Streptomyces coelicolor has long been known to exert a powerful negative influence on the production of the antibiotics actinorhodin, undecylprodiginine and the Calcium-Dependent Antibiotic (CDA). Here we report the analysis of a ΔabsA2 deletion strain, which exhibits the classic precocious antibiotic hyper-production phenotype, and its complementation by an N-terminal triple-FLAG-tagged version of AbsA2. The complemented and non-complemented ΔabsA2 mutant strains were used in large-scale microarray-based time-course experiments to investigate the effect of deleting absA2 on gene expression and to identify the in vivo AbsA2 DNA-binding target sites using ChIP-on chip. We show that in addition to binding to the promoter regions of redZ and actII-orfIV AbsA2 binds to several previously unidentified sites within the cda biosynthetic gene cluster within and/or upstream of SCO3215—SCO3216, SCO3217, SCO3229—SCO3230, and SCO3226, and we relate the pattern of AbsA2 binding to the results of the transcriptomic study and antibiotic phenotypic assays. Interestingly, dual ‘biphasic’ ChIP peaks were observed with AbsA2 binding across the regulatory genes actII-orfIV and redZ and the absA2 gene itself, while more conventional single promoter-proximal peaks were seen at the CDA biosynthetic genes suggesting a different mechanism of regulation of the former loci. Taken together the results shed light on the complex mechanism of regulation of antibiotic biosynthesis in Streptomyces coelicolor and the important role of AbsA2 in controlling the expression of three antibiotic biosynthetic gene clusters.The AbsA1-AbsA2 two component signalling system of  Streptomyces are notable for their ability to undergo morphological differentiation and for their ability to synthesize a wide variety of secondary metabolites. The model streptomycete, Streptomyces coelicolor A3(2), produces several antibiotics, including the non-ribosomally synthesised lipopeptide Calcium Dependent Antibiotic, CDA [i.e. cdaR [redZ [redD, [actII-orfIV, [absA locus was originally identified by isolating mutants unable to synthesize antibiotics but unaffected in their morphological development [absA1 and absA2 which comprise a two-component system, with AbsA1 possessing similarity to other streptomycete antibiotic biosynthetic cluster associated histidine-kinase sensor-transmitter proteins and AbsA2 displaying similarity to DNA-binding response regulator proteins [absA1/2 within the CDA biosynthetic gene cluster was only demonstrated after the determination of the S. coelicolor genome sequence [The bacteria of the genus tic, CDA , the bluicolor A3, produceicolor A3, producetic, CDA  each of .e. cdaR , redZ [6Z [redD,  and actII-orfIV, –9. InducI-orfIV, . The abselopment . Sequencproteins . The locsequence , 13–14.absA mutants by the Champness laboratory, together with suppressor mutants [absA1/2 transcript was more abundant in the original absA1 mutant C542 [absA2 mutant C570, in which the phosphorylated aspartate residue has been changed to a glutamate residue [absA2 is positively autoregulated and that the autoregulatory form is phosphor-AbsA2 [Sequencing of the original  mutants , and int mutants , strongl mutants . The effant C542  which poant C542  and less residue , relativor-AbsA2 .absA1 and absA2 were expressed as a single bi-cistronic leaderless transcript from the promoter upstream of absA1 (P1) [absA2 is also expressed from a second promoter (P2) located inside absA1 [absA2 is consequently higher than that of absA1. Significantly, the relative abundances of the two transcripts appear not to vary throughout growth, or with growth media type [S1 nuclease transcript mapping indicated that sA1 (P1) . Howeverde absA1 , which idia type et al., [cda cluster in J1501 and its derivative mutant absA C542 and detected expression from the promoters upstream of SCO3230 (cdaPSI), SCO3249 (acp), SCO3229 (hmaS) and SCO3215 (glmT) in J1501 but not in the C542 mutant. Expression from the promoters upstream of SCO3225 (absA1) and SCO3224 (a putative CDA resistance gene), was detected in C542 but not in J1501. Significantly, this study revealed little evidence for the regulation of cdaR by AbsA1/2 although more conclusive evidence was found for the AbsA1/2 mediated regulation of redZ [absA locus in regulating actII-orfIV and redD has also been noted [SCO3217 (cdaR), SCO3226, (absA1/2) and SCO3230 (cdaPSI) were unable to show that either form of AbsA2 bound the respective promoter regions, which suggested that in vivo AbsA2 may bind cooperatively with another protein e.g. CdaR [cdaR, redZ and actII-orfIV [Ryding et al.,  investig of redZ , and theen noted . Gel retcda, act and red gene clusters [in vivo AbsA2 ChIP-on-chip study, was conceived to more comprehensively examine the role of AbsA2, particularly with respect to the CDA biosynthetic gene cluster where several promoters have been previously shown to be AbsA2 dependent [The results of McKenzie & Nodwell, (2007) suggest AbsA2 plays a role as a “master” regulator of antibiotic biosynthesis and regulates the SARPs for the clusters . The preependent .Streptomyces coelicolor strain used in this study was MT1110 [−, SCP2− derivative of the wild-type strain 1147 [ΔcdaR mutant is provided in [EcoRI fragment (+442 to +1174 bp) is described. Plasmid maintenance was done in E. coli JM109 , mcrA, relA1, supE44 λ-, Δ(lac proB), [F’ traD36, proAB+, lacIq, Δ(lacZ)M15). The E. coli strain ET12567 (F-dam-13::Tn9 dcm-6 hsdM hsdR recF143 zjj-202::Tn10 rspL136) [dam, dcm and hsd methylation and when containing the conjugative vector pUB307, was used in E. coli/S. coelicolor conjugations [The s MT1110  which isain 1147 . The convided in  where thugations .absA2 deletion mutant was constructed using homologous recombination to replace the wild-type gene with a truncated version containing an internal deletion of 618 bp out of a total of 669 bp. An XbaI site was substituted for the wild-type nucleotides between positions 3538686 and 3539309 [absA2, which terminates translation after incorporation of three amino acids (MIL). A 1,757 bp fragment containing part of the absA2 N-terminal coding sequence and upstream region was amplified by PCR using primers “absA2up forward” and “absA2up reverse” , together with the absA1-absA2 intergenic region and the absA2 ribosome binding site. This insert does not comprise the P2 promoter [BamHI–XbaI fragment into similarly cut pMT3226 [absA2 was under the control of the tandem glycerol-inducible (gyl) promoters. The resultant plasmid pMT3226::3xF-absA2 was used to transform ET12567 (pUB307). This construct, and the parent vector pMT3226 as a negative control, were introduced into MT1110 ΔabsA2 by conjugation [absA2 was confirmed in Apramycin resistant ex-conjugants by PCR using primers “3xFUp” and “3226Down”. The pigmented antibiotic phenotypes of the complemented mutant (MT1110 ΔabsA2 (pMT3226::3xF-absA2)) and non-complemented mutant (MT1110 ΔabsA2 (pMT3226)) strains were determined visually following growth on solid media plates i.e. Mannitol-Soya agar [gyl promoters.For the construction of a p insert  comprisepromoter . The ins pMT3226 , so thatoya agar  and OxoiStreptomyces coelicolor cultures grown on Mannitol-Soya agar [ΔabsA2 (pMT3226::3xF-absA2)) and ΔabsA2 (MT1110 ΔabsA2 (pMT3226)) strains were set up in parallel. Pre-germinated spores were used to inoculate two-litre siliconized spring flasks containing 500 ml of modified YEME [450 of 0.06. The modified YEME comprised 10% sucrose instead of 34% and glucose was replaced by glycerol. Apramycin (50 μg/ml) and 0.01% Antifoam 204 (Sigma) were also added. The cultures were incubated at 30ºC, with shaking (200 r.p.m) and growth was monitored by following the O.D.450. Samples were taken for transcriptomic, ChIP-on-chip and pigmented antibiotic assays at 14, 18 and 35 h after inoculation.Spores were produced from confluent lawns of oya agar  and wereied YEME  to give ΔabsA2 (pMT3226::3xF-absA2) and ΔabsA2 (MT1110 ΔabsA2 (pMT3226)), which are shown in Replicate small-scale cultures designed to illustrate the pigmented antibiotic phenotypes of MT1110 et al. [For transcriptomic studies 10 ml samples of culture were harvested as described in . For ChIet al.  and diviet al., [et al., [et al., [et al., [et al., [CDA bioassays were conducted according to the method of Chong et al., . Actinor[et al.,  modified[et al., , whilst [et al., , modifie[et al., .mirVana miRNA Isolation Kit” instructions. The quality of the RNA obtained was assessed by running a Bioanalyzer Prokaryote Total RNA Nano chip (Agilent) and only RNA having a RNA Integrity Number of 6.5 or above was used in subsequent work. Protocols for S. coelicolor MT1110 genomic DNA isolation and labelling (Cy5), together with the labelling of cDNA (Cy3) were as previously described [Streptomyces whole genome microarrays [For this study a protocol, based on the standard protocol “Total RNA extraction by Tissue Lyser”, described previously ,was deveescribed  and hybrescribed  using 2 roarrays . The traStreptomyces microarrays, as previously described by Lewis et al., [i.e. the sample processed in the same way as the antibody immunoprecipitated chromatin, but without use of the specific antibody. To compensate for any dye bias in the experiment replicate hybridisations were conducted on different arrays with chromatin samples labelled in opposite Cy-dye orientations. The ChIP-on-chip microarray data are deposited with ArrayExpress (Accession Number:- E-MTAB-3527).Protocols for the chromatin extraction and immunoprecipitation techniques for the ChIP-on-chip experiment were as previously described  with the et al., . The ant2 values were scale-normalized using the “normalizeMedianAbsValues” function in R Bioconductor package Limma [Pre-processing. Microarray image files were scanned and imported into the Agilent Feature Extraction software (v9.1) for image analysis and resulting files were processed in R. Logge Limma –35. Probge Limma . In eachΔabsA2) mutant sample in both replicates. Candidate probes with a fold-change  larger than 1.0 standard deviation from the mean fold-change across all candidate probes were defined as AbsA2-enriched probes. These probes were then clustered based on their genomic position and 1 kb contiguous segments in which at least two enriched probes were identified were considered to be ‘regions of AbsA2 binding’, and thus of interest. Lists of genes comprising such regions of binding are provided in Candidate probes for AbsA2 enrichment were defined as those for which their value in the complemented mutant (3xF) sample is larger than their value in the non-complemented  antibiotic phenotype on MT1110 with regard to CDA, ACT and RED biosynthesis, [ΔabsA2 (pMT3226) over-expressed ACT and RED relative to the wild-type strain on a number of different solid media, whilst the complemented mutant (MT1110 ΔabsA2 (pMT3226::3xF-absA2)) exhibited a hyper-repressed pigmented antibiotic phenotype (ΔabsA2 (pMT3226::3xF-absA2), exhibited a repressed pigmented antibiotic phenotype relative to the non-complemented mutant ΔabsA2 (MT1110 ΔabsA2 (pMT3226))  was not reversed by introduction of the N-terminally triple-FLAG tagged version of absA2. We will return to discuss this below.Surprisingly the CDA over-production phenotype,  seen in i.e. before the onset of pigmented antibiotic production, when