gene clusters for different secondary metabolites and the conservation of their operators suggest that the improvements observed in the activation of pimaricin biosynthesis by Streptomyces natalensis could be extrapolated to the production of different compounds by other species. Streptomyces natalensis  DNA-binding domain with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family (LAL)  . The C-tly (LAL) . PimR bily (LAL) . Such anperators , it doesperators , thus foperators .TGGCAAGAAAGCGGCAGGTGTTCGGCAAG [the heptameric repeats are underlined]) is exactly conserved in the intergenic region between scnRII and scnRI in the scn gene cluster of Streptomyces chattanoogensis  and alspinensis , 15, incpinensis  and PolRpinensis , which aSince PimR is the archetype of SARP-LAL regulators, it was of great interest to characterize the role of those conserved heptameric repeats in the process of transcriptional activation.pimM expression, we designed seven constructs containing the pimM gene under the control of different versions of its promoter   . The priMethods) . The P4pMethods) . The pro altered .S. natalensis ΔpimM, a strain deficient in the production of pimaricin  medium without sucrose. Samples were obtained every 24 h for growth and pimaricin production measurements. S. natalensis ΔpimM containing pSETpimM, a pSET152 derivative with pimM under the control of its native promoter, was used as a positive control , after which a decrease in biomass was observed up to 96 h, probably due to nutrient depletion and the onset of cell death processes. However, significant differences were observed in pimaricin production . The strpimM transcription, a quantitative analysis of pimM expression in the different recombinant strains was performed. Total RNA obtained from 48-h cultures, a condition under which pimaricin is actively produced, was used as the template , in concordance with the absence of pimaricin production .r2) of 0.8918 for the plot.Except for those with P3pimM, the transcription values showed good concordance with pimaricin production. Figure S2 in the supplemental material shows the correlation between both data, including a least-square line fit that resulted in a Pearson's correlation coefficient  with GST-PimRSARP  and DNA SARP. To discard the possibility that interactions could be produced by the GST moiety of the fusion protein, control reactions were performed under the same conditions but using pure GST (10 μM) instead of GST-PimRSARP. This experiment produced negative results in all cases, excluding the possible binding of the GST protein to the promoters (not shown). In the cases where retarded bands were observed, the intensity of the band(s) was diminished by the addition of the same unlabeled DNA (not shown), suggesting that binding is specific.Interestingly, the number of shifted bands observed with the different variants of the promoter was in agreement with the binding of a protein monomer to each of the heptamers, with a minimum requirement of two protein monomers for efficient binding (see below). Thus, the native promoter would be able to accommodate two or three monomers in the region containing the triplet of heptamers plus two in the doublet, and three retardation bands were observed . SimilarSARP binding to the different probes and the pimaricin production observed with the different promoters. Thus, while little interaction was observed with the probes containing P1pimM or P7pimM, strong band shifting was observed with the probes containing promoters that yielded the highest pimaricin production, such as P3pimM and P6pimM.In general, there was a clear correlation between GST-PimRpimM transcription could be the consequence of pimR binding to the different promoter versions and the subsequent recruitment of RNA polymerase.Altogether, these results indicate that the differences observed in SARP to the duplexes was then studied by EMSAs. Interestingly, the results showed that two heptameric repeats are necessary for GST-PimRSARP binding , only one retardation band was observed with probe P2 (containing two heptamers), and no binding was observed with probe P1 (one heptamer) or P0 (without any heptamer) under the same reaction conditions.To confirm our previous interpretation of DNA-binding results, we constructed different duplex DNA probes using the triplet of heptamers in the native promoter as a model . Thus, P binding . While tpimM expression.PAS-LuxR regulator-encoding genes are associated with several polyene macrolide biosynthetic gene clusters, thus constituting a possible landmark of this type of gene cluster. In this context, they are transcriptional activators essential for the biosynthesis of the polyene encoded within the cluster. Their expression is a bottleneck in the biosynthesis of the antifungal; thus, polyene production is easily increased upon gene dosage increase , 21. HerpimM and essential for the subsequent pimaricin biosynthesis. Thus, in the S. natalensis ΔpimM cultures containing pP1pimM or pP7pimM constructs, where the entire triplet of heptamers or just one of the heptamers is removed, the production of the polyene was not detected, whereas the pimM transcript level was approximately half that of the control strain. Such a reduction in pimM transcription turned out to be sufficient to completely abolish pimaricin production by S. natalensis. In S. natalensis ΔpimM::pP2pimM, which lacked the doublet of heptamers, the expression of the PAS-LuxR regulator decreased by 20%, whereas pimaricin production was reduced by ca. 50% compared to that of the strain containing the native promoter, indicating that the doublet is less relevant than the triplet. However, the addition of one heptamer to the triplet (P5pimM) did not substantially improve pimaricin production compared with that from the native promoter. This suggests that the optimum number of repeats of the heptamer recognized by PimR is three and that at least one triplet is required at the operator to activate pimM transcription.The three direct heptameric repeats have proven to be important for the transcription of pimM transcription, with four nucleotides preferable to three, as demonstrated by the increases in pimM expression and pimaricin production by the strain S. natalensis ΔpimM::pP4pimM compared to those of the control with the native promoter. The classical SARP binding sequence is 11 bp in length  was the modification that rendered the best results, increasing pimM transcription almost 5-fold and polyene production by 50%.The location of the triplet also appears to play an important role in the activation of transcription by PimR. Streptomyces coelicolor that consists of an N-terminal SARP domain, a central ATPase domain, and a C-terminal tetratricopeptide repeat , with a minimum requirement of two protein monomers for efficient binding. One heptamer is not sufficient for sustained PimR binding, and at least two heptameric repeats are required for proper binding. A similar mode of binding has been proposed for other proteins belonging to the SARP family, including AfsR, a global regulator of secondary metabolism in e repeat , or DnrIe repeat . This cotor OmpR . SARPs ator OmpR , and alltor OmpR , 24. AddpimM transcription. Due to the high similarity among ScnRI, PteR, FilR and PimR, as well as the high degree of conservation of their corresponding operators  was used for expression studies. E. coli ET12567::pUZ8002 was used as donor in intergeneric conjugations.is ΔpimM  was routis ΔpimM  without is ΔpimM  at 30°C.E. coli ET12567::pUZ8002 and S. natalensis ΔpimM was performed as described previously . DNA sequencing was accomplished by the dideoxynucleotide chain-termination method using the DYEnamic ET terminator cycle sequencing kit  with an Applied Biosystems ABI 3130XL DNA genetic analyzer .Intergeneric conjugation between eviously . pUC19  was used as the template. In the case of P6pimM, one of the pairs was PimM-F/PPM1-R and the other one was PPM6-F/PimM-R. Using as the template the purified fragments from these first two PCRs, we performed fusion PCR with the PimM-F/PimM-R oligonucleotides to obtain the pimM gene together with its terminator. In all cases, the mutations were introduced into the sequences of the primers so that they were conserved in the fusion PCR product. In contrast to the above cases, P7pimM was obtained by a single PCR with the primers PPM7-F and PimM-R, which gave rise to a 987-bp fragment containing pimM under the control of a promoter lacking the first heptamer of the triplet.All constructs were obtained by fusion PCR. The primers are indicated in E. coli ET12567::pUZ8002. One of the transformants was selected and introduced into S. natalensis ΔpimM by conjugation as described