Hox genes are expressed in overlapping patterns and function in a partially redundant manner. In vitro biochemical screens probing the HOX-TF sequence specificity revealed largely overlapping sequence preferences, indicating that co-factors might modulate the biological function of HOX-TFs. However, due to their overlapping expression pattern, high protein homology, and insufficiently specific antibodies, little is known about their genome-wide binding preferences. In order to overcome this problem, we virally expressed tagged versions of limb-expressed posterior HOX genes  in primary chicken mesenchymal limb progenitor cells (micromass). We determined the effect of each HOX-TF on cellular differentiation (chondrogenesis) and gene expression and found that groups of HOX-TFs induce distinct regulatory programs. We used ChIP-seq to determine their individual genome-wide binding profiles and identified between 12,721 and 28,572 binding sites for each of the nine HOX-TFs. Principal Component Analysis (PCA) of binding profiles revealed that the HOX-TFs are clustered in two subgroups , which are characterized by differences in their sequence specificity and by the presence of cofactor motifs. Specifically, we identified CTCF binding sites in Group 1, indicating that this subgroup of HOX-proteins cooperates with CTCF. We confirmed this interaction by an independent biological assay (Proximity Ligation Assay) and demonstrated that CTCF is a novel HOX cofactor that specifically associates with Group 1 HOX-TFs, pointing towards a possible interplay between HOX-TFs and chromatin architecture.Homeotic genes code for key transcription factors (HOX-TFs) that pattern the animal body plan. During embryonic development,  Hox genes encode transcription factors that determine the vertebrate body plan and pattern structures and organs in the developing embryo. Despite decades of effort and research on Hox genes, little is known about the HOX-DNA binding properties in vivo. This lack of knowledge is mainly due to the absence of appropriate antibodies to distinguish between different HOX transcription factors. Here, we adapt a cell culture system that allows us to investigate HOX-DNA binding on a genome-wide scale. With this approach, we define and directly compare the genome-wide binding sites of nine posterior HOXA and HOXD transcription factors. We report that the in vivo HOX binding specificity differs from the in vitro specificity and find that HOX-TFs largely rely on co-factor binding and not only on direct HOX-DNA binding. Finally, we identify a novel HOX co-factor, a genome architecture protein, CTCF suggesting a possible interplay between HOX-TF function and chromatin architecture. Hox genes) are key regulators of development. They encode homeodomain transcription factors (HOX-TFs) that are expressed in an overlapping fashion along the anterior-posterior axis in all metazoans [Hox genes are organized in clusters with their order reflecting not only their expression along the anterio-posterior body axis but also their temporal expression onset . In most vertebrates, two rounds of whole-genome duplication have resulted in four clusters of Hox genes, coding for a total of 39 HOX-TFs. All HOX-TFs show high levels of sequence conservation between paralog groups (e.g. HOXA9 and HOXD9) and to a lesser extent between genes of the same cluster (e.g. HOXA1 to HOXA13) [reviewed in The homeotic genes (etazoans . In the HoxA and HoxD clusters (Hox9-13) are expressed along the proximo-distal axis following a collinear strategy [Hox genes revealed a remarkable redundancy within paralog groups controlling the development of the proximal (stylopod), middle (zeugopod), and distal (autopod) parts of the limb [Hoxa11 nor Hoxd11 in mice leads to substantial malformations of the stylo-, or zeugopods. However, deletion of both Hoxa11 and Hoxd11 causes a severe truncation of the stylopod and loss of the zeugopod [Hoxd13 gene cause the adjacent Hoxd12 to be expressed in a Hoxd13-like pattern associated with the functional rescue of the Hoxd13 deficiency. A similar deletion, removing Hoxd13 and Hoxd12 causes Hoxd11 to be expressed in a Hoxd13-like pattern; however, Hoxd11 is not able to rescue the loss of its two adjacent paralogs [In the developing vertebrate limb, the posterior genes of the strategy . Geneticthe limb , 6. For zeugopod , 8. A sisters Hox-13 are evice versa [In spite of the insights gained by these elegant series of genetic experiments, the high degree of HOX protein similarity and the overlap of expression domains have hindered the elucidation of the individual HOX-TF functions. HOX-TFs were also included in large biochemical surveys to identify the specific binding sequence of transcription factors –12. Two ce versa , 14. Thuce versa , 15, 16.ce versa . HoweverHere, we have analyzed and systematically compared the effects of nine limb bud-expressed HOX-TFs (HOXA9-13 and HOXD9-13) on cell differentiation and gene regulation and compare their genome-wide binding characteristics. To mimic the natural HOX environment as closely as possible, we used mesenchymal chicken limb bud cells and moderate retroviral overexpression . In thisWe find that certain HOXA/HOXD paralog TFs have opposing effects on chondrogenic differentiation and induce distinct regulatory programs in transduced cells. Further, by comparing the genome-wide DNA binding of nine HOX-TFs in this experimental setting, we find that the posterior HOX-TFs can be separated into two groups (Group 1 and Group 2), with distinct binding motifs and distinct associations with cofactors. Finally, we characterized CTCF (the CCCTC-binding factor) as a novel cofactor of HOX-TFs and show that Group 1 but not Group 2 HOX-TFs bind thousands of CTCF-occupied sites in the chicken genome.To systematically compare the function of posterior HOX-TFs, we virally expressed FLAG-tagged versions of each TF in chicken micromass (chMM) cultures. After validating a reproducible, similar, and moderate expression of virally expressed HOX-TFs , we asseInterestingly, paralogue HOX-TFs did not always have the same general impact on the chondrogenic differentiation of the chMM. While HOXA9 stimulated chondrogenic differentiation, its paralog HOXD9 inhibited the same process. In contrast, HOXA10 and HOXD10 both promoted chondrogenic differentiation. HOXA11 and HOXD11 both inhibited chondrogenic differentiation, but to a very different extent. Finally, HOXD13 and HOXA13 both strongly inhibited cartilage formation; however, Eosin staining showed that the cell morphology of the HOXA13-expressing chMM was quite distinct from HOXD12 or HOXD13 cultures .The simple readout of the chMM morphology showed that the HOX-TFs induce distinct effects on cell differentiation. In order to comprehensively compare the effects on gene expression, we performed RNA-seq of HOX-TF expressing chMM cultures. We used DEseq2  to generJUN and FOS, were among the most strongly upregulated genes in all of the datasets, suggesting that they might be direct targets of HOX-TFs.The hierarchical clustering recapitulated some of the main differences found between HOX-TFs that were detected in chMM gross morphology. HOX10 and HOX11 paralogs clustered together, while HOX9 paralogs, which bore striking differences in chMM morphology, clustered apart. Furthermore, the clustering process classified the paralog groups in an order that partially corresponded to their known role in limb development. The clustering separated the stylo-/zeugopod expressed HOX-TFs  from the autopod expressed HOXD12/13. Two factors, HOXA9 and HOXA13, clustered separately from all other HOX-TFs. This indicates that despite comparable effects on chMM morphology, the regulatory