we would predict AbsA2 would be bound to the known targets cdaR, redZ and actII-orfIV), at late exponential phase (18 h) which correlated with the onset of RED biosynthesis  when we might expect AbsA2 binding of actII-orfIV only and stationary phase (35 h) when ACT biosynthesis had begun  when we would predict AbsA2 binding of all three known targets would be abolished. This sampling methodology meant that the cultures were sampled before full development of their pigmented antibiotic phenotypes, as results of the pigmented antibiotic assays on the liquid cultures indicate  absA2 transcriptomic study indicated that, as expected from the results of the antibiotic phenotypic assays, a large number of antibiotic biosynthetic genes were significantly differentially expressed in the 3xFabsA2 strain relative to the ΔabsA2 strain.The results . of the ΔabsA2 strain relative to the 3xFabsA strain (At stationary phase (35 h) sixteen genes of the actinorhodin biosynthetic gene cluster are up-regulated in the A strain  Tables wA strain  and the A strain .red biosynthetic gene cluster are represented amongst the significantly differentially expressed genes was expected given the similarity of the RED phenotypes of both the 3xFabsA2 and ΔabsA2 strains in liquid culture, , whilst nineteen cda genes are up-regulated in the 3xFabsA2 strain relative to the ΔabsA2 strain at the late-exponential phase time-point (18 h) (SCO3226 (absA2) whose differential expression was expected due to its deletion from the ΔabsA2 strain, rather than due to bona fide changes in its expression.The results of the CDA bioassay are consistent with the results of the transcriptomics experiment which demonstrate that many genes of the t (18 h) . Consideet al., [SCO3220-3224 (cda genes) none of these genes appear in the list of significantly differentially expressed genes.When the results of the transcriptomic study are compared with the AbsA2 “network module” of Castro-Melchor et al.,  with theSCO7717 which is up-regulated in the complemented ΔabsA2 strain relative to the non-complemented ΔabsA2 strain at the 18 h time-point. As cdaR is similarly up-regulated at this time-point this is consistent with CdaR being a positive regulator of SCO7717.The same study  also desi.e. regulation of antibiotic biosynthesis. Whilst we cannot exclude the possibility that AbsA2 does regulate these genes, an in depth visual analysis of the chIP binding data, in combination with the expression patterns, and knowledge of gene function, has been unable to establish meaningful correlations between AbsA2 binding and gene expression/function for the majority of these genes. We therefore confine further discussion/analysis of the results to known, validated AbsA2 targets  and to genes of the cda cluster, based on the results of a previous absA2 deletion study [To investigate whether the significantly differentially expressed genes  were diron study .cda genes it was unclear whether these effects were directly, or indirectly, mediated by AbsA2, therefore, the results of the ChIP study relating to the cda cluster were of particular interest. Indeed, the ChIP data indicated that AbsA2 does bind to several sites within the cda cluster, binding to a region upstream of SCO3217 (cdaR), to the intergenic region of SCO3215 (glmT) and SCO3216 (ATPase), to the intergenic region between SCO3229 (hmaS) and SCO3230 (cdaPSI), and lastly to another region within SCO3226 (absA2).Although the transcriptomic results indicate AbsA2 exerts a strong effect on expression of the SCO3215-SCO3216 intergenic region which comprises two divergent promoters , and presumably a consequential decrease in production of forms of CDA comprising methyl-glutamate, is unclear.The AbsA2 ChIP peak which can most obviously be linked to a change in expression of a particular gene at a certain time-point is the stationary phase peak seen in the romoters . This reSCO3229 (hmaS)–SCO3230 (cdaPSI)—intergenic region. Although there are AbsA2 ChIP peaks present at this locus at both the 14 and 18 h time-points in the 3xFabsA2 strain  .The presence of a strong ChIP peak upstream of SCO3217)  was expeSCO3230 and SCO3217 is consistent with the regulatory model proposed by Ryding et al., [that the AbsA2 protein directly regulates the cda biosynthetic promoters and in its negatively regulating form overrides any positive regulation that may be exerted by cdaR” and they go on to suggest that “the negative regulatory effect of AbsA2 on CDA synthesis may serve to modulate cda expression in the wild-type, perhaps in competition with cdaR putative activation”.The situation observed here with regard to  et al.,  who suggcdaR and cdaPSI promoters the triple-flag tagged version of AbsA2 present in the complemented mutant is able to bind DNA in a normal wild-type fashion giving rise to the AbsA2 ChIP peaks observed. However, we suggest that unlike the wild-type AbsA2 the tagged version of the protein is unable to interact with, and repress, the transcription-activating effect of CdaR presumably due to the presence of the N-terminal triple-flag tag. Therefore, although we see clear evidence of AbsA2 binding at these promoters we do not see AbsA2 mediated transcriptional repression, as presumably CdaR is still active. This model satisfactorily explains the non-complementation of the pha phenotype with regard to CDA by the triple-flag tagged version of AbsA2  at the mid exponential phase time-point,  and that Sheeler et al., [SCO3225 promoter region in vitro.It is tempting to interpret these binding events in terms of a negative autoregulatory mechanism as AbsA2 has been shown to negatively regulate its own transcription, although an indirect mechanism where AbsA2 regulates its own expression excluded . We also et al.,  also didabsA2 ChIP peak is that, in contrast to the ‘monophasic’ peak pattern observed upstream of glmT, cdaPSI and cdaR, the absA2 ChIP peak is located internal to the gene and exhibits a curious asymmetrical, biphasic, form with a larger peak closest to the start of the gene overlapping with or followed by a smaller peak near the end of the gene.The most interesting aspect of the redZ at the mid-exponential growth phase time-point, and although the AbsA2 ChIP peak persists into the 18 h late exponential phase time-point in the 3xFabsA2 strain, here it does not satisfy the ChIP enrichment scoring criteria (redZ AbsA2 ChIP biphasic peak pattern resembles that seen targeting absA2 (SCO3226), with one large peak upstream of the gene and the second smaller peak within the gene. The fact that this unusual pattern is repeated at both the 14 h and 18 h time-points suggests that this is a genuine phenomenon and not an experimental artefact.As expected from the literature , AbsA2 bcriteria . The redredZ at the mid exponential time-point there is little difference in RED biosynthesis between the 3xFabsA2 and the ΔabsA2 strains during the course of the experiment, although it is likely that differences in RED production would have become more obvious had the incubation of the culture been continued for a longer time as was done in the small-scale culture experiment  where AbsA2 only binds in the 3xFabsA2 strain sample at the mid-exponential time-point  the redZ/actII-orfIV/absA2 genes in terms of the respective morphologies of the ChIP peaks, where AbsA2 associated with monophasic ChIP peaks exerts an indirect repressive effect on transcription by inhibiting activating transcription factors (e.g. CdaR), whereas the biphasic pattern of AbsA2 binding is associated with direct repression by AbsA2. Indeed, the fact that AbsA2 binding covers large portions of redZ, actII-orfIV and absA2, would suggest that the genes are more tightly constrained by AbsA2 occupancy and therefore less accessible to other transcription factors, than those with a single binding site.It is tempting to interpret the differences in the mechanism of action of AbsA2 at (a) the redZ, actII-orfIV and cdaR promoters correlate with the fact that AbsA2 has been shown to bind more weakly to the cdaR promoter than to the redZ and actII-orfIV promoters [in vivo AbsA2 binding patterns at the target loci also perhaps explain why previous investigators [i.e. in the case of redZ and actII-orfIV the double-peak binding pattern may be due to the effect of at least two widely spaced binding motifs, and possibly long-range DNA topological factors which influence DNA coiling/looping. In the case of the cda genes the presence/absence of binding DNA proteins (e.g. CdaR) may determine where AbsA2 binds. Indeed, it has been suggested previously that AbsA2 binding is dependent on the presence of CdaR [We also note that the differences in AbsA2 ChIP peak form at the romoters . Our obstigators  have bee of CdaR .absA2 and the redZ and actII-orfIV AbsA2 ChIP peaks also suggests an interesting hypothesis as to how AbsA2 acquired the status of “master regulator” of antibiotic synthesis. It is possible that initially AbsA2 was merely a ‘pathway specific’ regulator of the cda biosynthetic gene cluster, in which it is located. However, due to the similarity in sequences of the absA2, redZ and actII-orfIV genes when, through horizontal gene transfer, the biosynthetic gene clusters were brought together in S. coelicolor, AbsA2 was able to bind the SARP genes of the ACT and RED clusters and so extend its control over their gene clusters, in addition to the cda cluster. This hypothesis is consistent with the fact that within the cda cluster AbsA2 operates as an integral part of a regulatory system in conjunction with the activator, CdaR, whilst due to it being a recently acquired regulator of redZ and actII-orfIV it acts independently at these loci, reflecting its recent integration into the endogenous transcription factor network.The similarity of the Streptomyces coelicolor by the transcriptional regulator AbsA2. Despite myriad genetic and molecular biological studies of this transcription factor the present study represents the first attempt to integrate a transcriptomic study with chIP-on-chip technology to explore in vivo interactions of AbsA2 with the genome. The results  and suggest that it exerts a repressive influence on transcription through negatively regulating CdaR activity. Additionally, we show that AbsA2 also binds to its own gene (SCO3226), providing evidence for an autoregulatory mechanism, and to the SARP genes of the act (actII-orfIV) and red (redZ) clusters; differences between the patterns of the AbsA2 ChIP peaks at these genes and those within the cda cluster suggest that AbsA2 represses transcription of these regulatory genes directly and raises the intriguing possibility that AbsA2 displays two different mechanisms of binding DNA and of mediating repression.The present study used the powerful tool of high-density microarrays to investigate the regulation of antibiotic production in The results of this study go some way towards explaining and illuminating aspects of previous genetic and molecular studies of AbsA2. They also raise new questions as to the precise mode of action of AbsA2 and it is hoped that these genome-wide results will serve to inform and stimulate future work into this fascinating transcription factor.S1 FigabsA1- absA2 intergenic region, the red sequence represents the DNA which encodes the triple-flag tag and the blue sequence represents the absA2 coding