previously .To assay pimaricin in culture broths, 1 ml of culture was extracted with 4 ml of methanol and further diluted with methanol to bring the absorbance at 319 nm in the range of 0.1 to 0.4 units. Control solutions of pure pimaricin (Sigma) were used as the control. To confirm the identity of pimaricin, a UV-visible absorption spectrum  was routinely determined in a Hitachi U-2900 spectrophotometer. The quantitative determination of pimaricin was performed as previously described (RNA was extracted as described previously . BrieflyTaq DNA polymerase (Invitrogen) and 150 ng of total RNA as the template. The conditions were as follows: first-strand cDNA synthesis at 45°C for 40 min followed by heating at 94°C for 2 min and amplification consisting of 30 cycles at 94°C for 40 s, 63°C for 30 s, and 72°C for 30 s. Primers 5′-GCCTGCCCGACACGACCG-3′ and 5′-TCCACGACCTCGGGCGGC-3′ were designed to cover the 3′-end region of pimM, and 5′-GGGGTGGATTAGTGGCGAACG-3′ and 5′-CAACCCGAAGGCCGTCATCC-3′ were used for 16S rRNA, generating PCR products of 304 bp and 340 bp, respectively. Negative-control reactions were carried out with each set of primers and Platinum Taq DNA polymerase in order to confirm the absence of contaminating DNA in the RNA preparations. The identity of each amplified product was corroborated by direct sequencing of the PCR product.Transcription was studied by using the SuperScript one-step reverse transcriptase PCR (RT-PCR) system with Platinum Ex Taq (TaKaRa), 200 to 300 nM each primer, and the template cDNA diluted 1:2 and run on a StepOnePlus real-time PCR system (Applied Biosystems). The reactions were carried out in two biological replicates with three technical replicates each, and appropriate controls were included to verify the absence of gDNA contamination in the RNA samples and primer-dimer formation. Primers 5′-TTGGAGAATTGACGCACCAG-3′ and 5′-ATCCAGCAGGCGAACCAG-3′ for pimM and 5′-GACGCAACGCGAAGAACC-3′ and 5′-TGCGGGACTTAACCCAACATC-3′ for rrnA1 were designed to generate PCR products of 125 and 137 bp, respectively, near the 5′ ends of the mRNA. The PCRs were initiated by incubating the sample at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 66 to 70°C (depending of the set of primers used) for 34 s. To check the specificity of real-time PCRs, a DNA melting curve analysis was performed by holding the sample at 60°C for 60 s, followed by slow ramping of the temperature to 95°C. Baseline and threshold values were determined by the StepOnePlus software. TC values were normalized with respect to rrnA1 mRNA (encoding 16S rRNA). The relative changes in gene expression were quantified using the Pfaffl method (E) of one cycle in the exponential phase was calculated according to the equation (E = 10(−1/slope) (n = 5 or 6 with three replicates for each dilution), with a coefficient of determination (R2) of >0.99 .Reverse transcription of total RNA was performed on selected samples with 5 μg of RNA and 12.5 ng/μl of random hexamer primer (Invitrogen) using SuperScript III reverse transcriptase (Invitrogen). All RNA samples were analyzed with the Agilent 2100 Bioanalyzer , and only those with RNA integrity number (RIN) values  ranging l method  and RESTl method . The corSARP protein as described previously (DNA binding was tested by using electrophoretic mobility shift assays (EMSAs). The DNA fragments used for EMSAs were amplified by PCR using the primers PMF (5′-CGCTCCTGTGGATCGTTGCATAG-3′) and PMR (5′-TACAGGATCCGTTCTATCAAGGCTCGCCATGCAC-3′) for probes PpimM to P6pimM, or P7F (5′-CGTATGTTGTGTGGAATTGTGAGCG-3′) and PMR for probe P7pimM, using the corresponding construct as the template. The amplification products were then sequenced to confirm the absence of any mutations and then labeled at both ends with a digoxigenin (DIG) oligonucleotide 3′-end labeling kit (2nd generation [Roche Applied Science]). Binding assays were performed with GST-PimReviously .To obtain DNA duplexes for the validation of the binding site, one oligonucleotide pair   was anne


IbNAC1 expression to balance plant vitality and survival remains unclear. In this study, a wound-responsive G-box cis-element in the IbNAC1 promoter from -1484 to -1479 bp was identified. From a screen of wound-activated transcriptomic data, one transcriptional activator, IbbHLH3, and one repressor, IbbHLH4, were selected that bind to and activate or repress, respectively, the G-box motif in the IbNAC1 promoter to modulate the IbNAC1-mediated response. In the early wound response, the IbbHLH3-IbbHLH3 protein complex binds to the G-box motif to activate IbNAC1 expression. Thus, an elegant defense network is activated against wounding stress. Until the late stages of wounding, IbbHLH4 interacts with IbbHLH3, and the IbbHLH3-IbbHLH4 heterodimer competes with the IbbHLH3-IbbHLH3 complex to bind the G-box and suppress IbNAC1 expression and timely terminates the defense network. Moreover, the JAZs and IbEIL1 proteins interact with IbbHLH3 to repress the transactivation function of IbbHLH3 in non-wounded condition, but their transcription is immediately inhibited upon early wounding. Our work provides a genetic model that accurately switches the regulatory mechanism of IbNAC1 expression to adjust wounding physiology and represents a delicate defense regulatory network in plants.IbNAC1 is known to activate the defense system by reprogramming a genetic network against herbivory in sweet potato. This regulatory activity elevates plant defense potential but relatively weakens plants by IbNAC1-mediated JA response. The mechanism controlling  sporamin expression against herbivory in sweet potato. However, it simultaneously injures the plant by IbNAC1-induced JA response. Thus, dynamic regulatory networks to coordinate the expression of IbNAC1 are essential for appropriate growth and defense in sweet potato. Here, two bHLH transcription factors, IbbHLH3 and IbbHLH4, function antagonistically in activating the IbNAC1 promoter. Notably, IbbHLH3 coordinates activity of IbNAC1 promoter by interaction with several proteins in different time-course of wounding to activate/repress its transcriptional activation function. The dynamic interaction networks of IbbHLH3 improve defense ability against wounding, and timely prevent the injury by IbNAC1-regulated responses.Plants develop sophisticated defense systems against insect attack. IbNAC1 is a transcriptional activator, which up-regulates  Plants have developed sophisticated defense systems to cope with this frequent challenge. Among these systems, transcription factors play essential roles by modulating the expression of defense genes directly. Typically, transcription factors bind to a specific DNA motif in the promoter region of downstream genes based on their DNA binding domain. Several studies indicate that transcription factors have been characterized to bind with unusual  regions ,2. Follo[ATAF1/2 , PI-II [2 [PI-II , WIPK [5sporamin ,7 have bVSP1 and VSP2 against herbivory. Interestingly, other MYC-type transcription factors, including JA-associated MYC2-like gene 1 (JAM1), JAM2 and JAM3, show repressive functions in herbivore defense, in contrast to MYC2 [Jasmonic acid (JA) is involved in plant defense against insect herbivory . In resp to MYC2 –18. MYC2 to MYC2 ,17,19,20 to MYC2 ,22. Basesporamin in Brassica and tobacco effectively increases resistance to insect feeding [sporamin promoter by binding the specific cis-acting element, TACAATATC, immediately upon wounding in leaves. In addition to enhanced resistance against herbivory, IbNAC1 simultaneously modulates several JA responses, including growth inhibition, ROS overproduction, and precocious senescence, to influence plant physiology [Sweet potato is an important food crop, economic crop, and forage crop in Asia. Notably, it shows high resistance to insect feeding among crops in the field. A unique protein in sweet potato, sporamin, is abundant in tuberous roots as a storage protein for tuber sprouting . In prev feeding –27. ThusIbNAC1 expression. In the IbNAC1 promoter, a G-box motif acts as a switch in response to wounding signals. Two bHLH MYC-type transcription factors, IbbHLH3 and IbbHLH4, were identified as binding proteins of the G-box switch in the IbNAC1 promoter. These transcription factors function at different stages of the wounding response to synergistically coordinate IbNAC1 expression. Furthermore, with the temporal changes that occur in response to wounding, the function of IbbHLH3 factor was activated or repressed through interactions with different proteins, including bHLHs, JAZs, EIL1, and MAPKs. This activation or repression of IbbHLH3 helps plants to cope with IbNAC1-mediated injury.In this study, we further investigate the signal transduction