programs induced by HOXA9 and HOXA13 are distinct from the other posterior HOX-TFs. Moreover, the HOX11 paralogs induced transcriptional programs so similar to one another that the clustering algorithm was not able to separate the two replicate datasets from each factor. Interestingly, two genes coding for subunits of the AP1 class of transcription factors, HOX cross-regulation. In fact, we find that all posterior HOX-TFs, with the exception of HOXA9, where able to induce HOXA/D13 expression at least two fold in comparison to mock-infected chMM cultures. In contrast, HOXA9 had generally a repressive effect on the other HOX genes, especially toward its genomic neighbours in cis, HOXA10 and HOXA11  others can have opposing effects (HOXA13/HOXD13 and HOXA9/D9).Taken together, our analysis shows that, despite high homology and functional redundancy We next wanted to assess whether analogous differences could be observed between paralog groups in their genome-wide binding preferences. We generated ChIP-seq profiles from two biological replicates of virally expressed HOX-TFs in chMM cultures using the αFLAG antibody. We identified between 12,721 and 28,572 binding sites for each of the nine HOX-TFs . We firsWe then mapped location of HOX-TF binding sites relative to genes. The genomic binding of HOX-TFs respective to genes was similar for all nine factors. The majority of binding sites were either intronic (17–33%) or intergenic (53–60%) and only 9–21% of the peaks were located in annotated promoters (-5kb to +2kb of known TSS).Next, we performed a principal components analysis (PCA) to compare the datasets in an unbiased way, using the identified peaks as input . PCA shode novo motif analysis using the peak-motifs algorithm [in vitro and ChIP-seq derived motifs. However, several sequence features had not been detected in the previously published datasets. We found a prominent G at the 5’ end of all Group 1 motifs , which had also been detected using SELEX-seq [To find a possible cause for this separation, we first tested whether the grouping could be attributed to the sequence-specificity of the TFs. For this we performed lgorithm  with thein vitro datasets. Specifically, the CCATAAA motif (HOXA/D11) we observed was highly similar to a change in sequence specificity that HOXA10 undergoes when co-binding with PBX4 [The motifs identified for HOXA13 and HOXD13 were identical to the ones detected in PBM/SELEX-seq. In contrast, the primary motif of HOXA11 and HOXD11 did not overlap with those detected in the corresponding de novo motif analyses, HOXA13 was a notable exception of the Group 1 HOX-TFs, as it contained a high number of peaks carrying binding sites. To exclude the effect of weak and maybe indirect binding sites from the analysis, we performed the same analysis for the 10,000 and 1,000 strongest peaks  Fig 3)de novo mIn Group 2 of HOX-TFs, we were not able to detect any clear cofactor motif. In contrast, we found three putative cofactor motifs in five out of the six Group 1 HOX-TF peak sets. The first motif was the well-characterized TGANTCA AP1 binding site  Fig 3A)Fig 3A). de novo discovery of cofactor motifs can be masked by the strong overrepresentation of the primary motif. To exclude this possibility, we performed a reverse search and identified and counted all matches to CTCF  as a representative example. We first tested whether the CTCF-HOXA10 co-bound sites were enriched for promoters or intergenic regions but found no differences between HOXA10-only, HOXA10-CTCF shared or CTCF-only peaks . We thenin situ, in a quantifiable and sensitive manner. We expressed FLAG-tagged HOXA10 (Group 1) in chicken DF1 cells and performed the PLA assay using αFLAG antibody and an endogenous αCTCF antibody. We readily detected CTCF-HOXA10 interaction in the nucleus that was almost as strong as the interaction of CTCF with RAD21, which we used as a positive control . The PLA control . We also control .Hoxd12, but not Hoxd11 is able to substitute for a loss of Hoxd13 [HOX9 and HOX13 paralog groups are the only posterior HOX-TFs which retained all four copies of the genes, thereby reducing the selective pressure on each paralog and allowing their neo-functionalization [HOX genes, especially its direct genomic neighbours in cis, HOXA10 and HOXA11, further confirming the observation from hierarchical clustering that this factor is different from the other studied HOX genes. HOXA/D13 were not negatively regulated by HOXA9 but upregulated by all other HOX-TFs, demonstrating some degree of auto-regulation.In this study, we systematically compared the effect of nine limb-bud expressed HOX-TFs on the differentiation and gene regulation of primary mesenchymal limb bud cells. Hierarchical clustering of the regulated genes delineated two groups of HOX-TFs: HOX10/11/D9, and HOXD12/HOXD13 that, during limb development, are expressed in the stylo/zeugopod and autopod, respectively. The distinction between these two groups is in accordance with genetic experiments in mice demonstrating that Hoxa13 and Hoxd13 are partially redundant in loss-of-function experiments [Finally, although eriments , we herein vivo, but are rather co-expressed in overlapping patterns and exert their specific function in this biochemical context. Although HOX-TFs induced distinct effects in our experiments, their combinatorial or antagonistic action in vivo might play an important role in the developing embryo. Investigation of the in vitro sequence specificity of individual HOX-TFs showed that their homeodomains bind largely similar sequences [Drosophila, where low-affinity binding sites for the HOX-TF Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) were shown to be biologically more significant [in vitro determined monomer specificity and rather revealed a composite binding site like the one bound by a HOXA10-PBX4 dimer [JUN and FOS were also strongly upregulated by all HOX-TFs, suggesting a mechanism of cofactor cross-regulation. To our knowledge, AP1 has not been linked to limb patterning or HOX-TFs. However, these factors are known to be involved in a wide array of developmental and cell differentiation processes [HOX-driven limb patterning.Our systematic comparison focused on the effects of individual HOX-TFs and their genome-wide binding. However, HOX-TFs are rarely expressed alone equences , 12. Subequences , 16, 17.rocesses  and our HoxD locus during limb development, where two adjacent TADs regulate the gene expression in the proximal and distal limb, respectively [Drosophila for CTCF/Cohesin and Smad-TFs, which are the transcriptional effectors of TGFß/BMP signaling [PCA analysis separated the HOX-TF binding sites in two subgroups along PC2. We tried to identify the underlying cause for this distinction between HOX-TF binding sites and found co-binding with CTCF to correlate with Group 1 HOX-TF binding. We also describe CTCF as a novel cofactor of Group 1 HOX-TFs. CTCF/cohesin are now well-established factors with important functions in the spatial organization of the genome into topologically associating domains (TADs) –31. Amonectively , 35. Speignaling . The SmaHOX and CTCF coding sequences were amplified from chicken embryonic limb buds cDNA (HH27) and cloned into RCASBP-viruses as previously described [8 viral particles/ml or higher. The infection of chMM cultures and the histological assessment were performed as described elsewhere [escribed . DF1 cellsewhere . For theChromatin Immunoprecipitation was performed as described previously . BrieflyCells from harvested chMM cultures were separated prior to fixation of the ChIP samples and RNA was isolated from these cells using an RNaeasy Qiagen kit. RNA-seq libraries were constructed as