sequence.The green sequences represent restriction enzyme cut sites incorporated into the termini for ease of cloning. The black sequence represents the (PDF)Click here for additional data file.S2 FigabsA genomic region and the location of the absA2 deletion. The locations of the PCR primer target sequences and sizes of the MT1110 wild-type PCR product  and the ΔabsA2 PCR product (460 bp) are shown. Lower panel: Agarose gel photograph illustrating PCR products of the expected sizes generated from MT1110 wild-type and the ΔabsA2 strain.Upper panel: Schematic diagram illustrating the (PDF)Click here for additional data file.S3 FigΔabsA2 (pMT3226) and MT1110 ΔabsA2 (pMT3226::3xFabsA2) used in the main chIP/transcriptomic experiment: panels (A) & (C) and from the independent small-scale pigmented antibiotic production experiment: panels (B) & (D).Actinorhodin (ACT) and undecylprodiginine (RED) assays from replicate cultures of strains of MT1110 (PDF)Click here for additional data file.S4 FigΔabsA2 (pMT3226::3xFabsA2) relative to equivalent probe data from MT1110 ΔabsA2 (pMT3226).List of genes (and clusters of genes) identified at the 14 h time-point as containing two or more enriched microarray probes within 1 kb contiguous segmentsin MT1110 (TXT)Click here for additional data file.S5 FigΔabsA2 (pMT3226::3xFabsA2) relative to equivalent probe data from MT1110 ΔabsA2 (pMT3226).List of genes (and clusters of genes) identified at the 18 h time-point as containing two or more enriched microarray probes within 1 kb contiguous segments in MT1110 (TXT)Click here for additional data file.S6 FigΔabsA2 (pMT3226::3xFabsA2) relative to probe data from MT1110 ΔabsA2 (pMT3226).List of genes (and clusters of genes) identified at the 35 h time-point as containing two or more enriched microarray probes within 1 kb contiguous segments in MT1110 (TXT)Click here for additional data file.S1 Table2 format. In the column headings “3xF” is used as an alternative to “MT1110 ΔabsA2 (pMT3226::3xFabsA2)” and “pMT” is used as an alternative to MT1110 ΔabsA2 (pMT3226)”. Genes considered to be activated or repressed by AbsA2 are highlighted in green and red, respectively .The probe signals from for each coding sequence were averaged; data in all columns is in log(XLS)Click here for additional data file.S2 TableΔabsA2 (pMT3226) and MT1110 ΔabsA2 (pMT3226::3xFabsA2) at the 14 h, 18 h and 35 h time-points (Rank Product pfp <0.15). Genes in red are repressed in MT1110 ΔabsA2 (pMT3226::3xFabsA2) relative to MT1110 ΔabsA2 (pMT3226). Genes highlighted in green are activated in MT1110 ΔabsA2 (pMT3226::3xFabsA2) relative to MT1110 ΔabsA2 (pMT3226). Genes which possess clusters of ChIP-enriched microarray probes in MT1110 ΔabsA2 (pMT3226::3xFabsA2) relative to MT1110 ΔabsA2 (pMT3226) are highlighted by green/red background shading; the method used for clustering and scoring these clusters or ‘regions of interest’ is detailed in Materials and Methods. Genes of the act biosynthetic gene cluster are highlighted by red borders and genes of the cda biosynthetic gene cluster are highlighted by green borders.List of genes which are significantly differentially expressed between MT1110 (PDF)Click here for additional data file.S3 Table2 format. The method used for identifying clusters of probes, designated ‘regions of interest’ (listed in ΔabsA2 (pMT3226::3xFabsA2); MT, MT1110 ΔabsA2 (pMT3226); Fold_change = Average (log 2 (WT)) − Average (log 2 (MT)).All candidate AbsA2-binding probes are listed and the clustered probes used to identify regions of interest are included within this comprehensive list. The criteria for scoring candidate probes are detailed in Materials and Methods. The three separate sheets in the Excel spreadsheet list the AbsA2-enriched probes identified at each of the respective three time points of 14 h, 18 h and 35 h. The Cy3 and Cy5 dyes were swapped for the two biological replicates of each strain and the probe data was averaged; all data is presented in log(XLSX)Click here for additional data file.


Epigenetics classically refers to the inheritable changes of hereditary information without perturbing DNA sequences. Understanding mechanisms of how epigenetic factors contribute to inheritable phenotype changes and cell identity will pave the way for us to understand diverse biological processes. In recent years, the emergence of CRISPR/Cas9 technology has provided us with new routes to the epigenetic field. In this review, novel epigenetic techniques utilizing the CRISPR/Cas9 system are the main contents to be discussed, including epigenome editing, temporal and spatial control of epigenetic effectors, noncoding RNA manipulation, chromatin in vivo imaging, and epigenetic element screening. Epigenetics classically refers to the inheritable changes of hereditary information without perturbing DNA sequences. DNA methylation, demethylation, hydroxyl-methylation, histone modification, chromatin remodeling, gene imprinting, and noncoding RNA are the central mechanisms involved. They play important roles in diverse biological processes including gene regulation, iPSC reprogramming and maintenance, genomic imprinting, X-chromosome inactivation, aging, neurodegeneration, autoimmune modulation, and tumorigenesis –4.Adapted from a natural immune defense system in bacteria, the clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (Cas9) system, abbreviated as the CRISPR/Cas9 system, is a site-specific genome editing tool that could be implemented to target and mutate specific genomic regions in eukaryotic cells, especially in mammalian cells . The ratNotably, since the emergence of CRISPR/Cas9 technology, diverse applications have been explored beyond genome editing. Here, we will focus on the new toolkit that CRISPR/Cas9 has provided to us for epigenetic research.Fosb locus in the mice brain reward region, nucleus accumbens, could affect relevant transcription network and thus control behavioral responses evoked by drug and stress [Epigenome editing refers to the targeted rewriting of epigenetic markers , 12. On d stress –15. On td stress –19. DNA-d stress –23. On tTo achieve CRISPR/Cas9-mediated epigenome editing, the main strategy is fusing the Cas9 protein with a transcription repressor or activator domain, which was known as an epigenetic effector (epieffector) . To be sHS2 enhancer region, researchers successfully silenced multiple globin genes in K562 cells. Off-target perturbation in global gene expression seemed not to be an issue in this study [For example, when the fused epigenetic effector domain was Krüppel-associated box (KRAB), using the dCas9-KRAB complex to target and induce locus-specific deposition of H3K9me3 at the is study .Tbx3, a gene implicated in the maintenance of pluripotency, researchers observed downregulation of Tbx3, loss of H3K27Ac at the enhancer, and impaired pluripotency in embryonic stem cells. In addition, this study compared specificity for enhancer targeting between dCas9-LSD1 and dCas9-KRAB complexes. Interestingly, it was concluded that dCas9-LSD1 was enhancer-specific; however, the specificity of the dCas9-KRAB complex was questionable, for it may actually be silencing the promoter or changing the chromatin structure instead of targeting the enhancer to achieve Tbx3 downregulation. The difference in the experimental methods, such as origins of Cas9 nuclease, cell types of interest, and genes to be manipulated, might account for such contradictory findings [When the fused domain was LSD1, using the dCas9-LSD1 complex to target findings .Myod gene at a regulatory region distal to the promoter, upregulated the Oct4 gene from a regulatory region proximal to the promoter, and induced transcription in three-fourths of downstream hemoglobin genes by targeting several DNase hypersensitive sites within the β-globin locus control region. These findings updated the conventional view that controlling gene expression with engineered transcription factor at sites other than promoters was limited [When the fused domain was p300 core, which was an acetyltransferase, using this dCas9-p300 core complex, researchers activated the  limited .Oct4 gene in both human and mouse cells [Ngn2 and Neurod1 genes, the dCas9-VPR complex was shown to be capable to aid induced pluripotent cell (iPSC) neuronal differentiation [The fusion domain could also be the transactivation domain VP64 or VPR, the DNA methyltransferase 3A (DNMT3A), or the DNA demethylase TET. The dCas9-VPR complex is an escalated version of dCas9-VP64, where VP64 was further fused by two other transcription factors p65 and Rta to increase the transactivation efficiency. Both of them have well been demonstrated to be reliable gene activation tools. The dCas9-VP64 complex could directly activate the silenced se cells . By targntiation , 29. Thentiation . The dCantiation .Hoxa5 gene with fairly low off-target to on-target ratio [Foxp3 expression significantly using the dCas9-TET1 complex, although demethylation at the targeted enhancer region was induced successfully, while switching to the dCas9-p300 complex sustained Foxp3 expression even under inflammatory condition by inducing acetylation at the targeted promoter region [Efforts have been made to improve epigenome editing efficiency. The first strategy is incorporating SunTag into the dCas9-epieffector complex. SunTag is a repeating peptide array that can simultaneously bind with multiple copies of a certain protein. Several studies have highlighted the utility of the dCas9-SunTag system in improving transactivation robustness. For example, via recruiting multiple transactivation domains, the dCas9-SunTag complex has been proved to be able to augment a transactivation effect significantly compared to the conventional dCas9-single activator domain complex . In anotet ratio . The secet ratio . Similarr region .Briefly, the dCas9-epieffector complex could achieve methylation and demethylation at DNA level, rewriting histone marks by inducing methylation or acetylation at nucleosome level, and be optimized to improve the editing efficiency Figures , Table 1In a broad sense, epigenetics refers to the temporal and spatial control of gene expression, of which the total effect would determine cell phenotypes during development, aging, and disease pathogenesis , 37. ThuAscl1 gene promoter using a CIB1-TALE fusion construct as the locus-specific binding complex. When DNMT3A catalytic domain was the rewriter, the Ascl1 promoter was hypermethylated, resulting in decreased gene expression. When TET1 catalytic domain was the rewriter, the Ascl1 promoter was hypomethylated, resulting in increased gene expression. By controlling the gene expression temporally, researchers were able to affect the differentiation preference of neural stem cells [Studies prior to the emergence of CRISPR have proved the utility of optogenetics in temporal control of gene expression. Basically, there need to be two components, a DNA-binding protein fusing to a light-sensitive cryptochrome protein, and a chromatin modifier fusing to a cryptochrome protein interaction partner, vice versa. , 40 Whenem cells . Moreoveem cells .By the same principle, the dCas9 DNA-binding domain paired with optical-inducible proteins could also be utilized to recruit the epieffector domain to the targeted DNA site in an inducible and reversible manner described above.In