mechanism of wounding in the stimulation of IbNAC1 promoter region  was isolated by genome walking (Clontech). Promoter structure analysis revealed that several cis-regulatory elements, including TATA-box, G-box motif, W-box motif, ASF-1 binding site, CBF binding site and LTRE-1 motif, were predicted by PLACE [PIbNAC1::GUS) and transformed into sweet potato and Arabidopsis. As shown in IbNAC1 promoter region was activated by both local and systemic wound responses. In addition to wounding-induced activity, several wounding-related signals, including methyl jasmonate (MeJA), ethylene (ET) and reactive oxygen species (ROS), also triggered the IbNAC1 promoter  harbors cis-acting element(s) for the wound response.Sweet potato IbNAC1 is known to reprogram the defense mechanism in response to herbivory and wounding . Howeverby PLACE  and Planby PLACE  Fig 1A)IbNAC1 prpromoter . These r  in the IbNAC1 promoter region, a 5’ serial deletion of the IbNAC1 promoter was performed. Several truncated fragments of the IbNAC1 promoter  fused to the GUS reporter were delivered into Arabidopsis plants to rapidly screen wound-responsive cis-elements (4 transgenic Arabidopsis lines were established for each deleted promoter construct to determine the promoter activity. FL (-1665 to -1 bp) transgenic plants exhibited obviously higher GUS activity in response to wounding. However, GUS activity was abolished in response to wounding in Del-1 (-1395 to -1 bp), Del-3 (-618 to -1 bp) and Del-4 (-346 to -1 bp) plants but not in Del-2 (-1037 to -1 bp) plants (cis-element(s) were localized to the region from -1665 to -1395 bp. Furthermore, the promoter region (from -1665 to -1395 bp) with strong activity in response to wounding was dissected into three segments, -1665 to -1395 bp (Del-5), -1506 to -1395 bp (Del-6) and -1463 to -1395 bp (Del-7), and then fused to the 35S minimal promoter (35Sm) .To define the wound-responsive elements . Four in) plants . In Del-r (35Sm) . T4 tranonstruct . This inIbNAC1 promoter is a wound-responsive region, the 43-bp fragment was amplified, fused to the 35Sm and the GUS reporter gene (NWRE-35Sm::GUS) and transformed into Arabidopsis. T4 NWRE transgenic plants were used to study the wound-responsive activity. As shown in cis-element (CACGTG), G-box, was found in the NWRE region. The G-box has been identified as a key motif in activating promoters in responses to wounding, JA and ABA [mNWRE-35Sm::GUS) and then transformed into Arabidopsis. Three different independent lines were selected to determine the GUS activity. In contrast to intact NWRE transgenic lines, the wounding-responsive activity was completely aborted in all of the mNWRE lines, suggesting that the G-box motif is essential for the wound signal response in the NWRE of the IbNAC1 promoter (To verify that the NWRE region (-1506 to -1463 bp) in the  and ABA . To confpromoter .NWRE-35Sm::GUS was transformed into sweet potato. Three independent transgenic sweet potato lines with NWRE-35Sm::GUS showed strong GUS activity in the vicinity of the region wounded by tweezers compared to 35Sm::GUS lines  expression were correlated to IbNAC1 expression pattern. Therefore, both were cloned for further investigation. IbbHLH3 and IbbHLH4 belong to groups IIIe and IIId of MYC2-related bHLH transcription factors, respectively , ET and ROS are important elicitors that amplify the wound signal in plants. In our previous studies, and H2O2 ,7. Thus,ignaling .pADH1::AD-IbbHLH3) and pHIS2.1-NWRE (NWRE-TATAbox::HIS3) were grown on SD-L/-T/-H medium with 100 mM 3-AT. This means that IbbHLH3 was capable of binding to the NWRE region of the IbNAC1 promoter. To determine whether IbbHLH3 interacts with the G-box motif in the NWRE region, a G-box mutational assay was performed. In the mutated assay, the sequence of the G-box in the NWRE (GCGTTCTTACTTTTCGTAAGGGCACGTGAAGCACCCCAACAAA) was replaced with GCGTTCTTACTTTTCGTAAGGGAAAAAAAAGCACCCCAACAAA (mNWRE) and cloned into the pHIS2.1 vector (pHIS2.1-mNWRE). The transformant co-expressing pHIS2.1-mNWRE and pGADT7-IbbHLH3 was unable to grow on SD-L/-T/-H 3-AT medium. The absence of an interaction between mNWRE and IbbHLH3 indicated that IbbHLH3 binds to the NWRE region of the IbNAC1 promoter via the G-box motif. Additionally, the transformants co-expressing pGADT7-IbbHLH4 (pADH::AD-IbbHLH4) and pHIS2.1-NWRE grew well, but a lack of growth of the transformants with pGADT7-IbbHLH4 and pHIS2.1-mNWRE on SD-L/-T/-H 3-AT medium was observed, suggesting that the HIS3 gene driven by the NWRE was activated by the binding of AD-IbbHLH4 to the G-box motif . IbbHLH3 and IbbHLH4 fused to the GAL4-binding domain (BD), forming BD-IbbHLH3 and BD-IbbHLH4, respectively, were transferred into AH109. After a β-GAL filter assay, visible blue precipitates were seen in BD-IbbHLH3, suggesting that IbbHLH3 functions as a transcriptional activator to activate LacZ reporter  or mNWRE driving the FLUC gene as the reporter. The truncated IbNAC1 promoter and mNWRE abolished most of the elicitation activity of IbbHLH3 and the repression activity of IbbHLH4  driven by NWRE-TATAbox  or the fpromoter  as repor:IbbHLH4 . As show:IbbHLH4 . The fulgonistic . To conf IbbHLH4 . These rin vivo, IbbHLH3 and IbbHLH4 driven by 35S promoter were overexpressed in NWRE-A Arabidopsis plant, respectively. The GUS activity assay of double transgenic plants resulted that IbbHLH3 is a transcriptional activator of NWRE promoter in planta, while IbbHLH4 acts as a negative role  with IbbHLH3, which can bind to Gal4BS DNA sequence. The Gal4DB transactivation expression assay was carried out by interacting two effectors, 35S::BD-IbbHLH3 and 35S::IbbHLH4, and one reporter, 4xGAL4BS-TATA::FLUC. In this transient activation system, the enhanced reporter activity by IbbHLH3 was decreased by the co-expression with IbbHLH4  family proteins repress JA signaling by interacting with MYC2/3/4 transcription factors. Here, we are interested in determining the function of wound-responsive JAZs genes in sweet potato leaves. Using transcriptomic analysis, several wound-related JAZ proteins were selected to examine the interaction with IbbHLH3. Among these genes, the expression of wounding . To examwounding . The growounding . These rwounding .IbEIL1, was cloned and used for BiFC and Y2H. The BiFC results revealed that a strong YFP signal was reconstructed by nYFP-IbbHLH3 and cYFP-IbEIL1 in the cell nucleus  was grown at 25°C/20°C with Light/Dark photoperiod of 16 h/8 h. The third leaves of sweet potato were wounded using tweezers. For hormone treatment, the third expended leaves were sprayed with 0.1 M sodium phosphate buffer or sodium phosphate buffer containing treated chemicals  at 25°C for 1 hour.Sweet potato  and the consecutively truncated regions, such as Del-1 (1395 bp), Del-2 (1037 bp), Del-3 (618 bp) and Del-4 (346 bp), amplified by PCR were respectively cloned into pCAMBIA1391z to drive the GUS reporter gene. Furthermore, the selected region from -1665 to -1395 bp was further divided into three different fragments named Del-5 (-1665 to -1395 bp), Del-6 (-1506 to -1395 bp), and Del-7 (-1463 to -1395 bp). These fragments were respectively fused to the 35Sm promoter and GUS reporter. In parallel, the 35Sm promoter fused to the GUS reporter was used as a negative control in the wounding treatment. These reporter constructs were transformed into Arabidopsis and sweet potato plants to analyze the promoter activity.To construct a reporter vector to analyze the regulatory IbACTIN7  to generate the plasmid pHIS2.1-NWRE as bait. The coding regions of IbbHLH3 and IbbHLH4 were separately cloned into pGADT7 as effectors. Lithium acetate-mediated transformation  was perfIbbHLHs proteins were examined for transactivation function using a yeast system. The coding region of IbbHLH3 and IbbHLH4 were cloned into pBGKT7 (BD). Then, the BD-IbbHLH3 and BD-IbbHLH4 were delivered into yeast strain AH109. The transformants were screened by tryptophan-deficient SD medium, and determined its reporter activity by a filter assay using X-gal  as substrate.E. coli Cordon. The GST fusion proteins in bacterial cultures were induced by 0.1 mM IPTG for 4 hours. The cell pellets were lysed, and the recombinant proteins were purified using GSTrap FF . Then, the concentration of the extracted proteins was determined by the Bradford method [The open reading flame of IbbHLH3 and IbbHLH4 was cloned behind the GST-tag in the pGEX4T-3 vector . The fusion plasmids were transferred into d method .Single strands of NWRE fused to 6FAM were synthesized . The two strands of NWRE were mixed together in equimolar ratios and reacted at 95°C and cooled slowly to 25°C. The double-stranded NWRE fused to 6FAM were used as fluorescent probes for the EMSA experiment.The EMSA binding reaction contained appropriately 5X binding buffer , 1 μg of poly (dI-dC), a 6X dye, 1 μl of 6FAM-labeled NWRE probe, and added proteins in dosage-dependent manner. The reaction mixtures were reacted at room temperature, and then loaded into a 6.6% native bis-polyacrylamide gel in 0.5X TBE buffer. The shift of the probes in the gels was observed using LAS3000.The amino acid sequences were retrieved from GenBank. Multiple sequence alignments of the bHLH-MYC domain proteins were conducted using the MEGA 4.1 program. Phylogenetic analyses were generated by the neighbor-joining method. A bootstrap analysis of 1000 resampling replications was performed in MEGA 4.1.The p2YGW7 vector, which contains the 35S promoter, was used to drive the YFP-IbbHLH fusion proteins. Fluorescence images were obtained by confocal microcopy (Leica TCS SP5).IbNAC1 promoter fused to a TATA box and firefly LUC, respectively, were used as reporter plasmids. The reporter plasmids (5 μg) and the effector plasmids (5 μg), 35S::IbbHLH3 and 35S::IbbHLH4, were used for protoplast transactivation analysis. In the competing assay, an extra 5 μg of competitors were co-transformed with effectors and reporters. 