described previously , by seleDF1 cells were transfected with RCASBP(A)-3x FLAG-HOXA10, RCASBP(A)-3x FLAG-HOXD13, or RCASBP(B)-HA-CTCF, respectively. The cells were cultured for at least 6 days to ensure a high cellular infection rate. Upon confluency cells were transferred to 10 mm cover slips and further incubated for one day. Cells were fixed for 10 min with 4% PFA, blocked with TSA  and incubated with appropriate primary antibodies (in 10% horse serum in 1x DPBST) overnight at 4°C. Primary antibody combinations were: 1) FLAG-HOX and CTCF interaction: m-αFLAG M2 and rb-αCTCF; and 2) HA-CTCF and RAD21 interaction: m-αHA and rb-αRAD21. Antibody concentration for PLA were tested and used as follows: m-αFLAG M2 1:20,000 , m-αHA.11 1:8,000 , rb-αCTCF 1:20,000  and rb-αRAD21 1:1,000 .After primary antibody incubation, the PLA assay was performed using the Duolink In Situ Fluorescence Kit  according to manufacturer’s instructions. Protein-protein interactions were analyzed by using confocal imaging on a Zeiss LSM700 and the Axiovert Zen software.For the quantification of PLA experiments, the contacts in several independent frames were counted using ImageJ and divided by the number of nuclei in the frame. The PLA experiments were performed in at least two independent experiments.alGal4 reference genome. Reproducible peaks were identified using the MASC2 [Quality filtering and read mapping were performed as described previously . Reads whe MASC2  peak calhe MASC2 , 40. Forhe MASC2 . Summitshe MASC2 .De novo motif analysis was performed using the peak-motifs algorithm [lgorithm  and the lgorithm  to obtailgorithm . We then multiplied the HOX-TF RPKM with this ratio to obtain the effective RPKM and compared it to the HOX-RPKMs from HH23 wing buds, where all nine relevant HOX-TFs are at least partially expressed .HOXA genes we had to manually alter the chicken Galgal4 annotation. In the Galgal4 annotation  the HOXA cluster and its genes are annotated in triplicate. We manually changed the annotation so that every additional copy of the gene would be considered a new gene. We then mapped the RNA-seq samples to the altered annotation and used it only to assess the overexpression levels and the HOX autoregulation.In order to generate a correct estimate of the expression values for the S1 Fig(A) Schematic of the RCASBP(A) virus carrying a 3xFLAG tag and a HOX gene. RCASBP(A) virus produces viral polycistronic mRNA that can be either unspliced or spliced in two different variants. Only the SD-SA2 isoform gives rise to the mRNA that will be translated into HOX protein.HOX in chMM to the endogenous limb bud HOX expressions levels, posterior-distal chicken wing buds at the HH23 were dissected as indicated in the drawing. According to Nelson et al. all tested HOXA and HOXD genes are expressed the dissected tissue in at least ~20% of the cells (HOXA13) or in almost all cells (HOXD11) .(B) In order to compare the expression levels of viral HOX in chMM overexpression experiments on top and RPKM values from distal-posterior wing bud RNA-seq data on bottom.Bottom panel: the fold change of RPKM between posterior-distal wing bud at HH23 and individual chMM experiments for each replicate and averaged between the replicates. Note that HH23 limb buds are a cell population where not all cells express any given HOX gene, whereas in chMM every cell expresses the HOX gene of interest. HOXD9 and HOXA13 are the two lowest expressed factors in our wing bud derived RNA-seq data. HOXD9 expression is uniform but low at HH23 and HOXA13 is only expressed in a fraction of all cells .(C) Estimated degree of overexpression by RCASBP(A). Top panel: The fraction of viral mRNA giving rise to HOX protein as calculated from RNA-seq data see . Middle HOXD9 was cloned in to the RCASBP(A) it produces the protein that gives two distinct and reproducible bands. Therefore, we conclude that this represents the HOXD9 protein and a post-translationally modified version rather than the degradation product.(D) Western Blot (αFLAG) of chMM cultures at day 6 shows uniform expression levels of all nine HOX-TFs. Even though the correct CDS region of (E) Reproducibility analysis (Irreproducible Discovery Rate) for all ChIP-seq experiments for the HOX-TFs. Self-Consistency Analysis (IDR > 0.01%) for the biological replicates. Bottom: Reproducibility between biological replicates or between “pseudo-replicates” of the pooled replicates. Bold numbers represent the number of reproducible binding sites for each HOX-TF.(F) log10 IDR plots for the two biological replicates of the nine HOX-TF ChIP-seq experiments. The IDR analysis has been performed according to ENCODE ChIP-seq guidelines .(EPS)Click here for additional data file.S2 FigHOXA9-13 and HOXD9-13 expression was examined in the chMM control and each of the chMM overexpressing cultures. The numbers in the matrix represent expression fold change between the chMM experiments in comparison to the control. Red colour represents upregulation and blue downregulation.(A) HOX autoregulation derived from data displayed in A). Only the expression fold change of at least 0.9 fold are plotted in the scheme. Dark lines represent an up- or down regulation of at least one fold and the light lines represent the expression fold change between 0.9 fold to 1 fold.(B) A schematic of (EPS)Click here for additional data file.S3 FigPromoters are annotated as 5kb upstream and 2kb downstream of an annotated TSS. Intergenic regions are annotated as all regions that are outside promoter, exon and intron regions. The peaks were examined in a binary fashion and the affiliation to a specific genomic location was determined and plotted.(EPS)Click here for additional data file.S4 Fig(A) Pairwise peak overlap of the top 10,000 binding sites for each examined HOX-TF ChIP-seq.De novo motif analysis. Sequences underlying the 5,000 most significant peaks of all reproducible peaks (left) were used as input for the motif analysis. The three most significant motifs from the analysis are shown and in the black boxes (right) the number represents the number of the HOX binding sites detected in the top 1,000 peaks  (HOXD9 and HOXA9 have a second motif that is not a primary HOX motif and HOXA10 has third motif that is not a primary HOX motif and have been excluded from the counting). On the left is the comparison to the best fitting HOX motif detected by SELEX-seq .(B) (C) Presence of the binding sites from (B) were counted in the top 1,000, top 10,000 and all reproducible peaks and shown in the bar diagram for Group 1 HOX-TFs (blue) and Group 2 HOX-TFs (black).(EPS)Click here for additional data file.S5 Figde novo motif analysis. The five most significant motifs identified with the Oligos algorithm (top) and Positions algorithm (bottom) with RSAT suite are shown from left to right . RSAT parameters were used as default, oligo length = 7. Group 1 HOX-TFs results are shown in (A) and Group 2 HOX-TFs results are shown in (B). Sequences (peak summit +/- 75 bp) underlying all reproducible peaks were used as input for (EPS)Click here for additional data file.S6 Fig(A) Binding sites matching the published CTCF motif  were counted in the HOX top 1,000, top 10,000 and all peaks . Peaks carrying one or more CTCF binding site were counted only once.de novo motif analysis, published CTCF-motif, and primary HOX motif are shown left; top to bottom. Centrimo analysis of de novo HOX motif (as seen left of the graph) and the published CTCF motif for each HOX ChIP-seq dataset .