a light-activated CRISPR/Cas9 effector (LACE) system, CRY2 and CIB1 were fused to transactivation domain VP64 and catalytically deactivated Cas9, respectively. Cotransfection of these fusion protein pairs with guide RNA resulted in detectable levels of gene activation in the presence of blue light. Interestingly, when the N-terminal fragment of CIB1 was fused to both N- and C-terminus of dCas9, gene activation level in response to blue light was significantly increased, which is consistent with previous observation that simultaneous recruitment of VP64 domains to the target site had a synergistic effect on gene activation .Another similar strategy from a different angle is to split Cas9 into fragments and further tether them with optical inducible protein pairs. Upon light stimulation, Cas9 fragments would be brought together via light-induced dimerization to reconstruct nuclease activity. Interestingly, in a photoactivatable CRISPR/Cas9 system, when split Cas9 fragments were fused with the CRY2-CIB1 pair, no light-induced Cas9 activity was induced. Given the steric block effect may be at play, a different pair of proteins with much smaller molecular weight called magnets was used. The nuclease activity was successfully reconstructed upon light stimulation when Cas9 fragments were tethered with positive and negative magnets, respectively . In addiDPY30 gene, an epigenetic modifier, during the iPSC differentiation process revealed that DPY30 not only was indispensable for germ layer specification but also played dynamic roles in iPSC differentiation and maturation of specific lineages, which exemplified the utility of OptiKO in an epigenetic study [As to the chemical-controlled strategy, the optimized inducible gene knockdown or knockout (OptiKO) system was a good example. It was a strategy harnessing the CRISPR/Cas9 technology and tetracycline-inducible expression cassette in human iPSC. The OptiKO system has two main components. Cas9 was constitutively expressed while guide RNA (gRNA) was tetracycline-inducible. Clonal lines incorporating both the CAG-Cas9 cassette and the TET-ON/CAG-gRNA cassette was first isolated. In the following experiments, the addition of tetracycline to the isolated clonal cells at different time points could achieve targeted region knockdown or knockout flexibly. In particular, inducible knockdown of the ic study .Although more and more evidence is being reported regarding the feasibility of optical or chemical controllable epigenome editing, the detailed mechanisms remain to be elusive. Whether the local chromosomal or nucleosome context affects the binding affinity of the DNA-binding protein remains to be determined. What are the exact effects of locus-specific epigenome editing on DNA epigenetic profiling, histone modifications, and chromosomal interactions? What are the transcription factors participating in transcription activation or repression mediated by epigenome editing? Do we have the confidence to ensure every shot with unfailing accuracy? Such questions need to be answered to achieve reliable epigenome editing consequence and bona fide conclusions.Elusive as the mechanism is, studies discussed above indeed enlightened us with new possible routes to perturb epigenome precisely. It is conceivable that through temporal and spatial control of genes playing indispensable roles in epigenetic dynamic changes, we may acquire new insights into the underlying mechanisms .Rian, in mouse models. With a simultaneous delivery of multiple guide RNAs spanning the targeted region, the knockout rate could reach 33%. The Rian−/− mouse model facilitated functional studies of the gene knockout impact on nearby relevant gene expression in different tissues [Noncoding RNA plays important roles in gene imprinting and chromatin remodeling; thus, it is an indispensable topic in the epigenetic research field , 51. Acc tissues . In addi tissues . Moreove tissues . Convers tissues .Most recently, Feng Zhang Lab developed a RNA editing tool, named as the RNA Editing for Programmable A to I Replacement (REPAIR) system. It was adapted from the type IV CRISPR/Cas13 system, where Cas13b was deactivated for its RNA cleavage capacity while the RNA-binding ability was reserved. Deactivated Cas13b was further fused with an adenosine deaminase enzyme called ADAR. After being directed to the targeted RNA transcript, the Cas13b-ADAR complex would deaminize adenosine to inosine, which is an analog to guanine in diverse biological processes, resulting in A to I conversion. Although this CRISPR/Cas13-based RNA editing tool seemed to have no overwhelming advantage over the CRISPR/Cas9-based DNA editing method in terms of specificity due to a substantial number of off-target events across the transcriptome, it still presented some promising merits. For example, no PAM sequence constraints within the RNA target meant more flexible target binding capacity of Cas13b compared to the classical Cas9. Direct conversion of RNA base without reliance on endogenous repair mechanism also rendered it applicable in mitotic cells such as neurons. Additionally, the transient nature of RNA editing may provide a basis for the temporal control of targeted RNA transcription and downstream translation .Altogether, it seems that Cas9 instead of dCas9 is playing important roles in noncoding RNA manipulation, as more and more Cas proteins are being characterized, such as cpf1 , 61. It DNA wraps around histones to form nucleosomes. Nucleosomes continue to assemble in a spiral pattern. Through several rounds of such repeated twisting, DNA and protein are finally packaged into the superhelix structure, chromatin. Chromatin has two statuses, the tightly packed heterochromatin and the loosely packed euchromatin , 62. TheLive cell imaging is a valuable method for studying structure changes and interactions of chromatin in epigenetic regulation. Efforts have been made to adapt the CRISPR/Cas9 system for the in vivo imaging experiments. Generally, the design rationale is tagging the guide RNA loops or the dCas9 protein with fluorescent dyes to form a dCas9-gRNA-fluorescent dye complex. In a recent study, a dCas9 expression cassette was first incorporated into cells of interest. Guide RNA scaffold was redesigned, with two new stem loops protruded. One of the loops was tethered by protein MS2; the other was bound by protein PP7, which were able to recruit EGFP-conjugated MCP and mCherry-conjugated PCP, respectively. Clonal cells expressing dCas9, EGFP, and mCherry were selected as the tested platform. The delivery of guide RNAs into these cells would bring dCas9 and fluorescent proteins together at targeted loci. Colocalization and separation of the two-color fluorescent signals indicated possible interactions between the targeted loci on chromosomal level. Using this dual-color system, researchers achieved real-time labeling of major satellites, minor satellites, and two single loci on chromosomes . In anotβ-globin gene expression. Chromatin interactions were isolated at high resolution, unbiased analysis of which suggested spatial features that may be involved in transcription regulation [In addition to live cell imaging, CRISPR/Cas9 could also be used to characterize locus-specific regulatory composition. In a recent study, the deactivated cas9 nuclease was biotinylated to capture telomeric factors and components of enhancers regulating human gulation . Anothergulation , 70.Chromatin structures and interactions are gaining increasing academic attention in recent years. It has been widely accepted that the dynamic chromatin changes play pivotal roles in the temporal-spatial regulation of genes. The in vivo imaging technique and several other attempts described above indicated that CRISPR/Cas9 is a promising research tool for this topic.The accumulation of work has demonstrated the capacity of CRISPR/Cas9 for screening both coding and noncoding DNA sequences on a genome-wide scale . AlthougMaspin, in lung cancer cells both transcriptionally and translationally, which limited cancer cell growth and apoptosis [Ube3a gene on both alleles [Ube3a gene epigenetically. Other representative candidates may include the Rett syndrome, Huntington's disease, and Friedreich ataxia, where dCas9-epieffector complex may be harnessed to restore or repress the responsible gene to cure the disease [By transcriptionally activating or deactivating a specific gene that is normally silent or active, epigenome editing exhibited therapeutic potentials. It is easy to imagine that diseases with aberrant epigenetic marks as the underlying pathophysiologic mechanisms would be excellent targets to test epigenome editing-based therapeutics. The disease spectrum may include cancers, neurodegenerative diseases, neuropsychiatric diseases, neurodevelopmental diseases, and imprinting disorders. For example, using a dCas9-SAM-VPR complex, researchers reactivated a heavily methylated tumor suppressor gene, poptosis . Anotherpoptosis . Althoug alleles . With a  disease , 76, 77.In addition, epigenome editing has the potential to facilitate stem cell therapy. In recent years, iPSCs have been a major source for cell replacement therapy due to its accessibility and pluripotency –82. iPSCTaken together, these studies demonstrated the therapeutic potential of CRISPR/Cas9-mediated epigenome editing in various diseases and stem cell therapy.In conclusion, all of the studies described above indicated that CRISPR/Cas9-mediated epigenome editing holds a great promise for epigenetic studies and therapeutics. However, there are still some limitations to be scrutinized. First of all, in terms of basic science studies, although most studies claimed high specificity in their experiments, however, the high specificity usually is the result of repeated optimization. A precise model that could predict deleterious off-target effects during the experiment design stage is still lacking. In addition, although transactivation or repression effects on multiple genes were well documented in publications, mechanisms underlying the phenomenon were not clear. Epigenetic mark profiling on epigenome scale was not sufficient. Local CHIP-seq data usually only focused on the characterization of one or few histone marks. Theoretically, we hope that epigenome editing could achieve targeted gene regulation by changing epigenetic marks specifically and freely according to our wills. To achieve this goal, high specificity and clarified mechanisms are the prerequisite. Therefore, more thorough off-target event assessments and more studies focusing on mechanisms underlying epigenome editing are needed.Moreover, in terms of clinical applications, several issues need to be addressed prior to successful clinical translation. Firstly, the endurance of gene activation or a repression effect mediated by CRISPR/Cas9 remains to be undetermined. It has been thought that epigenome-editing-induced gene activation or repression is short-term , 91. On