2 μg of reference plasmid (RLUC) was co-transformed for normalization of each reaction, after which the LUC activity was examined by a dual-luciferase assay system .The NWRE region and the IbbHLH3 and IbbHLH4 was amplified by PCR, and subsequently cloned into pCAMIBA1300, which harbored a 35S promoter. These plasmid constructs were delivered into T4 NWRE-A plants and T4 mNWRE-A plants, respectively. T3 double transgenic plants were used to determine the NWRE activity by GUS reporter assay.The coding region of 35S::YFP, 35S::YFP-IbbHLH3 or 35S::YFP-IbbHLH4 were delivered by using GDS-80 gene delivery system . Gold particles (0.4 μm diameter) coated with 1 μg plasmid DNA were shot by 50 psi of helium, the gas flow rate around 10~15 L/min, and 6 cm target distance. After bombardment for 3 days, the expression of IbbHLH3, IbbHLH4, and IbNAC1 were monitored by qRT-PCR.Full-length coding sequences of sweet potato IbbHLH3, IbbHLH4, JAZ1, JAZ2a, JAZ2b, IbEIL1, IbWIPK1, and IbWIPK2 were cloned into the nYFP or cYFP vector through a Gateway reaction with the pSAT4-DEST-nEYFP and pSAT4-DEST-cEYFP vector systems . The conpCAMBIA1300-35S::GST-IbbHLH4 and pCAMBIA1300-35S::GFP-IbbHLH3 were co-transformed into Agrobacterium tumefaciens LBA4404. The acetosyringone-activated Agrobacterium with was injected into N. benthamiana leaves. Total proteins from the transformation leaves were incubated with GST-Resin . After washing, the bound proteins were eluted and detected by western blotting using anti-GFP , and anti-GST antibodies .The plasmid constructs The recombinant protein GST-IbbHLH4 or GST was co-incubated with MBP-IbbHLH3 and amylose resin . After washing five times with MBP column buffer, the MBP-binding protein was eluted and separated by SDS-PAGE. Anti-MBP and anti-GST antibodies were used for western blotting analysis to detect the MBP-IbbHLH3 and IbbHLH3-interacting proteins (GST-IbbHLH4 or GST). For GST pull-down assay, the GST-fusion proteins (GST-JAZs and GST-MAPKs) were co-incubated with MBP-IbbHLH3 and GST-Resin . After washing with PBS buffer, the GST-binding protein was eluted and detected by western blotting using anti-GST and anti-MBP antibodies .The coding region of IbbHLH3 was inserted into pGADT7 to generate the plasmid AD-IbbHLH3. The coding regions of IbJAZs, IbWIPKs, and IbEIL1 were separately cloned into pGBKT7 . Lithium acetate-mediated transformation  was perft-test was used to determine the statistical significance of the experiments in Excel 2010 at P<0.05 and P<0.01. Statistical analysis of one-way ANOVA was performed to determine the significant differences in SPSS at P<0.05.The experiments in this study were examined at least three individual biological replicates. Student’s Gene sequences from present study can be retrieved at GenBank/EMBL as following: IbNAC1 (GQ280387), IbbHLH3 (KU589265), IbbHLH4 (KU744525), IbJAZ1 (KX147234), IbJAZ2a (KX147232), IbJAZ2b (KX147233), IbEIL1 (KX147235), IbWIPK1 (HQ434622), IbWIPK2 (KU744642), AtMYC2 (AT1G32640), AtMYC3 (AT5G46760), AtMYC4 (AT4G17880), AtJAM1 (AT1G01260), AtJAM2 (AT1G01260), and AtJAM3 (AT4G16430).S1 Table(PDF)Click here for additional data file.S1 FigThe phylogenetic relationships were analyzed by the neighbor-joining method. A bootstrap analysis of 1000 resampling replications was conducted in MEGA 4.1.(TIF)Click here for additional data file.S2 FigThe sequences of IbbHLH3, IbbHLH4 and bHLH-MYC-type transcription factors, including AtMYC2, AtMYC3, AtMYC4, and JAM1, were aligned by GeneDoc. The transcriptional activation domain is shown in the gray region, and the underlined region is the bHLH-conserved domain. NLS are marked by a red frame.(TIF)Click here for additional data file.S3 FigThe expression levels of IbbHLH3 and IbbHLH4 were monitored by qRT-PCR under treatments of 50 μM MeJA for 1 hour, 2 mM SA treatment for 1 hour, and 1% H2O2 treatment for 1 hour. Error bars indicate SDs from three biological replicates.(TIF)Click here for additional data file.S4 Fig35S::YFP-IbbHLH3 and 35S::YFP-IbbHLH4, respectively. Simultaneously, AtMYC2 was used as a positive control for nuclear fluorescence observation under confocal microscopy.IbbHLH3 and IbbHLH4 were cloned into the p2YGW7 vector, forming (TIF)Click here for additional data file.S5 Fig(A) IbbHLH3 and IbbHLH4 were cloned into the yeast expression vector pGBKT7, forming BD-IbbHLH3 and BD-IbbHLH4, respectively. The yeast AH109 transformants containing BD-IbbHLH3 and BD-IbbHLH4 were analyzed by a β-GAL filter assay. (B) Expression analysis of the LacZ gene in EV, BD-IbbHLH3, and BD-IbbHLH4 transformants. Error bars indicate SDs (n = 5).(TIF)Click here for additional data file.S6 FigThe truncated IbNAC1 promoter (Del-1) (A) and the mutated NWRE region with 35Sm (B) were separately fused to FLUC as reporters. Renilla LUC (RLUC) was used as an internal control for normalization. Error bars indicate SDs (n = 10).(TIF)Click here for additional data file.S7 FigGST–IbbHLH4 or GST was incubated with MBP-IbbHLH3 and amylose resin. The reacted proteins were eluted from the resin and examined by western blotting using anti-MBP or anti-GST antibody, respectively.(TIF)Click here for additional data file.S8 FigIbNAC1 and IbbHLH3 in sweet potato overexpressing IbbHLH3ΔHLH. 35S::YFP-IbbHLH3ΔHLH or 35S::YFP was transiently expressed in sweet potato leaves by particle bombardment. After bombardment for 3 days, the expression of IbNAC1 and IbbHLH3 were monitored by qRT-PCR. Error bars indicate SDs (n = 5). Asterisks represent significant difference from YFP .(A) A schematic representation of HLH domain in IbbHLH3. (B) The expression of (TIF)Click here for additional data file.S9 Fig(A) A schematic model of the truncated DNA binding domain of IbbHLH4 (ΔIbbHLH4). (B) NWRE binding ability of truncated IbbHLH4. ΔIbbHLH4 was cloned into the yeast expression vector pGADT7, forming AD-ΔIbbHLH4. pHIS2-NWRE was used as bait to examine the binding ability of ΔIbbHLH4. (C) BiFC assay. ΔIbbHLH4 was fused to cYFP in the N-terminal region, and the interaction with IbbHLH3 analyzed in Arabidopsis protoplasts. (D) IbbHLH3 competing assay of ΔIbbHLH4 in regulation of the NWRE activity. Error bars indicate SDs (n = 10). Asterisks represent significant difference from reporter alone .(TIF)Click here for additional data file.S10 FigJAZ1, JAZ2a, JAZ2b, EIL1, IbWIPK1, and IbWIPK2, was monitored by qRT-PCR under a wounding time-course analysis. Error bars indicate SDs from four independent replicates.The expression of putative IbbHLH3-binding factors, including (TIF)Click here for additional data file.S11 FigThe purified MBP-IbbHLH3 protein were incubated with the GST-fusion proteins , respectively. Bound proteins were precipitated with the anti-GST agarose and further analyzed by western blot using anti-GST antibody and anti-MBP antibody. The input lane represents the protein level of IbbHLH3 before reaction.(TIF)Click here for additional data file.S12 FigThe amino sequence alignment of bHLH group IIIe transcription factors, including AtMYC2/3/4 and IbbHLH3, was analyzed by GeneDoc software. The HLH-domain (blue line) was conserved in bHLH-IIIe transcription factors. The putative phosphorylation site in IbbHLH3 was labeled as red underline.(TIF)Click here for additional data file.S13 FigThe amino sequence alignment of bHLH group IIId transcription factors, including AtJAM1/2/3 and IbbHLH4, was analyzed by GeneDoc software. The HLH-domain (blue line) was conserved in bHLH-IIId transcription factors, expect for AtbHLH14.(TIF)Click here for additional data file.