(B) CTCF-like motifs discovered in (EPS)Click here for additional data file.S7 Fig(A) Binding sites matching the published JDP2 (AP1) motif  were counted in the HOX top 1,000, top 10,000 and all peaks . Peaks carrying one or more JDP2 binding site were counted only once.de novo motif analysis, published JDP2 (AP1) motif, and primary HOX motif are shown left, top to bottom. Centrimo analysis of de novo HOX motif (as seen left of the graph) and the published JDP2 motif for each HOX ChIP-seq dataset .(B) AP1-like motifs discovered in (EPS)Click here for additional data file.S8 Figde novo detected, GC-rich motif were counted in the HOX top 1,000, top 10,000 and all peaks . Peaks carrying one or more binding sites were counted only once.(A) Binding sites matching the de novo motif analysis, consensus GC-rich motif from all HOX-TF ChIP-seq, and primary HOX motif are shown left, top to bottom. Centrimo analysis for de novo HOX motif (as seen left of the graph) and the consensus GC-rich motif for each HOX ChIP-seq dataset .(B) GC-rich motif discovered in (EPS)Click here for additional data file.S9 Fig(A) Overlap between the top 1,000, top 10,000 and all HOX-TF peaks with all CTCF peaks.(B) Overlap between the top 1,000, top 10,000 and all HOX-TF peaks with all RAD21 peaks.(C) Left: Venn diagram depicts individual and shared binding sites between CTCF and RAD21 genome-wide. Right: Venn diagrams depict the overlap between two co-bound fractions, CTCF-HOX and RAD21-HOX in yellow and green, respectively.(EPS)Click here for additional data file.S10 FigAll HOXA10, all CTCF and only HOXA10-CTCF co-bound peaks and their genomic location distribution. Promoter regions encompass 5kb region upstream and 2kb region downstream of the annotated TSS.(EPS)Click here for additional data file.S11 Fig Triplicate PLA experiments were performed probing the protein-protein interaction of 3xFLAG-HOXA10 and CTCF (top left) and 3xFLAG-HOXD13 and CTCF (top right) with αFLAG and αCTCF antibody. Control PLA experiments were done probing: 1) for POSITIVE control (bottom right): the protein-protein interaction of HA-CTCF and RAD21 using m-αHA and rb-αRAD21 antibody and 2) for NEGATIVE control (bottom left): the protein-protein interaction of FLAG-protein and CTCF using m-αFLAG and rb-αCTCF antibody in DF1 cells not expressing any FLAG-tagged protein. On the right: Contacts were counted with ImageJ and calculated to represent average contacts per nuclei. Standard deviation represents variation between different frames (pictures) counted from the same sample. n = number of frames counted.(EPS)Click here for additional data file.S1 Table(TXT)Click here for additional data file.


The mouse gene Krox20 encodes a transcription factor required for the specification of two segments (rhombomeres) of the developing hindbrain. In rhombomere 3, Krox20 is subject to direct positive feedback governed by an autoregulatory enhancer, element A. In contrast, a second enhancer, element C, distant by 70 kb, is active from the initiation of transcription independent of the presence of the KROX20 protein. Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility. This work uncovers a novel, asymmetrical, long-range mode of cooperation between cis-acting elements that might be essential to avoid promiscuous activation of positive autoregulatory elements.Developmental genes can harbour multiple transcriptional enhancers that act simultaneously or in succession to achieve robust and precise spatiotemporal expression. However, the mechanisms underlying cooperation between  The formation of multicellular organisms from the egg to the adult stage is largely under genetic control. The activation of specific genes is governed by regulatory DNA sequences present nearby on the chromosome. Most of these sequences promote activation and are called enhancers. In this paper, we study two enhancers governing the expression of a gene involved in the formation of the posterior brain in vertebrates. One of these enhancers is involved in a positive feedback loop: it is itself activated by the protein product of the gene that it regulates. The other enhancer was thought to be only involved in the initial accumulation of the protein, necessary for the subsequent activation of the feedback loop. Here we show that the second enhancer directly cooperates with the autoregulatory enhancer to increase its accessibility and its activity. Our work uncovers a novel, long-range mode of cooperation between enhancers that restricts the domain of action of autoregulatory enhancers within embryos and might be essential to avoid their inappropriate activation. A major class of cis-regulatory elements are transcriptional enhancers, which can recruit combinations of transcription factors (TFs) to modulate transcription initiation from (a) cognate gene promoter(s), in general independently of their relative distance and orientation ; ii; iiKrox2Krox20 autoregulation, together with element A. For this purpose, we first analysed the expression of Krox20 in composite heterozygous embryos, Krox20ΔA/ΔC, carrying deletions of element A on one allele and of element C on the other  [Krox20ΔA/ΔC embryos the level of expression of Krox20 is not a limiting factor for the activation of the only wild type allele of element A. Therefore, the most likely explanation for the defect in Krox20 maintenance is that, in r3, the deletion of element C impairs the activity of element A located on the same chromosome and that the two elements synergistically cooperate, in cis, for the establishment and/or maintenance of the autoregulatory loop. A more conventional, partial redundancy between elements A and C appears much less likely.The similarity of the phenotypes observed in r3 upon deletion of elements A or C led us to investigate the possibility of an involvement of element C in he other . Although stages . In r5,  embryos . This deKrox20 activation, or whether it is required during the autoregulation phase as well, we generated a genetic condition in which element C is initially active, but is deleted at a later stage. This was achieved by combining the Krox20Cflox allele  on multiple viewpoints in the locus [Krox20 is no more transcribed [Krox20 gene and its distant regulatory element A (separated by over 200 kb) show highly similar distributions of 4C-seq signal  spreads its interactions about equally over the two neighboring TADs  to shape the pattern of expression of the gene. Among these enhancers, element C performs a dual function, as a classical enhancer and as a potentiator in cis of element A. We propose that this latter role may constitute a general means to prevent promiscuous activities of autoregulatory elements.In the present study, we have made a further step in the understanding of the molecular mechanisms governing the expression of a master developmental regulator, using both enhancer knock-outs and investigations of chromatin structure. This analysis reveals that Krox20 gene. Element C was responsible for the initiation of its expression in r3, whereas element B, possibly together with element C, was in charge of the initiation in r5. Subsequently, element A governed the maintenance of the expression through a positive feedback loop towards a definitive engagement into an odd-numbered rhombomere fate [Krox20 expression in r3  clearly distinguishes this function from classical enhancer cooperative activities  and possibly from other hierarchical logic modes of interactions [Our study also revealed an unexpected function of element C: it is required for autoregulation, which was thought to be only dependent on element A. Using genetic approaches, we showed that an interaction must occur in ractions ,45.cis to promote the expression of the target gene [Krox20 expression by reducing the accessibility of the KROX20 protein to element A [At this point, it is not known whether this additional enhancer potentiator function