Drosophila embryos. We demonstrate that increasing the number of Zelda binding sites accelerates the kinetics of nuclei transcriptional activation regardless of their transcriptional past. Despite its known pioneering activities, we show that Zelda does not remain detectably associated with mitotic chromosomes and is neither necessary nor sufficient to foster memory. We further reveal that Zelda forms sub-nuclear dynamic hubs where Zelda binding events are transient. We propose that Zelda facilitates transcriptional activation by accumulating in microenvironments where it could accelerate the duration of multiple pre-initiation steps.Pioneer transcription factors can engage nucleosomal DNA, which leads to local chromatin remodeling and to the establishment of transcriptional competence. However, the impact of enhancer priming by pioneer factors on the temporal control of gene expression and on mitotic memory remains unclear. Here we employ quantitative live imaging methods and mathematical modeling to test the effect of the pioneer factor Zelda on transcriptional dynamics and memory in  Drosophila embryos. Here, the authors use quantitative live imaging and mathematical modeling to measure the effect of Zelda on transcriptional dynamics and memory, observing temporal control of gene expression by Zelda through transient interactions in hubs.Zelda is a pioneer factor that regulates transcription in  Drosophila, the transcriptional activator Zelda (Zld) plays an essential role in zygotic genome activation1.During the first stages of metazoan embryogenesis, the zygotic genome is largely quiescent, and development relies on maternally deposited mRNAs and proteins. Transcriptional activation of the zygotic genome occurs only hours after fertilization and requires specific transcription factors. In cis-regulatory elements prior to gene activation, which establishes competence for the adoption of cell fates, and (ii) opening chromatin, which facilitates subsequent binding by classical transcription factors. Indeed, hours before their activation, Zelda binds to thousands of genomic regions corresponding to enhancers of early-activated genes3. Zelda binding is associated with modifications of the associated chromatin landscape and overall chromatin accessibility5.Zelda exhibits several pioneer factor properties such as (i) priming e.g Bicoid and Dorsal)7. Consequently, target gene response is strengthened by Zelda binding, both spatially and temporally, for developmental enhancers8 as well as for synthetic enhancers where input parameters are tightly controlled9.Moreover, this ubiquitously distributed activator is able to potentiate the action of spatially restricted morphogens , regardless of past transcriptional states. By monitoring transcription in Zelda-maternally depleted embryos, we reveal that Zelda is dispensable for mitotic memory. On the contrary, we found that Zelda allows the temporal disadvantage of arising from a transcriptionally inactive mother to be bypassed. Using a mathematical modeling framework, we propose that mitotic memory requires long-lasting transitions, which are accelerated by Zelda, thus overriding mitotic memory of silent states. By analyzing Zelda protein kinetics during many cell cycles, here we report that while it is not retained on chromosomes during mitosis, this transcription factor exhibits a highly dynamic behavior. We observe that intra-nuclear distribution of Zelda is not homogeneous, and that it accumulates in local hubs, where its residence time is surprisingly transient (in the second range). Taken together, our results provide insights into Zelda-mediated temporal control of transcription during zygotic genome activation in vivo.snail (sna) gene expression, a proximal (primary) and a distal (shadow enhancer)13. Both enhancers are bound by Zelda (Zld) at early nuclear cycles (nc)3. When driven by its native enhancers, endogenous expression of snail is extremely rapid, precluding analysis of the impact of Zelda on fine-tuning the timing of its activation. We therefore used a previously described truncated version of the sna shadow enhancer (snaE) that leads to a stochastic activation, compatible with the tracking of transcriptional memory across mitosis14.In the blastoderm embryo, two enhancers control enhancer < promoter < MS2-yellow transgenes, with a unique minimal promoter (sna promoter) and an MS2-yellow reporter 16 were added to the snaE in three locations, either close to the promoter (Zld 3’), in the middle (Zld mid) or at a distance (Zld 5’)  Fig. .Fig. 1ZesnaE-extraZld transgenes was dictated by the regulatory logic of the sna enhancer, and expression was indeed evident in the presumptive mesoderm, similarly to the endogenous sna pattern  as a landmark to define dorso-ventral coordinates  and Twist18.First, we implemented an automatic image analysis pipeline that segments nuclei in nc14 Fig.  and tractes Fig. . Unless 16, our dynamic data showed that increasing the number of Zld-binding sites resulted in precocious transcriptional activation , referred to as synchrony19 , whereas the number of active nuclei at this stage increased upon the addition of extra Zld-binding sites  was examined  while ‘b’ is the mean duration. Thus, ‘a’ = 1 corresponds to the exponential distribution. A mixture of gamma distributions covers the case when the number of transitions (parameter ‘a’) is random. The cumulative distribution function for the mixture is formulated in Fig. a’ in the mixture) can be computed from the first two moments of the distribution of waiting times   Fig. .a’ and ‘b’, from populations of nuclei (descendants of active and descendants of inactive nuclei)  is correlated to the ‘b’ values  tend to have a strong ainactive/ aactive ratio, whereas enhancers with small ‘b’ values have a vanishing memory bias  ≈4.98 μm2∙s−1)  experiments and determined that Zelda recovery was remarkably fast in interphase nuclei Fig. . Because−1) Fig. , thus vas−1 Fig. .koff) (see Methods) and deduced the estimated residence time (RT) (RT = 1/koff)  Fig. . Our FCS30. These transient bindings events seem to be compensated for by increased local concentrations, in particular nuclear microenvironments, referred to as subnuclear hubs8. We thus explored Zelda spatial distribution within nuclei in living Drosophila embryos.The low binding and fast diffusion rates for Zelda were unexpected given the large impact of a single extra Zld-binding site on accelerating the kinetics of gene activation Fig. . However8, multiple Zelda proteins seem to cluster in local dynamic hubs that exhibit a diversity of sizes and lifetimes 31. We therefore conclude that, at least with a synthetic transgene, long-lasting stable contacts between sites of transcription and Zelda dense regions are not detected. Given the important number of Zelda targets, rigorously connecting transcriptional activation to Zelda hubs would require a broader analysis with adapted imaging methods. However, these observations are reminiscent of a dynamic kissing model recently described between an active gene and mediator clusters in ES cells33.Immuno-FISH and live imaging experiments revealed that only a subset of our MS2 reporter nascent RNAs were located within a Zelda dense region Fig. . FurtherHaving demonstrated that Zelda was not homogeneously distributed, we wanted to determine if the dynamic properties of Zelda within hubs differed from its global dynamics.34.One possibility was that high local concentrations of Zelda would correspond to long-lasting chromatin-binding events that would result in increased residence time (long RT). Given the rapid movements of Zelda hubs  to seconds (likely specific binding) and this occurs regardless of its local concentration. How could Zelda exhibit a similarly transient chromatin binding at random positions and in regions where its concentration is high? An exciting possibility would be that dense regions could be enriched for Zelda target genes. Under this hypothesis, Zelda target genes could cluster together thereby increasing the probability to locally accumulate Zelda proteins. Alternatively, Zelda could spatially organize its targets in space through its pioneer factor properties and then maintain these clusters of targets possibly through its intrinsically disordered domainsDrosophila development.Using quantitative imaging approaches in living embryos combined with mathematical modeling, we have investigated a role for the pioneer-like factor Zelda in regulating mitotic memory. We demonstrated that Zelda is dispensable for transcriptional mitotic memory. While our modeling suggests that memory may be potentiated by a reduction in the number of steps required for post-mitotic transcriptional activation, we propose that Zelda accelerates this activation by decreasing the time spent at each preceding step. Our data support a model whereby Zelda binds transiently to chromatin in localized nuclear microenvironments; to potentially accelerate the timing of the transitions required prior to transcriptional activation . These dynamic properties allow the pioneer-like factor Zelda to act as a quantitative timer for fine-tuning transcriptional activation during early yw stock was used as a control. The germline driver nos-Gal4:VP16 (BL4937) was recombined with an MCP-eGFP-His2Av-mRFP fly line (gift from T.Fukaya). RNAi were expressed following crosses between this recombinant and UASp-shRNA-w (BL35573) or UASp-shRNA-zld4, virgin females expressing RNAi, MCP-GFP-His2Av-mRFP were crossed with MS2 containing transgenic constructs males. The GFP-zld and mCherry-zld strains were obtained by CRISPR25. GFP-ash128 and His2Av-mRFP (BL23651) and eGFP-bcd27.The snaE enhancer transgene was described in Ferraro et al14. The 24XMS2 tag was inserted immediately upstream of the yellow reporter gene coding sequence. We verified that our MS2 sequences do not contain any canonical binding site and we also compared synchrony of the snaE transgenes with our 24XMS2 to newly available MS2 loops, 24XMS2ΔZelda38, kindly provided by Pr.Dostatni  were acquired using a Zeiss LSM780 confocal microscope with the following settings: GFP and RFP proteins were excited using a 488 nm and a 561 nm laser respectively. A GaAsP detector was used to detect the GFP fluorescence; a 40x oil objective and a 2.1 zoom on central ventral region of the embryo, 512 × 512 pixels, 16 bits/pixel, with monodirectional scanning and 21z stacks 0.5μm apart. Under these conditions, the time resolution is in the range of 22–25 s per frame. Two and one movie respectively for snaE + 3Zld in white-RNAi and zld-RNAi background were acquired on a Zeiss LSM880, keeping the time resolution is in the range of 21–22 s per frame. Image processing of LSM780 and LSM880 movies of the MS2-MCP-GFP signal were performed in a semi-automatic way using custom made software, developed in PythonTM. Live imaging of GFP-zld/ + ; His2Av-mRFP/ + and GFP-bcd/ + ; His2Av-mRFP/ + embryos were performed using a Zeiss LSM780 confocal microscope with the following settings: GFP and RFP proteins were excited using a 488 nm and a 561 nm laser respectively. A GaAsP detector was used to detect the GFP fluorescence. 