Although it was nearly 70 years ago when transposable elements (TEs) were first discovered “jumping” from one genomic location to another, TEs are now recognized as contributors to genomic innovations as well as genome instability across a wide variety of species. In this review, we illustrate the ways in which active TEs, specifically retroelements, can create novel chromosome rearrangements and impact gene expression, leading to disease in some cases and species-specific diversity in others. We explore the ways in which eukaryotic genomes have evolved defense mechanisms to temper TE activity and the ways in which TEs continue to influence genome structure despite being rendered transpositionally inactive. Finally, we focus on the role of TEs in the establishment, maintenance, and stabilization of critical, yet rapidly evolving, chromosome features: eukaryotic centromeres. Across centromeres, specific types of TEs participate in genomic conflict, a balancing act wherein they are actively inserting into centromeric domains yet are harnessed for the recruitment of centromeric histones and potentially new centromere formation. Transposable elements (TE) are segments of DNA that can move, or transpose, within the genome. The existence of elements capable of intragenomic mobility was first discovered in maize by American scientist Barbara McClintock in the 1940s and described in her seminal 1950 paper , including retroviruses, and non-LTR elements. While there is no evidence for DNA transposon activity in humans in the past 50 million years   consists of a 5′ UTR with a bidirectional RNA polymerase II promoter, two open reading frames (ORF-1 and ORF-2), a 3′ UTR, and a polyadenylation signal followed by a poly-A tail (Fanning and Singer A full-length Alu (~ 300 bp) is derived from the signal recognition particle RNA 7SL (Ullu and Tschudi Alu) element (~ 2–3 kb) is a composite unit  gene, the 3′ LTR of HERV-K10, and a polyadenylation signal followed by a poly-A tail  pol-pro encoding the enzymes: protease, RT, and integrase; and (3) env encoding proteins allowing for horizontal transfer  is comprised of ancient remnants of endogenous retroviral sequences Ono  and inclDespite the observation that some mobile elements are still capable of encoding for proteins that facilitate mobility, it is the RNA transcript of a retroelement that is an integral component of its transposition via reverse transcription. For example, LINE1 is transcribed in the nucleus, after which both nascent LINE1 RNA and its translated protein form a ribonucleoprotein protein complex (RNP) in the cytoplasm. The RNP complex migrates back into the nucleus, where the ORF2 protein, containing endonuclease (EN) activity, makes a nick in genomic DNA at an insertion site. ORF2 also encodes for RT, which converts the RNA to DNA via target primed reverse transcription (TPRT). The result of this RT-mediated movement is the insertion of a full-length, or often 5′ truncated, LINE1 into the genome in a novel location   is a prime example of the use of RNAi as a primary mechanism for silencing. In C. elegans, Tc1 elements (a type of transposon) give rise to dsRNAs, which are cleaved into siRNAs that can mediate post-transcriptional degradation of the target TE transcript  mechanism. TE transcription can result in the formation of double-stranded RNAs (dsRNAs), which have been shown to trigger RNAi in a wide variety of organisms  , the mispairing of two stretches of highly similar DNA sequences, such as similar TEs  in mouse). While satellites appear virtually ubiquitous in regional centromeres that are fixed within species  of a latent centromere site, an evolutionary breakpoint associated with previous centromere activity and the potential for new centromere formation  have revealed much about the non-coding regions of the human genome interspersed across chromosome arms. While these advances have led to breakthroughs in understanding the genomic landscape for 80–90% of the human genome, the complex repeat structure of centromeres has relegated these chromosome regions to the last frontier of the human genome. Despite this, a recent and remarkable computational effort has led to the production of graphical models of human centromere sequences Miga , bypassiThe finding that human centromeres contain retroelements is not simply a recent discovery. Indeed, the first centromere-pericentromere boundary sequenced for human, the X chromosome, revealed that not only are retroelements present throughout, there was evidence that older elements resided farther from the core of the centromere, while recently inserted, and in some cases still active, retroelements were found within the higher order array of the centromere core . Interestingly, satellite DNA arrays were found that consist of TIR-220 repeats, thus demonstrating the potential ability of a TE to contribute to the formation of satellite arrays through the production of internal tandem repeats via its foldback mechanism . Recent work in two Arabidopsis species in which centromere-enriched retroelements are found indicates that specific TEs preferentially insert into centromeric regions. The ATCOPIA93 retroelement was found in low copy number scattered throughout the genome in A. thaliana, whereas retroelements related to ATCOPIA93 in A. lyrata displayed a high copy number specifically enriched in the centromeric regions , was transformed into A. thaliana to test whether this TE would preferentially insert into the centromere regardless of host genome environment. Whole-genome sequencing following transformation indicated that (1) new Tal1 insertions were found in the centromeric satellite arrays of the A. thaliana genome and (2) the sequences flanking the inserted elements were biased towards these centromeric satellite arrays. At face value, it would reason that the Tal1 TE targets centromeric regions by recognizing satellite arrays specifically. However, the satellite sequences between these two species share only ~ 70% identity . Following the formation of these neocentromeres, an invasion of CR2s, a centromere-specific retroelement, followed at a frequency that established CR2-rich neocentromeres  elements are actively transcribed  under strong, constitutive promoters juxtaposed to the α satellite arrays. Notably, centromere function of the HAC is reliant on transcriptional activity of these markers  has shown that while TE-free satellite arrays can support centromere function, active transcription is still a requisite for the stable propagation of the HACs (Bergmann et al. It is clear that centromeres are rapidly evolving and thus are found in nature at many different points along their lifecycle. At each point, we observe their dynamic nature as well as their intimate relationship with TEs in their host genome. Upon initial formation as a neocentromere following some chromosomal insult or rearrangement, a single TE may serve to initiate centromere histone recruitment via its nascent transcription. As centromeric histones spread to form a centromere within an equilibrium state, a complex centromere evolves that is characterized by retroelement insertions and the evolution of divergent satellites from newly inserted centromeric TEs Fig. . The gen


Accordingly the expression of these genes is elevated in gfi1b mutant and LSD1 inhibited hematopoietic cells, while during megakaryocytic differentiation, declining Gfi1b levels fostered the reciprocal upregulation of these cytoskeletal factors. Concordantly, manipulation of Kindlin3 and Talin1 expression demonstrated positive correlation with megakaryocytic differentiation with over-expression stimulating, and inhibition diminishing, this process. Co-operativity between these factors and integrins in promoting differentiation was further underscored by physical interactions between them and integrinβ3/CD61 and by stimulation of differentiation by the Talin1 head domain, which is necessary and sufficient for integrin activation. Therefore this study demonstrates the significance of Gfi1b regulated Kindlin3-Talin1 expression in driving megakaryocytic differentiation and highlights the contribution of cytoskeletal agents in the developmental progression of these platelet progenitors.Understanding the production and differentiation of megakaryocytes from progenitors is crucial for realizing the biology and functions of these vital cells. Previous gene ablation studies demonstrated the essential role of the transcriptional repressor Gfi1b (growth factor independence 1b) in the generation of both erythroid and megakaryocytic cells. However, our recent work has demonstrated the down-regulation of this factor during megakaryocytic differentiation. In this study we identify two new gene targets of Gfi1b, the cytoskeletal proteins Kindlin3 and Talin1, and demonstrate the inverse expression and functions of these cytoskeletal targets relative to Gfi1b, during megakaryocytic differentiation. Both  Gfi1b has also been implicated in lymphoid development  may may20] mCuriously, our Talin1 over-expression experiments demonstrate that the Talin1 head domain is relatively more potent than the full-length protein in promoting megakaryocytic differentiation. Since the THD is known to activate integrin β3 by tilting its transmembrane domain (TMD) , 37, ourBoth in platelets and in megakaryocytes, increased expression of integrins coupled with their heightened “inside out” activation upon association with abundant Kindlin3 and Talin1 then enables high affinity multi-meric ligand binding. Ligand engagement in turn produces “outside-in” integrin signaling which activates downstream cascades like the PI3K (phosphatidyl inositol 3-OH kinase)/Akt pathway . In megaof one or more of the most significant interactions and/or protein mediators, identified by these analyses.Curiously, our recent screen for Rgs18 (another robust gene target of Gfi1b which potently stimulates megakaryocyte differentiation) interacting proteins has uncovered extensive associations of this GAP (GTPase activating protein) with Kindlin3, Talin1, Rap1 (Ras-proximate-1) and multiple integrins and integrin associated proteins in megakaryocytes . Since Rap1, a Ras-like small GTPase and its associated protein RIAM (Rap1-GTP interacting adaptor molecule) are important for recruiting Talin1 to integrinβ tails , 42, theThese studies could produce a wealth of information on both megakaryocyte differentiation and platelet aggregation thus providing not only a more complete picture of these vital processes but also revealing more avenues for regulating them in disorders caused by their hypo- or hyper-activity or dysfunction. Moreover, since Kindlins, Talins and Integrins are expressed in a wide spectrum of cells, insights into their co-operativity and coordinated functions should be relevant in other developmental contexts and in diseases associated with the dysfunctions of these proteins or their paralogs in these tissues. Remarkably Talin1 is known to be significantly up-regulated in metastatic cells of several cancers such as prostate cancer, breast cancer, hepatocellular carcinoma etc. –45. TherS1 FigA. Murine talin1 promoter sequence (Accession # JN945242). Sequence included in promoter construct driving luciferase expression is indicated in black. Exon1 sequence is shown in bold letters with the transcription start site (tss) underlined. Primers used for ChIP qPCR are underlined and the putative Gfi1b binding sites shaded. Tln1p1 represents the 5’ and Tln1p2 the 3’ amplicons in B. Murine kindlin3 promoter sequence (Accession # JN958519). Sequence included in promoter construct is indicated in black. The initiator ATG is underlined. Primers used for ChIP qPCR are underlined and the putative Gfi1b binding site is shaded. The sequence is numbered relative to the tss (not shown).