of element C, which is functionally distinct from its classical enhancer activity, is dependent on enhancer activity. Several recent studies have shown that enhancers can be transcribed and that the products of this transcription can act locally in get gene . It is plement A , are alslement A . It is tKrox20 is almost normally expressed in the absence of element C, suggesting that cooperation between elements A and C is not essential in this rhombomere to support element A activity. It is possible that element B, which is likely to constitute the major initiator element in r5 and physically interacts with element A  is locked in a configuration that is not accessible to its cognate TF and therefore unable to activate transcription, despite its possible interaction with the promoter. This lock will be released when the potentiator function is provided by a second cis-acting element (element C). It is interesting to note that in transgenic constructs, element A is able to activate a promoter in the absence of element C in cis. This difference in behaviour might be explained by two types of reasons: in transgenic constructs, element A is very close to the promoter, in contrast to the endogenous enhancer, located far upstream to the promoter; the chromatin context of a transgene is likely to be different, possibly more permissive than that of a highly regulated endogenous locus.The introduction of an enhancer potentiator within a positive feedback loop may constitute an efficient prevention (safety lock) against inappropriate activation of autoregulatory elements. According to our model , in the cis-acting elements becomes essential for establishing the appropriate specificity of the positive feedback loop. As indicated in the model, such a feedback loop can be broken by mutation of either the TF or of any of the two cis-acting elements .Krox20Cre [Krox20ΔA [Krox20Cflox line was generated at the Institut Clinique de la Souris  by homologous recombination in ES cells; the Krox20ΔC allele was obtained as described in Krox20 [EphA4 [All mouse lines were maintained in a mixed C57BL6/DBA2 background. We used the following alleles: rox20Cre  and KroxKrox20ΔA ; the moun Krox20  and EphA0 [EphA4 .ChIP-seq experiments were performed as previously described . Brieflyhttps://github.com/GenomicParisCentre/eoulsan/tree/branch-chip-seq). First, reads were filtered out when they would not pass Illumina filters (module filterreads with option illuminaid). Files corresponding to technical replicates were merged , followed by trimming of the reads using Trim Galore! . Mapping was performed using STAR [Sequencing was performed on multiplexed samples using 50 bp single-end reads on an Illumina HiSeq system (E9.5 embryos) or using 42 bp paired-end reads on an Illumina NextSeq (E8.5 embryos) according to the manufacturer’s specifications. Chip-seq data were analysed as follows, using Eoulsan  with exting STAR  .4C-seq libraries were constructed as previously described  with smahttp://promoter.bx.psu.edu/hi-c/download.html. Directionality indexes were calculated as described using tools described previously [Position of mouse TADs in ES cells and associated 40 kb normalized Hi-C matrices  were obteviously . Interachttp://picard.sourceforge.net) . To consider only fragments coming from transcription factors protected DNA (and not from nucleosomes), only fragment with size lower than 100 bp were kept. Bigwig tracks were obtained using DeepTools BamCoverage (1.5.9.1). Peak calling was performed using MACS2 (2.1.0.20140616), using a q-value< = 0.01 threshold (other parameters as default). For quantification, we first defined a set of non-redundant enriched regions for all samples by taking the union of all peak summits from both replicates of all samples, grouped together all summits distant from less than 50 bp, and for each group kept only the summit with the lowest q-value . We then quantified the signal at all summits in each sample by counting the number of fragments . Normalisation and statistical analysis were performed using the bioconductor DESeq2 package (1.6.3). Library size factors were calculated on fragment counts in genomic bins of 10 kb. Comparison between wild type, Krox20ΔC/ΔC and Krox20ΔA/ΔA embryos was performed using negative binomial Wald Test (DESeq2).ATAC experiments were performed according to Buenrostros and colleagues , using ahttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE94716The data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE94716 and is available at the following address: S1 Fig(A) Sequence of the deleted element C and associated genomic coordinates (mouse genome assembly mm9). This sequence corresponds to the cloned sequence of the mouse element C described in [(B)In situ hybridization for EphA4 mRNA performed on control (Krox20+/ΔC) and homozygous (Krox20ΔC/ΔC) mouse embryos at the indicated somite stages. Note the reduction of the size of r3 in the homozygous mutant. Rhombomeres positions are indicated on the left of each embryo.ribed in . (B)In (TIF)Click here for additional data file.S2 Fig(A) The left boundary of the Krox20 TAD, as previously determined from Hi-C in ES cells [Krox20 and +1 TADs. The transition zone between the -1 and Krox20 TADs is highlighted in grey. CTCF ChIP-seq data in E14.5 mouse brain  Coordinates of the different regions used for the analysis of the distribution of 4C-seq signal. (C) Normalized distribution of 4C-seq signal for the Nrbf2, Element A and Krox20 viewpoints using previously determined TAD boundaries ([Nrbf2 gene and Element A (right). The ratio of signal between the -1 and Krox20 TADs (average of 2 replicates) is shown above for each viewpoint. When the previously determined TAD boundary is used (left), the 4C-seq signal of the Nrbf2 viewpoint is almost equally distributed over the -1 and Krox20 TADs, whereas a much more discrete distribution is observed when the cluster of CTCF sites is used (right). In contrast, the nearby Element A viewpoint always restricts its strongest signal to the Krox 20 TAD, similar to the associated but much more distant Krox20 gene.ES cells , spreadsES cells ). Hi-C iES cells  or by ou and genndaries  o(TIF)Click here for additional data file.S3 Fig(A-C) ChIP-seq was performed for the H3K27ac mark on wild type E8.5 embryo heads (light blue) in duplicates and only one replicate is shown. ATAC-seq was performed on dissected regions  from wild type (light blue), Krox20ΔC/ΔC (green) and Krox20ΔA/ΔA (red) E8.5 embryos. Genes, cis-regulatory elements (orange) and a genomic scale are indicated at the top. CTCF ChIP-seq data in E14.5 mouse brain (ENCODE) are indicated below each panel.(TIF)Click here for additional data file.S4 FigTg(NE:gfp), carrying a construct in which NE is linked to the gfp gene driven by a minimal promoter, was analysed by double ISH with krox20 (orange) and gfp (purple) probes at 3s and 10s stages.A zebrafish transgenic line (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