512 × 512 pixels, 16 bits/pixel, images were acquired using a 40x oil objective, with bidirectional scanning and 8 stacks 1μm apart. Under these conditions, the time resolution is in the range of 5–7 s per frame. Live imaging of GFP-zld embryos  embryos containing snaE + 1Zld3’  and green (MS2 nascent RNA spots) channels were first maximum intensity projected. The analysis was divided into three parts: before mitosis (nc13), during mitosis and after mitosis (nc14). During interphases, nuclei were segmented  and tracked with a minimal distance criterion. Chromosomal splitting at the end of anaphase was automatically detected and used to define a nucleus-by-nucleus mitosis frame. During mitosis, nuclei were segmented by intensities and tracked by an overlap in consecutive frames criterion. By merging these three parts, we could give a label to each nc13 mother nucleus, their daughters in nc14 and an extra label to recognize one daughter from the other. MS2 spots were detected with a blob detection method with a user-defined threshold constant value. Detected spots were associated to the closest nuclei, inheriting their label. Finally, for each tracked nucleus, the timing of first transcriptional activation was recorded. Analyses were done with the activation timing of the two daughters for synchrony and the first active daughter for memory.zld-RNAi embryos. We implemented a spot detection algorithm to export files with the detected MS2 spot. The thresholding of the MS2 spot is consistent with all the other automatic analyses. A spatial domain was determined, taking the pseudo-furrow as a landmark and focusing on a 25μm region around it (note that activation does not seem to occur outside of this area). Nuclei in this region are visually tracked frame-by-frame to detect when activation in nc13 occurs, when mitosis occurs and to recover the first activation frame in nc14. Approximately 2 to 3-fold more nuclei from inactive mothers, compared to descendants of active mothers, were analyzed.Upon high reduction of maternal Zelda, zygotic activation is perturbed, and major developmental defects occur. Abnormal nuclear shape  for 25 min on a shaker at 450 rpm; formaldehyde was replaced by 5 ml methanol and embryos were vortexed for 30 s. Embryos that sank to the bottom of the tube were rinsed three times with methanol. For immunostaining, embryos were rinsed with methanol and washed three times with PBT (PBS 1x 0.1% triton). Embryos were incubated on a wheel at room temperature twice for 30 min in PBT, once for 20 min in PBT 1% BSA, and at 4 °C overnight in PBT 1% BSA with primary antibodies. Embryos were rinsed three times, washed twice for 30 min in PBT, then incubated in PBT 1% BSA for 30 min, and in PBT 1% BSA with secondary antibodies for 2 h at room temperature. Embryos were rinsed three times then washed three times in PBT for 10 min. DNA staining was performed using DAPI at 0.5 μg.ml−1. Primary antibody dilutions for immunostaining were mouse anti-GFP (Roche IgG1κ clones 7.1 and 13.1) 1:200; rabbit anti-GFP (Life technologies A11122) 1:100; mouse anti-Ser5-P Pol II  1:100; rat anti-Ser2-P Pol II (3E10) 1:5; rabbit anti-GAF  1:250; Secondary antibodies ; anti-mouse Alexa 488-conjugated ; anti-mouse IgM Alexa 555-conjugated ; anti-rabbit Alexa 555-conjugated ; were used at a dilution 1:500. Fluorescent in situ hybridization was performed as described in14. A Dixogygenin-MS2 probe was obtained by in vitro transcription from a bluescript plasmid containing the 24-MS2 sequences, isolated with BamH1/BglII enzymes from the original Addgene MS2 plasmid (# 31865). Snail probe was described in19. Primary and Secondary antibody for Fluorescent in situ hybridization were sheep anti-Digoxigenin (Roche 11333089001) 1/375; mouse anti-Biotin  1/375; anti-mouse Alexa 488-conjugated  and anti-sheep Alexa 555-conjugated  1:500. Mounting was performed in Prolong Gold. Fluorescent in situ hybridization with yellow probes was performed as described in40, probe sequences will be available upon request.Embryos were dechorionated with bleach for 1–2 min and thoroughly rinsed with HIbl) was corrected by an unbleached ROI (Iunbl) of the same nucleus and another ROI outside of the nucleus (Iout) following the simple equation:Fluorescence recovery after photobleaching (FRAP) in embryos at nc14  was performed with the following settings: a Zeiss LSM780 using a 40x/1.3 Oil objective and a pinhole of 66 μm. Images (512 × 32 pixels), 16bits/pixel, zoom 6x, were acquired every ≈20 ms during 400 frames. GFP was excited with an Argon laser at 488 nm and detected between 507–596 nm. Laser intensity was kept as low as possible to minimize unintentional photobleaching. A circular ROI (16 × 16 pixels) 0.07 µm/pixels, was bleached using two laser pulses at maximal power during a total of ≈130 ms after 50 frames. Hub FRAP was performed in embryos from nc10 to nc13 with the following settings: FRAP was performed on a Zeiss LSM880 using a 40 × /1.3 Oil objective and a pinhole of 83 μm. Images (256 × 256 pixels), 12bits/pixel, zoom 8x, were acquired every ≈104 ms during 100 frames. GFP was excited with an Argon laser at 488 nm and detected between 496–565 nm. Laser intensity was kept as low as possible to minimize unintentional photobleaching. A circular ROI (8 × 8 pixels) 0.1 µm/pixel, was bleached using four laser pulses at maximal power during a total of ≈114 ms after 3 frames. To discard any source of fluorescence intensity fluctuation other than molecular diffusion, the measured fluorescence recovery in the bleached ROI region  as in their case.one exchanging population29.Ceq defined as above and Feq = koff / (koff + k*on). FD(t) is the fluorescence recovery due to diffusion and Fexc(t) the fluorescence recovery due to exchange.Finally, for the reaction-diffusion model, we started from the analytical expression developed in the supplemental (Supplementary Equation FD(t) is defined using a slightly modified version of the analytical equation of the 20th order limited development of the Axelrod model for Gaussian profile illumination and diffusion43:K is a constant proportional to bleaching deepness, M is the mobile fraction and τ is the half time of recovery. To minimize the effect of mobile fraction on Ceq, M was kept between 0.9 and 1.1.Since we used a Gaussian shape illumination profile, w the value of the radius at 1/e2 of the Gaussian beam  and β a discrete function of K tabulated in ref. 44.Diffusion coefficients of the different molecules were determined according toFexc(t) is defined as in ref. 29, slightly modified with respect to the Gaussian illumination, leading to the following equation:K defined as previously.45. Multiple measurements per nucleus, in multiple nuclei and embryos at 20 °C were used to generate multiple ACF, used to extract kinetic parameters. The FCS measurement volume was calibrated with a Rhodamine6G solution46 using Df = 414 μm2.s−1. Each time series was fitted with the reaction dominant model29:Feq = koff / (koff + k*on), Ceq = k*on / (koff + k*on), τDf = w2xy / 4 μ Df and ω = wz / wxy.FCS experiments were performed on a Zeiss LSM780 microscope using a 40x/1.2 water objective. GFP was excited using the 488 nm line of an Argon laser with a pinhole of 1 airy unit. Intensity fluctuation measured for 10 s were acquired and auto-correlation functions (ACFs) generated by Zen software were loaded in the PyCorrFit programk*on, koff and Df (reaction constants and diffusion coefficient) were estimated using the PyCorrFit program implemented with Eq. For each correlogram the value of ∞ value to account for long time persistent correlation during the measurements. Based on the average number of Zelda molecules (N), connected to the G(0) value following the formula G(0)=+/−90 nM.Photophysics of the dye was neglected and we introduced a GT0 is a deterministic incompressible lag time, the same for all nuclei, and Tr is a random variable whose value fluctuates from one nucleus to another. The decomposition in Eq. Tr is defined such that it takes values close to zero with nonzero probability. This property allows us to set the time origin to the instant when the first nuclei initiates transcription, in order to determine Tr. For large populations of nuclei, this stems to setting the time origin to T0 (see ).We are interested in the time needed for post-mitotic transcription (re)activation. We model this time as the sum of two variables:o T0 see .The stochastic part of the (re)activation time is modeled using a finite state, continuous time, Markov chain.A1, A2,…, An−1, An. The states Ai, 1 ≤ i ≤ n − 1 are metastable and OFF, i.e. not transcribing. The state An is ON, i.e. transcribing. Each metastable state has a given lifetime, defined as the waiting time before leaving the state and going elsewhere. For the purposes of this paper, we considered that each of the states has the same lifetime denoted τ. Also, the topology of the transitions is considered linear: in order to go to Ai+1 one has to visit Ai.The states of the process are We have also tested a more complex model, with uneven lifetimes and therefore, more parameters. However, the complex model did not provide a sensibly better fit with data. Furthermore, the determination of individual parameters of the more complex model is uncertain: many different combinations of parameter values lead to very close fit. Following Occam’s razor principle, we based our analysis on the simplest, homogeneous jump model. For consistency, detailed models are described in The image analysis software and associated graphical user interface developed for this study are available from the corresponding author upon request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Supplementary Movie 11Supplementary Movie 12Supplementary Movie 13Supplementary Movie 14


A G-quadruplex (G4) is a well-known nucleic acid secondary structure comprising guanine-rich sequences, and has profound implications for various pharmacological and biological events, including cancers. Therefore, ligands interacting with G4s have attracted great attention as potential anticancer therapies or in molecular probe applications. To date, a large variety of DNA/RNA G4 ligands have been developed by a number of laboratories. As protein-targeting drugs face similar situations, G-quadruplex-interacting drugs displayed low selectivity to the targeted G-quadruplex structure. This low selectivity could cause unexpected effects that are usually reasons to halt the drug development process. In this review, we address the recent research on synthetic G4 DNA-interacting ligands that allow targeting of selected G4s as an approach toward the discovery of highly effective anticancer drugs. A. A84]. A4TT-3′ from the other adoptable topologies and promoter G4s , as elucidated by an NMR structural analysis analysis c 86]. I. I4TT-3′A long-loop DNA sequence arranged by a monomeric G4 was also amenable to molecular recognition by hybridization of the complementary strand, which was demonstrated by an atomic-level NMR analysis . In partMYC encodes a multifunctional transcription factor that can act as a transcription activator of some genes involved in cell proliferation, while acting as a transcription repressor of other genes involved in growth arrest . T. TMYC G4MYC G4  over the G4s in the promoters for other genes , as demonstrated by fluorescence titration experiments. They further confirmed its biological activities extensively using western blotting, exon-specific RT-qPCR . T. TMYC G4  allowed for an excellent repression of c-MYC at low micromolar concentrations in cultures of colorectal carcinoma (HCT116) cells, and the G4-mediated action was biologically validated by an exon-specific RT-qPCR assay . . MYC G4 g ligand . AlthougCR assay ,94 in BuVEGF has a promoter region, in which the G4-forming sequences are located pyridazin-4(5H)-one derivatives were screened as a new class of G4-interacting ligands for BCL2-targeted therapeutics, and two hit compounds identified were found to bind to BCL2 G4 structures with clear preferences over c-kit, c-MYC, and telomere G4s, as well as dsDNA , and in turn stimulates proliferation, differentiation, and survival in hemopoietic precursor cells phenoxazine (BPO) derivatives as potent c-kit G4 ligands , which encodes the catalytic subunit of telomerase, has commanded considerable attention as a compelling biomedical target, particularly for cancer, as elevated TERT expression is observed in ~90% of human cancer cells, whereas it is normally silenced in most normal cells ,162. Abeferation ,168.The mutations that were identified in >70% of melanomas partially account for the elevated level of TERT expression . C to T We would like to highlight a unique approach to addressing the issue of hTERT downregulation on the basis of the mutations in a G4-mediated manner. The region located approximately -90 to -22 upstream of the transcription start site was abundant in GC base pairs and has multiple PQSs a 43]. I. I43]. IHRAS, NRAS, and KRAS, was first discovered in human tumors as driver oncogenes and has long been recognized as an important therapeutic target. Mutation of KRAS is one of the most oncogenic driver mutations in pancreatic, colorectal, and lung cancers, and plays a role in acquiring and increasing the drug resistance . . 