(PDF)Click here for additional data file.S2 FigA. Gfi1b, LSD1 and β-actin protein levels in immature (U) and mature (I) megakaryocytes  (left panel) and erythroid  cells (right panel). B. Time course of GPIIb, Gfi1b and Rgs18 (another Gfi1b target) message expression in fetal liver cells differentiated in culture into megakaryocytes (1° meg diff). These figures were reproduced from [ced from .(PDF)Click here for additional data file.S3 FigA. Kindlin3, Talin1 and CD61 protein expression in megakaryocytes. Western blot of Kindlin3, Talin1 and CD61 expression in uninduced (Un) and induced (In) L8057 cells. 60 μg of total protein was loaded per lane. B. Model of regulation of megakaryocytic differentiation by Gfi1b, Kindlin3, Talin1 and their downstream effectors.(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


RUNX3 single nucleotide polymorphisms (SNPs) associated with ankylosing spondylitis (AS).To explore the functions of Individual SNP associations were evaluated in 4230 UK cases. Their effects on transcription factor (TF) binding, transcription regulation, chromatin modifications, gene expression and gene interactions were tested by database interrogation, luciferase reporter assays, electrophoretic mobility gel shifts, chromatin immunoprecipitation and real-time PCR.rs4265380, which was robust (P=4.7×10−6) to conditioning on another nearby AS-associated RUNX3 SNP (rs4648889). A RUNX3 haplotype incorporating both SNPs was strongly associated with AS . In a large UK cohort, rs4265380 is associated with leucocyte counts (including monocytes). RUNX3 expression is lower in AS peripheral blood mononuclear cells than healthy controls (P<0.002), independent of rs4265380 genotype. Enhancer function for this RUNX3 region was suggested by increased luciferase activity  for reporter constructs containing rs4265380. In monocytes, there was differential allelic binding of nuclear protein extracts to a 50 bp DNA probe containing rs4265380 that was strongly augmented by lipopolysaccharide activation. TF binding also included the histone modifier p300. There was enrichment for histone modifications associated with active enhancer elements (H3K27Ac and H3K79Me2) that may be allele dependent. Hi-C database interrogation showed chromosome interactions of RUNX3 bait with the nearby RP4-799D16.1 lincRNA.We confirmed the independent association of AS with RUNX3 regulatory region involves at least two SNPs apparently operating in different cell types. Monocytes may be potential therapeutic targets in AS.The association of AS with this  RUNX3, a transcription factor involved in the maturation of several immune cell types.From genome-wide association studies, it appears that more than 100 genes are involved in susceptibility to ankylosing spondylitis (AS). One of these is RUNX3, which are strongly associated with AS lie in a putative genomic regulatory region. One of these SNPs, rs4648889, appears to be operative particularly in CD8+ T cells.Several single nucleotide polymorphisms (SNPs) upstream the promoter of RUNX3 region (rs4265380) that may be restricted to monocytes rather than T cells. The independent associations of AS with these two SNPS and the likelihood that they have functional effects in two different cell types suggest that both CD8+ T cells and monocytes could be therapeutic targets in AS. Understanding the functions of this RUNX3 could provide new insights into the pathogenesis of AS.We report potential functional effects from a second independently associated SNP in this By investigating the AS-associated mechanisms controlling RUNX3 transcription and the regulatory effects of RUNX3 itself on downstream gene expression, we expect to identify pathways that include credible therapeutic targets for future drug development.ERAP1 and IL23R.IL23R-IL12RB2 intergenic region, where they may influence gene expression.7–10Although genetic predisposition to ankylosing spondylitis (AS) is strongly associated with the human leukocyte antigen (HLA)-B27 immune response gene genome-wide association studies (GWAS) have clearly shown that it is polygenic.RUNX3 (Runt-related transcription factor 3) have previously been demonstrated by GWAS.RUNX3 promoter are strongly associated with AS (lead SNP rs6600247: P=1.3×10−14).rs4648889 within this block reduces recruitment of the interferon regulatory factor 4 (IRF4) transcription factor (TF), leading to reduced RUNX3 expression in CD8+ T cells in an allele-dependent fashion.18 19Convincing associations between AS and SNPs upstream of RUNX3 with AS. Having previously established the strong association of AS with rs4648889 in this region (P=1.3×10−14),rs4265380. We characterise some of the functional effects of this SNP and conclude that these two adjacent but independent signals probably exert their effects in different cell types.Here, we present evidence for the complexity of both the molecular and cellular associations of rs4265380 using TaqMan SNP assay .Historical genotype data from the UK subset of cases included in the AS Immunochip study (n=4230) and ethnically matched healthy controls (n=9200) were used in the association studies.Following written informed consent (COREC 06/Q1606/139 and OXREC B 07/Q1605/35), venous blood samples were obtained from 17 cases fulfilling either the modified New York criteria for AS or the  Assessment in SpondyloArthritis international Society  (ASAS) imaging criteria for axial spondyloarthropathy.rs4265380 was performed as previously described.Cloning of the 250 bp sequence flanking 6/mL in prewarmed Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum, penicillin/streptomycin and L-glutamine and rested overnight.CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using a monocyte isolation kit . Monocytes were resuspended at 1×105) were stimulated for 24 hours with or without ultrapure lipopolysaccharide  before harvesting for experiments.Monocytes (4×10rs4265380 was incubated with nuclear extracts obtained either from primary CD14+ monocytes stimulated with LPS for 24 hours or a monocyte cell line from histiocytic lymphoma (U937) stimulated with phorbol-12-myristate-13-acetate (PMA). Nuclear extracts from monocytes and U937 cells were prepared using the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction kit . EMSA was performed using LightShift Chemiluminescent EMSA Kit (Thermo Scientific) with no modifications. Single-stranded biotinylated oligonucleotides (prepared with biotin 3′ end DNA labelling kit) were mixed and annealed at room temperature for 1 hour. Five micrograms of nuclear extract and 0.5 ng biotin labelled double-stranded oligonucleotides  were used in each experiment. Unlabelled probes in 100-fold excess were used as competitor.A 50-bp DNA probe including The sequences of the synthetic single-stranded oligonucleotides are listed below:CGTTATGAGTCAGCTCAAGTAA-3′;C* s (sense): 5′-CTCCATGACGCAATTTGGGCTCTGTTATGAGTCAGCTCAAGTAA-3′;T* s: 5′-CTCCATGACGCAATTTGGGCTCGGAGCCCAAATTGCGTCATGGAG-3′;C* as (antisense): 5′-TTACTTGAGCTGACTCATAACAGAGCCCAAATTGCGTCATGGAG-3′.T* as: 5′-TTACTTGAGCTGACTCATAACrs4265380). and qPCR performed on immune complex-associated DNA using allele-specific primers for rs4265380 , detected with SYBR Green (Qiagen Ltd) on ABI ViiA 7 PCR device .ChIP was performed using the Low Cell# ChIP kit  as previously described.Relative occupancy was calculated as a ratio of specific signal over background: % input (specific loci)/% input (background loci). The following ChIP grade antibodies were used: p300 ((C-20), SC585X, Santa Cruz Biotechnology, Dallas, USA), H3K4Me1 , H3K79Me2 , H3K27Ac  and IgG .RNA was isolated with TRIzol  and reverse transcribed with Superscript III (Invitrogen) to synthesise cDNA as previously described.PCRs were performed in triplicate and the 2−ΔCt method was used to calculate the expression of each gene. All values were normalised to β-actin .miRNA quantitative real-time RT-PCR miRNeasy Mini Kit (Qiagen Ltd) was used to isolate total RNA, according to the manufacturer’s instructions. Ten nanograms of RNA was reverse transcribed and quantified by TaqMan real-time PCR using the TaqMan microRNA Assay . RNU6 was used as endogenous control . For detection, we used a vii7 PCR machine (Applied Biosystems).https://genome.ucsc.edu/ENCODE/) and Roadmap Epigenomics (http://epigenomegateway.wustl.edu/browser/roadmap/) projects and previously published GWAS association dataRUNX3 region.https://www.chicp.org/chicp).RUNX3 transcription in AS cases and controls was evaluated from historical data derived from RNA-seq in PBMCs from 72 AS cases and 62 healthy controls.19We used publicly available data from the ENCODE  package.Association analysis was performed using the logistic regression function in PLINK,RUNX3 contains potentially important regulatory sequences. However, the strongest association with AS in this region is with rs6600247 (P=4.23×10−9) that does not overlap with obvious functionally active sites. We therefore investigated SNPs in strong LD with rs6600247 that lie in a region associated with open chromatin for evidence of regulatory activity; these include rs4648889  was the most strongly associated  after conditioning on rs6600247 (lead IGAS SNP) or rs4648889  DNase I hypersensitivity sites; (2) H3K4Me1, H3K27Ac and H3K79Me2 histone modifications that are associated with regulatory regions and replication initiation sites and (3) ChIP sequencing peaks for TF binding, particularly p300 . This region, therefore, appears to contain a potential regulatory element upstream of RUNX3 specifically active in monocytes, in which the C allele at rs4265380 increases the risk of AS.The region upstream of s4648889 . Interros4648889  shows thrs4265380 and rs4648889—that were both independently associated with AS in a haplotype analysis (−8) compared