High expression of the transcription factor ZFX is correlated with proliferation, tumorigenesis, and patient survival in multiple types of human cancers. However, the mechanism by which ZFX influences transcriptional regulation has not been determined. We performed ChIP-seq in four cancer cell lines  to identify ZFX binding sites throughout the human genome. We identified roughly 9000 ZFX binding sites and found that most of the sites are in CpG island promoters. Moreover, genes with promoters bound by ZFX are expressed at higher levels than genes with promoters not bound by ZFX. To determine if ZFX contributes to regulation of the promoters to which it is bound, we performed RNA-seq analysis after knockdown of ZFX by siRNA in prostate and breast cancer cells. Many genes with promoters bound by ZFX were down-regulated upon ZFX knockdown, supporting the hypothesis that ZFX acts as a transcriptional activator. Surprisingly, ZFX binds at +240 bp downstream from the TSS of the responsive promoters. Using Nucleosome Occupancy and Methylome Sequencing (NOMe-seq), we show that ZFX binds between the open chromatin region at the TSS and the first downstream nucleosome, suggesting that ZFX may play a critical role in promoter architecture. We have also shown that a closely related zinc finger protein ZNF711 has a similar binding pattern at CpG island promoters, but ZNF711 may play a subordinate role to ZFX. This functional characterization of ZFX provides important new insights into transcription, chromatin structure, and the regulation of the cancer transcriptome. P-value <0.001) in gallbladder adenocarcinoma, and patients that survived <1 yr were found to have significantly higher ZFX expression than patients that survived >1 yr  . There aed >1 yr . Taken tZFX is encoded on the X Chromosome and highly conserved in vertebrates. Among the roughly 2000 site-specific DNA-binding TFs, the C2H2 ZNFs are the largest class encoded in the human genome. Although the biological functions of the majority of ZNFs are unknown, the molecular functions of ZNFs include not only sequence-specific binding to DNA but also protein–protein interactions and RNA binding . DNA-binAlthough the structure of ZFX suggests that it is a DNA binding transcriptional activator that is expressed at high levels in many different types of cancers, ZFX binding sites have not yet been mapped in cancer cells. To understand the mechanism by which ZFX may regulate the cancer transcriptome, we performed ChIP-seq, NOMe-seq, and RNA-seq assays with knockdown experiments in HEK293T kidney, HCT116 colon, C4-2B prostate, and MCF-7 breast cancer cells, identifying ZFX-binding sites and ZFX-regulated genes throughout the human genome.Supplemental Fig. S1 for ZFX antibody validation and Supplemental Table S1 for access information for all genomic data sets). We chose to use these cancer models because there is a strong link in these four cancer types between ZFX expression and cell proliferation, tumor development, or patient survival. For example, prostate cancer tissues exhibit significantly higher ZFX expression than benign prostatic hyperplasia and adjacent tissues and siRNA-mediated knockdown of ZFX suppresses the proliferation of prostate cancer cells and reduces the number of colonies in colony forming assays .We identified roughly 9000 reproducible ZFX binding sites in each cancer cell line . Annotation of the ZFX binding sites with respect to different genomic regions showed that ∼80% of the sites are located in a promoter region (±2 kb of a TSS). In each of the cell types examined, only about 1000 sites are located in distal elements  B. To furSupplemental Table S3E). In contrast, the distal sites bound by ZFX are not always the same in the different cell types. We note that both the common and the cell-type–specific ZFX binding sites are robust and reproducible . Therefore, we asked whether ZFX binding has a similar distribution or, as for most TFs, if ZFX binds mainly upstream of the TSS. We found that ZFX has a stronger preference for binding at +240 bp downstream from the TSS (n = 3961) and plotted heatmaps centered to each TSS . Most of the time, ZFX is bound at +240 bp of the TSS . However, there are a small number of promoters (<10%) that have a ZFX peak at −240 bp , and some promoters that have two ZFX peaks symmetrically located on either side of the TSS . We conclude that the presence of motif, which is symmetrically enriched ±240 bp from the TSS may be necessary, but is not sufficient, to recruit ZFX because ZFX has a higher binding frequency at +240 bp than at −240 bp.We further investigated the distribution of the AGGCCTAG motif relative to the TSS. We found that this motif is symmetrically enriched ±240 bp from the TSS, with CpG island promoters showing greater enrichment than non-CpG island promoters and CpG island promoters bound by ZFX showing greater enrichment than those not bound by ZFX G. The sy the TSS H. The reSupplemental Table S4A). Genes identified as responsive to changes in the level of a TF include both direct target genes and genes that are in downstream signaling pathways regulated by the direct target genes . One approach to identify direct ZFX target genes is to determine which of the deregulated genes have ZFX binding sites in their promoter regions. We found that the promoters of 744 of the 1271 down-regulated genes (58.5%), but only 143 promoters of the 1249 up-regulated genes (11.4%) are bound by ZFX in C4-2B cells . Although the number of deregulated genes in the MCF-7 knockdown experiments is less than in the C4-2B knockdown experiments, the down-regulated genes again have a higher percentage of promoter-bound ZFX (59.1%) than do the up-regulated genes (17.5%)  F.Supplemental Fig. S3A). An alternative explanation could be that another TF is functionally redundant with ZFX in MCF-7. C2H2 ZNFs comprise the largest class of site-specific DNA-binding proteins encoded in the human genome and have arisen through gene duplication followed by mutation. Specifically, ZFX is very similar to ZFY and ZNF711 ; ZFX and ZNF711 are both encoded on the X Chromosome, whereas ZFY is located on the Y Chromosome. Overall protein homology is 92% between ZFX and ZFY, with the zinc finger domains having 97% homology, suggesting that these two proteins may have fully redundant activities . To investigate the possible functional redundancy of these two TFs, in an independent set of siRNA experiments than shown in Supplemental Table S4C). We again observed an increase in ZNF711 expression when ZFX was knocked down  upon knockdown of both ZFX and ZNF711 in MCF-7 cells than in the combined single knockdown experiments .One explanation for the smaller effect on the transcriptome of MCF-7 cells could be inefficient knockdown of ZFX. However, the reduction in ZFX was similar in the siRNA-treated C4-2B and MCF-7 cells , 98.6% of which overlapped with ZFX binding sites in MCF-7 cells. As expected, ZNF711 binding sites were also enriched at CpG island promoter regions, both in MCF-7 cells and in SH-SY5Y cells . Unlike the ZFX binding pattern in the four cancer cell types, ZNF711 appears to have more cell-type–specific binding sites . A comparison of ChIP-seq tags shows enrichment of ZNF711 at the promoter regions bound by ZFX, but the ZNF711 ChIP-seq signals are weaker than the ZFX ChIP-seq signals in MCF-7 cells  but not in the single knockdown cells, and the promoters of the FAAH and LIPT2 genes are bound by both ZFX and ZNF711 in MCF-7 cells . Although Supplemental Fig. S6).The preferred location of ZFX at +240 bp is a unique position for a DNA binding TF. To further characterize the relationship between the bound ZFX and open chromatin surrounding the TSS, we used Nucleosome Occupancy and Methylome Sequencing (NOMe-seq). This genome-wide method identifies nucleosome-depleted regions (NDRs) and provides single molecule resolution for both accessibility and DNA methylation, which can very precisely identify specific TF binding sites . When weWe profiled ZFX binding sites genome-wide in kidney, colon, prostate, and breast cancer cell lines. Unlike many oncogenic TFs that bind to distal elements, ZFX binds to the majority of CpG island promoters that are active in cancer cells, and many genes with promoters bound by ZFX were down-regulated upon knockdown. Surprisingly, ZFX binds at +240 bp downstream from the TSS of ZFX-regulated promoters, in the open chromatin region between