219]. CBy taking advantage of the sequence-selectivity of PIPs, as mentioned earlier, a head-to-head-type polyamide dimer was developed for the selective targeting of a designated G4, which displayed an inducible effect toward G4 formation  220]. T. T220]. Although a number of biomedical targets in the approach toward tumor therapy are recognized and investigators are pursuing the development of drugs, results have been limited thus far. The G-quadruplex (G4) has been recently considered to be a potential biomedical target, particularly for tumor therapy, and a considerable body of evidence has been accumulating that G4-interacting drugs exhibit good antitumor activities. As protein-targeting drugs face similar situations, G4-interacting drugs displayed low selectivity to the targeted G4 structure, mainly because of the similar skeletons among different G4 forms observed in the genome. In this review, we addressed the ligands interacting with G4 that were devised to gain selectivity for a particular G4 structure and to exhibit selective bioactivity for tumor cells. The selectivity issues remain incompletely solved, but if accomplished, would substantially impact cancer therapy.The G4-driven oncogenes introduced here are known to be usually well-correlated and concertedly influence tumorigenesis, tumor growth, and malignant transition ,221,222.Importantly, G4 has an influence, not only on telomere or tumor-related genes, but also on differentiation- and neuron-related genes. As mentioned earlier, OCT4 expression may be governed to some degree by G4 formation at the proximal promoter in human embryonic stem cells (CCTL14), as shown by a reporter assay system , while t


In addition to transcription factor binding, the dynamics of DNA modifications (methylation) and chromatin structure are essential contributors to the control of transcription in eukaryotes. Research in the past few years has emphasized the importance of histone H3 methylation at lysine 27 for lineage specific gene repression, demonstrated that deposition of this mark at specific genes is subject to differentiation-induced changes during development, and identified enzymatic activities, methyl transferases and demethylases, that control these changes. The present review discusses the importance of these mechanisms during intrathymic αβ T cell selection and late differentiation. Pioneering studies in prokaryotes have led to the paradigm that adjusting gene transcription in response to environmental signals involves transcription factors, proteins that bind specific DNA sequences (cis-regulatory elements) close to the transcription start site. Such binding promotes DNA-templated RNA synthesis by the RNA polymerase. The same paradigm governs the control of transcription in eukaryotic cells, with added layers of complexity at virtually every step, including the multiplicity of RNA polymerases, the functional overlap among trans-acting factors, and the unsuspected promiscuity of transcription factors with cis-regulatory elements. Typical genes are controlled by multiple, often tissue-specific cis-regulatory elements, potentially distantly located relative to the transcription start site. Such elements are bound by transcription factor assemblies which themselves typically recruit cofactor complexes that mediate their action on the polymerase complex.In addition, eukaryotes use two important layers of controls of gene expression, DNA methylation and chromatin dynamics. Eukaryotic DNA is methylated on cytosines located upstream of a guanine, and stretches of such palindromic CpG dinucleotides  are frequently found in cis-regulatory elements. Their methylation status is inversely correlated with gene expression . The impSpecific histone modifications are associated with specific gene expression states or regulatory regions . AcetylaThere is compelling evidence that changes in H3 K27 methylation are not simply associated with gene expression status, but have a causative role in setting gene transcription levels –11. HoweT cell development in the thymus is a multi-step process combining cell proliferation, differentiation and survival-selection events . As a reThe earliest αβ lineage-committed thymocytes have successfully rearranged one of their TCRβ-encoding genes and express neither CD4 nor CD8 coreceptors (“double-negative” [DN] thymocytes) . After t+ T cells, whereas those recognizing MHC-I-bound peptides become CD8+ T cells  T cells, which recognize lipids bound to CD1d molecules –49. In mRegardless of the ligand they recognize, iNK T cells differ from conventional T cells in multiple respects . They arNucleosomes carrying trimethylated H3K27 are preferentially located at and near promoters of silent genes –58. TherIn vitro analyses suggest a strict correspondence between Jmjd3 and Utx catalytic activities and H3K27Me3 demethylation. That is, both molecules are highly specific for H3K27Me3, relative to other methylated histone residues  by Jmjd3 and Utx demethylases. These enzymes belong to a large family defined by the presence of a complex catalytic domain, called JmjC –77. Theiresidues –83, wherCd4 and Cd8 expression and T cell development  has a strong impact on cell differentiation and function in multiple experimental systems, including in ES cells and embryonic development, tumor development, and early hematopoiesis –96. This+ T cells  suggested that this modification was important for transcriptomic changes during late αβ T cell differentiation , 44. ChaExperimental assessments of these predictions have produced mixed results. Deletion of Ezh2, the predominant H3K27 methyltransferase in the T cell lineage, has no reported impact on the differentiation of SP from DP thymocytes, unlike at earlier stages of T cell development, during the differentiation of iNK T cells, or in mature T cells –71, 108.The reciprocal experiment, namely deletion of Jmjd3 or Utx targeted to DP thymocytes, showed at first glance similar results as mice lacking either or both enzymes had CD4 and CD8 SP thymocytes and T cells , 105. HoS1pr1 .Analyzing the impact of these enzymes on H3K27 methylation status and the transcriptome gave unexpected results. Even though DP and SP thymocytes are non-dividing cells, the inactivation of Jmjd3 and Utx had a highly specific impact on H3K27Me3 distribution . Unlike S1pr1 . This inS1pr1 . The latS1pr1, the impact on the transcriptome of differentiating SP thymocytes was limited to a small number of genes, many of which were normally up-regulated during the terminal differentiation of SP thymocytes, including Klf2  cells . Unexpec T cells . Thus, tStudies of histone demethylase functions provided a mirror image of these findings. In contrast to their selective impact on late thymic maturation in conventional thymocytes, Utx and to a lesser extent Jmjd3 were found to be important for multiple aspects of iNK T cell development , 70, 114Kdm6a) is located on chromosome X. Accordingly, female cells carry (and express) two Kdm6a alleles; in contrast male cells express Utx from their single Kdm6a allele and the Y chromosome-located Uty gene, encoding the Utx-related protein Uty. Although lacking demethylase activity, Uty is functionally redundant with Utx during the development of male mice  the genome-wide deposition of the mark and its implied pleiotropic impact, (ii) the multiplicity of protein and protein complexes involved in the “writing,” “reading,” and “erasing” of the mark, with various degree of functional overlap, and (iii) the multifunctional nature of many components, and specifically H3K27Me3 demethylases. Nevertheless, studies over the past few years have brought important clarifications on the function of this mark in T cell development, both on its impact on the transcriptome of differentiating cells and its biological consequences, and on the mechanisms that underpin this impact.S1pr1) indicates that such effects would be gene specific. A distinct and tantalizing possibility is that, even in non-dividing cells, H3K27Me3 is functionally erased by nucleosome replacement rather than (or in addition to) catalytic demethylation. Replacement mechanisms  abrogates H3 K27 methylation and results in a major disruption of cell homeostasis and differentiation, the consequences of the double Jmjd3-Utx disruption are less striking, both on H3K27Me3 and developmental fates. At the gene level, evidence in non-dividing thymocytes that H3K27Me3 is “erased” despite Jmjd3 and Utx disruption   indicatechanisms  deposit chanisms –121 and It will be important to integrate the dynamics of H3 K27 methylation in the broader context of epigenetic control of gene expression. Much progress has been made understanding the mutual relationships of activating and repressive histone marks. H3 K27 methylation and acetylation are biochemically mutually exclusive, and accordingly exert opposite effects on gene expression. More strikingly, evidence is accumulating that H3 K4 and K27 methylations, which are typically found in active vs. silent genes or enhancers, respectively, are the end products of enzymatic complexes that coordinate writing of one mark with erasure of the functionally opposite mark. That is, Ktm2/MLL complexes associate both an H3 K4 methyl transferase and H3K27Me3 demethylases, whereas PRC2 complexes associate H3 K27 methyl transferase activity and H3K4Me3 demethylases of the Jarid1-RBP2-Kdm5 family , 123.How these activities integrate with the other key histone repressive mark, H3 K9 methylation, has been addressed in various experimental systems  but remaCd4 and Cd8 loci. T cell development is accompanied by reduced methylation at CpG islands in both loci following commitment to the αβ lineage and onset of CD4 and CD8 expression, followed by partial, lineage specific, remethylation of the silenced coreceptor gene  and those ensuring the active repression of Cd4 in CD8-differentiating thymocytes  , 133. AlThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: The tissue‐specific transcriptional programs during normal development require tight control by distal cis‐regulatory elements, such as enhancers, with specific DNA sequences recognized by transcription factors, coactivators, and chromatin remodeling enzymes. Gata3 is a sequence‐specific DNA‐binding transcription factor that regulates formation of multiple tissues and organs, including inner ear, lens, mammary gland, T‐cells, urogenital system, and thyroid gland. In the eye, Gata3 has a highly restricted expression domain in the posterior part of the lens vesicle; however, the underlying regulatory mechanisms are unknown. Results: Here we describe the identification of a novel bipartite Gata3 lens‐specific enhancer located ∼18 kb upstream from its transcriptional start site. We also found that a 5‐kb Gata3 promoter possesses low activity in the lens. The bipartite enhancer contains arrays of AP‐1, Ets‐, and Smad1/5‐binding sites as well as binding sites for lens‐associated DNA‐binding factors. Transient transfection studies of the promoter with the bipartite enhancer showed enhanced activation by BMP4 and FGF2. Conclusions: These studies identify a novel distal enhancer of Gata3 with high activity in lens and indicate that BMP and FGF signaling can up‐regulate expression of Gata3 in differentiating lens fiber cells through the identified Gata3 enhancer and promoter elements. Developmental Dynamics 247:1186–1198, 2018. © 2018 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists Novel Gata3 lens‐specific enhancer is located 18 kb upstream of transcription start site.BMP and FGF signaling can regulate Gata3 expression during mouse lens development. Precise control of gene expression during development is crucial for establishing hundreds of different cell types forming the embryo, including their spatial organization. Many key developmental genes are regulated by a combination of proximal  and distal regulatory elements such as enhancers   Gata3 has been extensively studied in multiple systems, including inner ear, kidney, mammary gland, T‐cells, and thyroid gland  antibody  that also includes enhancer E8 located 4 kbp upstream of TSS. Given the proximal location of enhancers 1A and 1B, their sequences were combined to generate a 1AB/PROMOTER reporter construct (1AB). Enhancers E1D and E7 were tested together as the 1D7/PROMOTER reporter (1D7)  . Significantly, enhancer 1A alone was able to drive strong eGFP expression in the E14.5 lenses, with expression patterns very similar to that observed in 1AB embryos Fig. . In cont+ early lens fibers. Both cell populations are exposed to FGF and BMP from the vitreous humor, and both factors have been implicated in fiber differentiation  conditions, expression of a 5‐kb GATA3 promoter‐peGFP construct was ∼25 times greater than that of maternal peGFP vector Fig. A. ExpresThe present data provide novel insights into transcriptional regulation of Gata3 during mouse eye development through the identification of an evolutionarily conserved bipartite enhancer located ∼17–19 kb upstream from the TSS that is active in lens, cornea, and other ocular cells. Expression of EGFP driven by the 1AB construct in the lens recapitulates the endogenous pattern of Gata3 in mouse embryos, and studies of multiple reporters in differentiating lens cells support the idea that BMP and FGF signaling control expression of Gata3 in lens.GFP mouse model and expression studies of multiple Gata3 enhancer constructs driving expression of eGFP proteins are consistent with previous studies of Gata3 expression during mouse lens development   and Etv5/ERM  using Phusion High‐Fidelity PCR Master Mix . The purified fragments were subcloned into pGL3 vector  fused to a minimal 4ETATA promoter  as schematically depicted in Figure Staged embryos were fixed in 4% paraformaldehyde overnight, cryoprotected in 30% sucrose in phosphate‐buffered saline, and embedded in optimal cutting temperature Tissue Freezing Medium  for cryosectioning; 10‐μm transverse sections were collected, washed in PBS, and incubated for 30 min with Image iT FX Signal Enhancer . Slides were washed in PBS and incubated overnight at 4°C with rabbit anti‐GFP (1:2000) . Sections then were washed three times for 10 min in PBS and incubated for 2 hr with the secondary antibody, goat anti‐rabbit Alexa Fluor 488 (1:200) . Cell nuclei were counterstained with DAPI (1:1000) for 10 min . Slides were washed with PBS and mounted with Vectashield . Images were taken with ZEISS Axio Observer fluorescent microscope . Relative eGFP expression was measured as fluorescence intensity from three different sections using ImageJ software. eGFP staining of wild‐type lens was set as a reference point.Genomic DNA (gDNA) from transgenic animals was extracted by overnight digestion of the tail in 100 µl of DNA digestion buffer  with addition of proteinase K to final concentration of 0.5 mg/ml. Proteinase K was inactivated by incubation of the digested tail at 95°C for 15 min. DNA was precipitated by addition 100 µl of isopropanol, washed with 70% ethanol, air‐dried, and resuspended in 100 µl of nuclease‐free water. gDNA concentration was measured by NanoDrop ND‐2000 , and each sample was diluted to 5 ng/µl; 10 ng of gDNA was used for real‐time PCR reaction in each well of a 96‐well plate. Number of transgene integrations was measured by TaqMan qPCR in Universal PCR Master Mix using EGFP TaqMan assay (Mr00660654_cn) according to manufacturer's protocol . Quantitation of eGFP copy number was normalized on endogenous mouse Tfrc reference gene .Most of transcription factor binding sites were retrieved from the JASPAR database  with penicillin G and streptomycin. The day after plating, DCDML cultures were transfected using Lipofectamine 2000  and treated 5 hr later with 15 ng/ml FGF‐2 and 10 ng/ml BMP‐4 . The cells were lysed 6 days later in SDS‐PAGE sample buffer, and equal amounts of lysate were transferred to PVDF membranes. The blots were probed with primary anti‐GFP antibody  and secondary antibody conjugated to Alexa Fluor 680 . The staining was analyzed by the Odyssey infrared imaging system . The results of eGFP immunoreactivity were normalized to β‐actin staining in the same sample.Dissociated cell‐derived monolayer lens cultures (DCDMLs) were prepared from E10 chicken embryos as described previously (Le and Musil,


LOX gene promoter function can make an important contribution to elucidating resistance mechanisms. Here, we cloned the CmLOX08 promoter of melon (Cucumis melo) and identified the main promoter regions regulating transcription in response to signalling molecules and abiotic stresses.Lipoxygenases (LOXs) play significant roles in abiotic stress responses, and identification of CmLOX08 from melon leaves was cloned, and bioinformatic analysis revealed that it harbours numerous cis-regulatory elements associated with signalling molecules and abiotic stress. Five 5′-deletion fragments obtained from the CmLOX08 promoter—2054 (LP1), 1639 (LP2), 1284 (LP3), 1047 (LP4), and 418 bp (LP5)—were fused with a GUS reporter gene and used for tobacco transient assays. Deletion analysis revealed that in response to abscisic acid, salicylic acid, and hydrogen peroxide, the GUS activity of LP1 was significantly higher than that of the mock-treated control and LP2, indicating that the − 2054- to − 1639-bp region positively regulates expression induced by these signalling molecules. However, no deletion fragment GUS activity was induced by methyl jasmonate. In response to salt, drought, and wounding treatments, LP1, LP2, and LP4 promoted significantly higher GUS expression compared with the control. Among all deletion fragments, LP4 showed the highest GUS expression, indicating that − 1047 to − 1 bp is the major region regulating promoter activity and that the − 1047 to − 418-bp region positively regulates expression induced by salt, drought, and wounding, whereas the − 1284 to − 1047-bp region is a negative regulatory segment. Interestingly, although the GUS activity of LP1 and LP2 was not affected by temperature changes, that of LP3 was significantly induced by heat, indicating that the − 1284- to − 1-bp region is a core sequence responding to heat and the − 2054- to − 1284-bp region negatively regulates expression induced by heat. Similarly, the − 1047- to − 1-bp region is the main sequence responding to cold, whereas the − 2054- to − 1047-bp region negatively regulates expression induced by cold.The 2054-bp promoter region of CmLOX08 promoter and demonstrated that it is a signalling molecule/stress-inducible promoter. Furthermore, we identified core and positive/negative regulatory regions responding to three signalling molecules and five abiotic stresses.We cloned the The online version of this article (10.1186/s12870-019-1678-1) contains supplementary material, which is available to authorized users. TomloxD in transgenic tomatoes clearly indicated that this gene is involved in endogenous jasmonic acid (JA) synthesis, and in turn regulates the expression of plant defence genes and resistance to high temperature  pre-warmed to 37 °C and incubated at 37 °C for 30 min. A portion of this mixture (100 μL) was then added to 900 μL stop buffer (0.2 M Na2CO3). Fluorescence was measured using a Cary Eclipse Fluorescence Spectrophotometer  at excitation and emission wavelengths of 365 nm and 455 nm, respectively. Stop buffer and 10 nM to 80 nM 4-methylumbelliferone (4-MU) were used for calibration and standardization. Protein concentrations were determined using a Bradford protein assay kit  following the manufacturer’s instructions with bovine serum albumin used as a standard. GUS activity is presented as nM of 4-MU generated per min per mg soluble protein. GUS measurements were repeated three times.Transient expression of GUS activity in the treated tobacco leaves was measured as described previously . Tobaccot test. A P value ≤0.05 was considered significant. Charts presenting data were generated using Origin software (version 8.0).Data are expressed as the mean values ± standard deviation (SD) of three independent experiments and were analysed using SPSS statistical software  using an independent sample Additional file 1:CmLOX08-pro and GeLOX08-pro. CmLOX08-pro promoter: oriental melon (Cucumis melo var. makuwa Makino); GeLOX08-pro promoter: the melon (Cucumis melon L.) genome database. Identical and dissimilar nucleotides are shown on a background of blue and gray, respectively. The two primers LOX08pro-F and LOX08pro-R which cloned the CmLOX08 promoter are indicated by arrows. The translation initiation codon (ATG) is framed and marked for “+ 1”. (PDF 498 kb)Alignment of the nucleotide sequences of Additional file 2:GUS histochemical staining of the p121GUS tobacco leaves as negative control. (PDF 125 kb)Additional file 3:CmLOX08 at various time points under 50 mM NaCl (a) and drought (b) treatments. (PDF 148 kb)Expressions of Additional file 4:CmLOX08 full length promoter. Lane M: DL5000 DNA Marker; lane 1 to 5: CmLOX08 full length promoter fragment. (PDF 123 kb)PCR amplification of Additional file 5:The five different length recombinant vectors were verified by plasmid PCR. Lane M: DL5000 DNA Marker. Lane 1 to 4: LP1 (2054 bp); lane 5 to8: LP2 (1639 bp); lane 9 to 12:LP3 (1284 bp); lane 13 to 16: LP4 (1047 bp); lane 17 to 20: LP5 (418 bp). (PDF 126 kb)