with the most common CG haplotype. The OR of CA relative to TA is 5.35 (P=2.7×10−7).We included the two SNPs—analysis . Both SNhttp://geneatlas.roslin.ed.ac.uk/), a public database including more than 400 000 individuals (from the UK Biobank), to investigate a plausible role for rs4265380 associations with leucocyte cell counts. The results for rs4265380 showed a significant reduction in monocyte (P=0.006), neutrophil (P=6.4×10−7) and eosinophil (P=8.3×10−7) counts associated with the C allele. No association was apparent for lymphocyte counts (P=0.2), consistent with our hypothesis that rs4265380 is acting specifically in monocytes.We interrogated the Roslin Gene Atlas (RUNX3 mRNA in PBMCs measured by RNA-seq showed significantly reduced expression (P<0.002) in AS cases compared with healthy controls. When these results were stratified on rs4265380, there was no apparent influence of this SNP on expression levels  compared with the minimal promoter alone, which was consistent with its having enhancer activity. Since there was no apparent difference between constructs containing either the rs4265380 C (risk) or T (protective) allele in this system  and also the SYF2 locus, which is 240 kb upstream of the RUNX3 promoter. By interrogating the Capture Hi-C plotter database (https://www.chicp.org/chicp),RUNX3 bait (overlapping rs4265380) and other regions of chromosome 1, specifically with RP4-799D16.1 and miR4425, but not with the more distant SYF2 locus (SYF2 (data not shown). rs4265380 AS-risk C allele alters enrichment for specific histone marks in stimulated monocytes.The F2 locus . ExpressF2 locus . miR-442We assessed the enrichment of specific histone marks associated with active chromatin in this region by ChIP-qPCR (n=3) on LPS-stimulated CD14+ monocytes. There was relative enrichment for H3K79Me2  and H3K27Ac  in the presence of the C (risk) allele online , while Hrs11209026, encoding an R381Q loss of signalling function dimorphism in the cytoplasmic tail of the IL23 receptor, highlights the importance of the IL23/IL17 axis in AS.et al identified 113 possible non-HLA-B27 AS associated loci, of which only 13 were exonic.RUNX3 locus is unlikely to be an exception.Finding mechanistic explanations for the multiple genetic associations of common diseases, such as AS, is challenging. A small minority of such SNP associations alter protein function that have yielded insights with potential therapeutic implications. Thus, the AS association with rs4648889, an AS-associated SNP in this regulatory region upstream of RUNX3, influenced TF binding and gene transcription in CD8+ T cells.RUNX3 association with AS. Although rs4265380 is only ~500 bp from rs4648889, not only is its association with AS independent of other AS-associated SNPs in this region but also it likely acts in a different cell type. DNA sequences in the vicinity of rs4265380 show regulatory activity (demonstrated by luciferase reporter activity in vitro) and allele-specific effects on TF binding and histone modifications that could potentially alter gene expression. The relevant TF/DNA complexes include p300, a histone acetyltransferase associated with open chromatin that is involved in transcriptional activation.35Previously, we showed that rs4265380 association with AS is probably explained by operational differences in monocytes rather than CD8+ T cells. First, interrogation of epigenetic databases shows relevant areas of open chromatin, TF binding and enrichment for chromatin markers of active transcription limited to monocytes. Second, the allele-specific effects on TF binding appear to be restricted to nuclear extracts from monocytes (particularly after stimulation with LPS).Our data suggest that the rs4265380 was allele specific, but ChIP in heterozygous C/T monocytes from AS cases indicates that p300 actually binds to both rs4265380 alleles in the context of native chromatin. The allelic difference we observed in EMSA is unlikely to be driven by p300 alone but more likely by a multicomponent TF complex recruiting the relevant transcriptional machinery. It is not yet clear whether these findings translate to influences on the expression of nearby genes, including RUNX3. The complexity of the RUNX3 associations and their multiple potential physical chromosomal interactions is further highlighted by the possible interaction of RUNX3 bait (including rs4265380) with a neighbouring non-coding RNA (RP4-799D16.1 lincRNA) of unknown function  and monocytes in AS. However, substantial further work will be necessary to confirm this.In summary, we have identified a regulatory region upstream of


CCCTC-binding factor (CTCF) is largely responsible for the 3D architecture of the genome, in concert with the action of cohesin, through the creation of long-range chromatin loops. Cohesin is hypothesized to be the main driver of these long-range chromatin interactions by the process of loop extrusion. Here, we performed ChIP-seq for CTCF and cohesin in two stages each of T and B cell differentiation and examined the binding pattern in all six antigen receptor (AgR) loci in these lymphocyte progenitors and in mature T and B cells, ES cells, and fibroblasts. The four large AgR loci have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the end of each locus near the enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus that also shows significant lineage-specificity (T vs. B cells) and developmental stage-specificity (pre-B vs. pro-B) in CTCF binding. We show that cohesin binding shows greater lineage- and stage-specificity than CTCF at most AgR loci, providing more specificity to the loops. We also show that the culture of pro-B cells in IL7, a common practice to expand the number of cells before ChIP-seq, results in a CTCF-binding pattern resembling pre-B cells, as well as other epigenetic and transcriptional characteristics of pre-B cells. Analysis of the orientation of the CTCF sites show that all sites within the large V portions of the Igh and TCRβ loci have the same orientation. This suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D-J-enhancer end of each locus. The V region portions of the Igκ and TCRα/δ loci, by contrast, have CTCF sites in both orientations, providing many options for creating CTCF-mediated convergent loops throughout the loci. CTCF/cohesin loops, along with transcription factors, drives contraction of AgR loci to facilitate the creation of a diverse repertoire of antibodies and T cell receptors. The evolutionarily conserved, ubiquitously expressed zinc finger protein CCCTC-binding factor (CTCF) plays many important roles in gene activation and/or gene repression . Many yeCohesin is often found bound at CTCF sites genome wide –18, and −CD8− “double-negative” (DN) thymocyte stage, at which time the TCRβ, TCRγ, and TCRδ rearrangements all take place. If both productive TCRγ and TCRδ rearrangements occur, the cell differentiates into a γδ T cell. However, if a productive TCRβ rearrangement precedes productive rearrangement of both the TCRγ and TCRδ loci, the DN thymocyte progenitor will differentiate into a double-positive (DP) thymocyte, at which stage the TCRα genes undergoes rearrangement and the DP thymocyte will then differentiate into an αβ T cell  genes in T and B cells occurs at precisely defined stage of lymphocyte development . The proβ T cell .in situ hybridization (3D-FISH) that the Igh locus has a rosette-like structure made by multiple long-range interactions , in order to assess the extent of lineage- and stage-specificity of CTCF and cohesin binding at AgR loci. Although much of the CTCF binding in the large V region part of the AgR loci is lymphoid specific, our data presented here show that the extent of lineage (T vs. B lineage precursors) and developmental stage-specificity  of CTCF and cohesin binding varies significantly from locus to locus, with the Igκ locus being the only one of the 6 AgR loci with clear lineage- and stage-specificity of the onset of most CTCF and cohesin binding. Also, we show that the pattern of CTCF and cohesin binding observed in the most mature B or T cell precursor population is maintained in mature B or T cells, respectively. MEFs show many fewer sites of binding while ES cells are intermediate. In contrast to the large V gene portion of each locus, the CTCF sites between the V and D/J genes and near enhancers tend to display conserved CTCF and cohesin binding among all of the cell types examined including non-lymphocytes. We hypothesize that the acquisition of binding of many CTCF/cohesin complexes within the V gene portion of each of the four large AgR loci establish many long-range loops, and provide a semi-contracted locus upon which transcription factors can act to direct full locus contraction so that V genes throughout each locus will have approximately equal opportunity to rearrange to create a diverse set of V(D)J rearrangements.+ cells from BM of RAG1−/− mice are pro-B cells. All CD19+ cells from BM of RAG−/− mice bearing a rearranged IgM transgene are pre-B cells  cells, and thymocytes from RAG−/− mice bearing a Vβ transgene are all at the subsequent stage of “double-positive” (DP) cells. IL7-cultured pro-B cells were obtained from day 7 cultures of RAG1−/− pro-B cells cultured in media containing supernatant from J558 cells stably expressing IL7 as previously described J rearrangements that will result in unique deletions in each individual B or T cell precursor. RAG deficiency also provides a precise genetic block in differentiation, so the cell populations are free of contamination from later stages of differentiation. All CD19-B cells . Likewisescribed . All micChIP was done with antibodies against H3K4me3 , H3ac , and H3K4me1 (Abcam) as previously described . PrimersRNA was extracted from cells using TRIzol , and cDNA was made with QuantiTect Reverse Transcription kit, and genomic DNA was elminated using genomic DNA wipeout buffer .−/− progenitors and the ChIP-seqs were from GEO GSM987801 and GSM987802 . This was done since we know that over half of CTCF sites are constant among different cell types, and that TADs, which are bounded by CTCF sites, are also relatively conserved across cell types , 15.Preliminary quality control over raw sequence data was performed with FastQC 0.11 . DuplicaSignal tracks normalized to 1M reads were generated from alignment (bam) files using MACS2  accordinp-value (1E-5) as specified in Ref.  , with a  in Ref. . Downstr in Ref.  and R/Bi in Ref. . Custom homer.ucsd.edu/homer/data/genomes/mm9.v5.7.zip. Peak overlaps were computed using the HOMER mergePeaks tool .In particular, peaks were annotated with genomic features obtained from RefSeq release 66 through The above pipeline was used to uniformly process all of our ChIP-seq data and raw sequencing data obtained from GEO. For the Rad21 and Smc1 (another cohesin subunit) ChIP-seq on ES and MEF, we used the called peaks provided by the authors.