the TSS and the first downstream nucleosome. Genome-wide analyses of open chromatin and DNA methylation demonstrate that promoters bound by ZFX have a more accessible region, with lower levels of DNA methylation near the TSS and more highly phased nucleosomes downstream from the TSS, compared to promoters that are active but not bound by ZFX. Taken together, these findings support the hypothesis that ZFX may act as a transcription activator and play an important role in maintaining a nucleosome-free promoter region and/or in positioning nucleosomes at many CpG island promoters in the human genome.Supplemental Fig. S7). For example, cell division cycle 27 homolog (CDC27), a component of the anaphase promoting complex/cyclosome that ubiquitinates Cyclin B (MLH3), which is implicated in maintaining DNA replication and mismatch repair . Although the ZFX binding pattern in normal prostate cells is very similar to the ZFX binding pattern in the prostate cancer cell line, the ChIP-seq peaks in PrEC were considerably smaller, suggesting that high ZFX expression in cancer cells may result in stronger binding and higher expression of genes involved in cell proliferation.In accordance with findings from previous studies , 2015, wCyclin B , and Muth repair , both haAlthough ZFX binds to approximately 8000–9000 promoters in a given cell type, siRNA-mediated knockdown of ZFX resulted in altered activity of only a subset of these promoters. Although the ZFX motif is enriched symmetrically ±240 bp from the TSS, our results suggest that ZFX acts as a transcriptional activator only when bound at +240 bp. However, not all promoters with a ZFX bound at +240 bp responded in the knockdown experiments. There are several possibilities that can explain why reduction of ZFX levels only affected a small percentage of promoters to which it is bound. First, it is possible that the incomplete knockdown of ZFX by siRNA treatment may have prevented the identification of all ZFX-regulated genes. In the future, knockout of ZFX by CRISPR/Cas9 could be performed to determine if a larger set of ZFX-regulated genes is identified upon complete removal of ZFX from the cell. It is also possible that cobinding of ZFX with other TFs is required for ZFX to regulate transcription. Finally, there is the possibility that other TFs are functionally redundant with ZFX. We tested the possibility that ZNF711, a TF that shares high homology and a similar DNA binding motif to ZFX, can substitute for ZFX. Indeed, ZNF711 binding sites are shared by ZFX binding sites, and we identified several hundred additional ZFX-bound target genes that are down-regulated upon knockdown of both ZFX and ZNF711; perhaps complete loss of both proteins is required to observe the full effect of ZFX on the transcriptome.There are two main types of transcriptional regulatory elements, promoters and enhancers. Unlike enhancers, which are located far from a TSS, are cell-type specific, and are closely linked to cellular identity , 2016, pSupplemental Fig. S9); these findings are consistent with a role for ZFX in transcriptional  regulation. ZFX does appear to be uniquely placed in relation to the phased nucleosomes located downstream from the TSS, and ZFX-bound promoters have a more open region near the start site than do promoters that are active but not bound by ZFX. Therefore, we postulate that perhaps ZFX is involved in creating a nucleosome-depleted region in CpG island promoters by recruiting the transcription preinitiation complex and/or in positioning the downstream nucleosomes . The human prostate cancer C4-2B cells were obtained from ViroMed Laboratories. The human normal prostate epithelial cells (PrEC) were obtained from Lonza (Cat# CC-2555). The corresponding medium of each cell line  was supplemented 10% fetal bovine serum (Gibco by Thermo Fisher Scientific) and 1% penicillin and streptomycin at 37°C with 5% CO2. PrEC cells were grown with PrEGM Bullet Kit , which were obtained from Lonza (Cat# CC-3166). All cell stocks except primary cells (PrEC) were authenticated at the USC Norris Cancer Center cell culture facility by comparison to the ATCC and/or published genomic criteria for that specific cell line; all cells are documented to be free of mycoplasma. Preauthentication was performed at Lonza for PrEC, and the first passage from the cultured cells was used for the ChIP assay.The human kidney HEK293T (ATCC# CRL-3216), colon HCT116 (ATCC# CCL-247), and breast cancer MCF-7 (ATCC# HTB-22) cells were obtained from ATCC . ZNF711 ChIP-seq experiments in MCF-7 cells were performed using antibodies from two different rabbits that were generated against ZNF711 amino acids 1–358; these antibodies have been previously used in ChIP-seq and were provided by Dr. Kristian Helin (https://github.com/nboley/idr) after preprocessing data with the ENCODE3 ChIP-seq pipeline (https://www.encodeproject.org/chip-seq/). ZFX and ZNF711 binding sites are listed in Supplemental Tables S3 and S5. A detailed description of ChIP-seq analyses can be found in Supplemental Methods.ZFX ChIP assays were performed in HEK293T, HCT116, C4-2B, MCF-7, and PrEC cells using a ZFX antibody  according to ENCODE standards . The ZFXan Helin . H3K4me3an Helin  with thehttp://homer.ucsd.edu/homer/) .To discover de novo motifs enriched in the ChIP-seq peaks, we collected sequences of 20-bp windows of the ZFX peak summits and used MEME version 4.10.1  with a m/homer/)  was orig/homer/)  and ZFX /homer/) . Therefo/homer/) A. The moSupplemental Table S4.For transient knockdown, cells were transfected in triplicate with 100 nM of siRNA oligonucleotides targeting human ZFX (Cat# L006572000005), ZNF711 (Cat# L008444020005), or control oligonucleotides (Cat# D0018101005) using SMART pool DharmaFECT transfection reagent 3 (Cat# T200301) for C4-2B and reagent 1 (Cat# T200101) for MCF-7 (Dharmacon). Cells were incubated for 24 h and transfected again with the same concentration of siRNAs, and the incubation was continued for an additional 24 h. RNA was extracted using TRIzol reagent  following the manufacturer-suggested protocol. cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit . RNA-seq libraries were made using KAPA Stranded mRNA-Seq Kit with KAPA mRNA Capture Beads  and sequenced on an Illumina HiSeq. To remove batch effects, matched controls and knockdown samples were prepared and sequenced at the same time. Differentially expressed genes were selected using the Gene Specific Algorithm from Partek Flow software with the upper quartile normalization method (Partek Inc.). An FDR cutoff of 0.05 was used to select statistically significantly differently expressed genes. Differentially expressed genes with absolute fold change >1.5 are listed in m does not occur in the human genome  and therefore there is no endogenous background of GpCm. The enzyme can only methylate GpC dinucleotides that are accessible in the context of chromatin, i.e., not protected by nucleosomes or other proteins that are tightly bound to the chromatin. The second step of the method involves bisulfite treatment of the M.CviPI-methylated chromatin, which converts unmethylated Cs to Ts. This allows the distinction of GpC from GpCm and CpG from CmpG. Using this method, NDRs are defined as regions having increased GpCm methylation over background  that are at least 140 bp in length. Because the bisulfite treatment also allows the distinction of CpG from CmpG, the endogenous methylation status of the genome can also be obtained in the same sequencing reaction. It is important to note that in contrast to the induced GpCm, which represents nucleosome-free, open chromatin that is available for TF binding, the endogenous CmpG represents nucleosome-bound chromatin