(1)de novo) . InsulatorDB motifs were converted to motif matrices (input for subsequent HOMER processing steps) using HOMER seq2profile tool with 0 mismatches. Motif matrices for HOMER-detected motifs were downloaded directly from the motif finding report. Finally, a custom motif file was created following HOMER guidelines.Assemble a library of CTCF motifs. A total of nine CTCF motifs were considered—five motifs retrieved from InsulatorDB, and four motifs detected in pre-B and pro-B ChIP-seq with HOMER findMotifsGenome.pl. (three known motifs and one (2)Run annotatePeaks.pl (HOMER) on confident ChIP-seq peaks in a specific region (bed file) against the above library of CTCF motifs . This will generate a peak annotation file with instances of CTCF motifs found within the confident peaks in the region.(3)For each peak  annotated to motif(s), assign a (+) (forward) or (–) (reverse) orientation to each motif, by checking the strand on which the motif is detected. If multiple motifs are detected, take the consensus sign. Ambiguous cases are discarded.(4)Using the data in the annotation file, assemble a bed file with strand (orientation) information.In order to assess directionality of CTCF-binding sites, we devised the following procedure:We have found that the common method of analyzing ChIP-seq data by mapping only unique reads is not appropriate for AgR loci which have arisen by extensive gene duplication of individual V genes. It is especially important for the TCRα/δ locus, which has a relatively recent triplication of a large portion of the locus in C57BL/6 mice, the genome on which all of these data are mapped . This beTo determine the lineage and stage-specificity of CTCF and cohesin binding, we performed ChIP-seq for CTCF and Rad21 (a subunit of cohesin) for two sequential stages in B lymphocyte differentiation (pro-B and pre-B) and two stages in thymocyte differentiation (DN and DP). We compared these data to published data for binding in ES cells and MEFs. The four largest AgR loci  have a similar organization with a large number of V genes spread over a major percentage of the loci, followed by the (D)J-C genes tightly clustered at one end of the locus. The much smaller Igλ and TCRγ loci have a very different organization and many fewer V genes, and will be discussed later. All four large AgR loci have 1–4 CTCF bound sites between the V genes and the D and/or J genes, and the majority of these sites in the Igh, Igκ, and TCRα/δ loci are bound by CTCF and cohesin not only in all four lymphocyte subsets but also in MEF and ES cells. Nevertheless, in some cases, such as Cer and Sis in the Vκ-Jκ intervening region , 55, onl−/− early progenitors) show many fewer bound CTCF peaks, suggesting that entry into the B progenitor stage is important for the initiation of CTCF binding in Ig loci. Only the Igκ locus shows marked lineage- and stage-specificity for CTCF binding.In contrast to the fairly uniform binding of CTCF/Rad21 in the D-J-C regions among diverse cell types, CTCF and Rad21 binding within the large V portion of these loci shows greater lymphocyte specificity  of CTCF and cohesin binding at these sites. Often binding was highest in the developmental stage during which rearrangement normally occurs. The predominant reason for a higher peak at any specific location in one cell type vs. another is that a larger proportion of the cells used for the ChIP-seq lysate had CTCF (or Rad21) bound at that site. Within a locus, one reason for different heights of the various CTCF peaks could also be that a different proportion of cells had CTCF bound at the different sites, or it could reflect differences in the binding strength of CTCF to the individual sites, since the motif for CTCF is quite variable .We examine each of the four large AgR loci separately in the sections below. The analysis of the two smaller AgR loci that do not have the same V, D, and J gene organization pattern will be presented later.The Igh locus rearranges in the pro-B cell stage. T cells display some Dh-Jh rearrangements which may have occurred before the B and T cell lineages have bifurcated from their common progenitor . The 121The Igκ locus rearranges in pre-B cells and displays maximal CTCF and cohesin binding at that stage Figure . Only on−/− pro-B cells resembles that of pre-B cells, not pro-B cells, in that all of the binding sites in the proximal half of the locus are now strongly binding CTCF  , 64. TheTwo of the large AgR loci, Igh and TCRβ, have rather simple CTCF orientation patterns, with all bound CTCF sites in the large V gene portion of the loci pointing toward the D-J-C regions, and most of the CTCF sites in the D-J-C-enhancer regions oriented toward the V region CTCF sites  Figures and 8A. CTCF sites are located in both orientations in the V part of the locus, although the majority of sites (129/144) are pointing toward the J-C regions Figure B as prevThe Igκ locus has four CTCF sites in between the V genes and the J genes. The set of two sites closest to the V genes, called Cer, plays an important role in locus conformation and regulation, and both CTCF sites are oriented toward the V genes . The setvia a long-range loop between the 2 CTCF sites as indicated in Figure The TCRγ locus is a small locus, encompassing 200 kb, and is composed of three clusters of V-J-C genes, one of which contains only pseudogenes Figure . The maiThe Igλ locus, like the TCRγ locus, is organized in clusters, and V genes only rearrange to J genes within their cluster Figure  71). Th. Th71). The ChIP-seq data presented here show that the CTCF and cohesin bound sites within the four largest AgR loci can be divided into two categories. All four large AgR loci have CTCF sites between V genes and D-J genes, and some have CTCF sites near the enhancers that are usually located downstream of J or C genes and, thus clustered at one end of the locus. With some exceptions, the CTCF sites in these very small D-J-C-enhancer regions of each locus tend to be bound by CTCF in ES cells, and sometimes in MEF, the two non-lymphoid cell types we examined. Some of these CTCF sites have been reported to have insulator function, preventing the spread of active chromatin and keeping germline transcription of proximal V genes low, and deletion of these sites results in preferential rearrangement of proximal V genes , 73. DesBy contrast, the majority of the CTCF sites within the large V gene portion of each of those loci, which comprise >85% of the total size of each locus, show lymphocyte specificity in that very few sites are bound in ES cells or MEFs. However, only the Igκ locus shows both lineage (T vs. B cell) and also developmental stage-specificity (pre-B vs. pro-B) for the majority of its bound CTCF sites. This enhanced specificity of CTCF binding may be because, of the four large loci, this is the only one that does not undergo any rearrangement at the first stage of T or B cell differentiation , and thus pre-B cells are characteristic of a later stage of lymphocyte differentiation. ES cells, although having few CTCF sites bound in the AgR loci, often show more binding than the end-stage differentiated MEFs, suggesting that, just as ES cells often have low level transcription of a wide variety of cell type-specific genes, their CTCF binding, although limited, becomes more restricted as ES cells differentiate into non-lymphoid cells. Since DNA methylation has been reported to inhibit CTCF binding, it is possible that the CTCF sites in the AgR loci might be CpG methylated in non-lymphocytes, and only become demethylated in an early lymphoid progenitor stage –76. HoweImportantly, we show that Rad21, a component of the cohesin complex, shows greater lineage and developmental stage-specificity in general than CTCF for most of the large AgR loci analyzed for both the V portions of the loci as well as for the several of the CTCF sites in the D-J-C-enhancer regions. Since the loop extrusion model proposes that cohesin is critical to the creation of CTCF-mediated loops, the greater developmental specificity of Rad21 binding to AgR loci suggests substantial developmental specificity in AgR looping , 67, 77.Over the past 3 years, it has become clear that the orientation of the non-palindromic CTCF motif is important in the creation of the long-range loops that mediate most of CTCF’s architectural functions as well as its insulator and enhancer-blocking functions , 64, 78.The Igh locus is the subject of the most detailed 3D-FISH analyses. The Igh locus has been demonstrated to be in a rosette-like structure, with three rosettes present in pre–pro-B cells that further contract into one in pro-B cells, the stage at which rearrangement of that locus takes place . It is lThe much smaller TCRβ locus, such as the Igh locus, has all of the CTCF sites within the V region part of the locus oriented toward the D-J gene segments Figure . There a−/− pro-B cells shows many interactions within the Igκ locus, but there is a paucity of interactions from Cer/Sis/iEκ region to a large portion of the proximal half of the locus, from ~Vκ12-46 to Vκ4-83. Although CTCF sites throughout the locus are bound in IL7-cultured pro-B cells, this portion of the locus with the fewest Hi–C interactions within the proximal part of the locus lack any CTCF sites oriented toward Cer/Sis J genes to which one V gene will rearrange. We have previously shown that CTCF is important for the contracted structure found at the Igh locus in pro-B cells, since knock-down of CTCF shows a more extended Igh locus . HoweverIn order to efficiently utilize the large number of V genes in each loci, it seems reasonable that there should be heterogeneity in the long-range looping interactions that bring the V genes close to the (D)J gene such that different V genes are closest to the (D)J gene in the population of precursor lymphocytes undergoing V(D)J recombination. This need for heterogeneity could suggest that long-range loops are constantly forming and reassembling in each precursor lymphocyte to create a structure in which different V genes are closest to the (D)J genes at different times, and there is evidence that CTCF binding, and by inference chromatin looping, is very dynamic . AlternaThis study was carried out under approval of our protocol by The Scripps Research Institute’s IACUC.SL performed all of the bioinformatic analyses. SL, EB-M, and AF analyzed all of the data. EB-M performed some of the ChIP. H-YS and MK provided the data in Figure S2B in Supplementary Material. AF and EB-M prepared the figures. AF, EB-M, and MK wrote the manuscript with input from all the authors, especially SL.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.