that is not available for TF binding. Open chromatin is expected to have high levels of GpCm but low levels of CmpG; thus, each of the two separate methylation analyses serve as independent (but opposite) measures that should provide matching chromatin designations (open versus closed). C4-2B NOMe-seq data were generated as previously described (https://github.com/dnaase/Bis-tools) , the findNDRs function in the aaRon R package was used (https://github.com/astatham/aaRon).The NOMe-seq method is a combination of the genome-wide identification of open chromatin regions plus whole-genome bisulfite sequencing (to identify methylated DNA). The first step of the method is based on the treatment of chromatin with the M.CviPI methyltransferase. This enzyme methylates Cs in the context of GpC dinucleotides. GpCescribed  and sequescribed . Each FAescribed  and procescribed . To idenescribed  program s-tools) . For ideSupplemental Table S4), and genes bound by ZFX were selected for Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) analysis. The differentially expressed genes were used to identify enriched gene sets using the GSEA tool  <0.05 was used to select significantly enriched gene sets. The same differentially expressed genes were analyzed for enrichment in particular GO categories using the TopGO program (https://bioconductor.org/packages/release/bioc/html/topGO.html). A Fisher's exact test was performed, and an adjusted P-value cutoff 0.05 was used to select statistically significantly enriched GO categories .Differentially expressed genes upon ZFX knockdown are selected using FDR cutoff 0.05 and fold change cutoff 1.5 in C4-2B cells (SEA tool . Hypergehttp://www.ncbi.nlm.nih.gov/geo/) under accession number GSE102616. Access to other publicly available data sets from GEO or ENCODE (Supplemental Table S1.All ChIP-seq, RNA-seq, and NOMe-seq data generated in this study have been submitted to the NCBI Gene Expression Omnibus (GEO; r ENCODE  used in


Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitary gonadotropes, most notably to induce synthesis of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), but also to ensure the appropriate functioning of these cells at the center of the mammalian reproductive endocrine axis. Aside from the activation of gene-specific transcription factors, GnRH stimulates through its membrane-bound receptor, alterations in the chromatin that facilitate transcription of its target genes. These include changes in the histone and DNA modifications, nucleosome positioning, and chromatin packaging at the regulatory regions of each gene. The requirements for each of these events vary according to the DNA sequence which determines the basal chromatin packaging at the regulatory regions. Despite considerable progress in this field in recent years, we are only beginning to understand some of the complexities involved in the role and regulation of this chromatin structure, including new modifications, extensive cross talk, histone variants, and the actions of distal enhancers and non-coding RNAs. This short review aims to integrate the latest findings on GnRH-induced alterations in the chromatin of its target genes, which indicate multiple and diverse actions. Understanding these processes is illuminating not only in the context of the activation of these hormones during the reproductive life span but may also reveal how aberrant epigenetic regulation of these genes leads to sub-fertility. Expr export , 22, 41. by GnRH , 43.loci to provide elaborate cross talk  in yeast SAGA/SLIK HAT complexes is recruited to gene promoters in this way, thereby facilitating HAT activity  while alpromoter , 48. Thicription –51.Cga promoter. The phosphorylation of H3S28 at the Cga 5′end is via GnRH-activation of MSK1, which is a downstream target of ERK and p38 MAPK  in the coding regions of all three gonadotropin genes also increases following exposure to GnRH . This isMonoubiquitinated H2B at lysine 120 (H2BK120ub) is generally associated with actively transcribed genes, as first reported in yeast, although its precise function in mammals has been controversial. It is reportedly required for recruitment of the Set1/COMPASS complex to gene promoters due to its recognition by one of the complex subunits. This ubiquitin is later removed by a component of the SAGA HAT complex to allow recruitment of the kinase that phosphorylates RNAPII at S2, thus signaling promoter escape and elongation , 73. HowThe apparent lack of requirement for H2BK120ub at some mammalian promoters is likely due to the more numerous complexes that can catalyze H3K4me3 than found in yeast. As described above, these complexes contain distinct subunits which allow recruitment of the lysine methyl transferase (KMT) complex through diverse proteins including transcription factors. Accordingly, there is no apparent correlation between levels of promoter H2BK120ub and expression levels of the three gonadotropin genes, nor with levels of H3K4me3 at their promoters. However, GnRH induces a major increase in H2BK120ub levels globally, and specifically in the coding regions of the gonadotropin genes, in keeping with the changes in coding region H3K36me3 described above . The facHistone citrullination, in which histone tail arginine residues are converted by peptidylarginine deiminase (PAD) enzymes to citrulline has been observed but is still poorly understood. The citrullination is thought to induce chromatin decondensation, and was recently shown to be particularly crucial for transcriptional activation during early embryonic development , 78. NotDNA methylation is generally considered a stable mechanism to repress gene expression, often in concert with repressive histone modifications. However, since the discovery of the TET family of enzymes , 81, it Lhb gene in association with promoter H3K27 methylation, possibly playing a role in recruitment of the KMT enzyme to this locus. Tet1 expression is downregulated to allow the precursor gonadotropes to complete their differentiation, following exposure to GnRH and also in response to estrogens or androgens via steroid receptors that bind the Tet1 promoter (Lhb gene promoter by TET2 which hydroxymethylates the methylated CpGs on this gene promoter so, in concert with the GnRH-activated transcription factors, facilitating Lhb expression (Tet1 and the ensuing elevation in gonadal steroids provides the feedback to keep Tet1 repressed.In this context, TET1, which is highly expressed in gonadotrope-precursor cells, was found to repress the promoter . With depression . In thisCga and Lhb gene promoters in functional gonadotropes differs markedly, in accordance with their distinct expression levels and means of regulation. In the gonadotropes, the Cga proximal promoter, similar to that of many other highly expressed genes but unlike its state in non-gonadotropes, is exposed and accessible to transcription factors, such that transcription may be initiated quite easily (Cga expression by GnRH is, therefore, likely directed primarily at the level of RNAPII promoter escape and elongation such that after exposure to GnRH it is quickly released from the promoter (Cga than Lhb promoter in unstimulated mature gonadotropes, and the fact that the first nucleosome in the coding region, characteristic of genes with high RNAPII occupancy (Lhb gene. RNAPII transition through this nucleosome is clearly facilitated by the incorporation of a histone H2A variant which allows greater nucleosomal mobility (For all three gonadotropin genes, nucleosome levels drop following GnRH treatment, reflecting histone displacement , 31. Howe easily . Upregulpromoter , 48. Thiccupancy , is posiCga gene differs fundamentally from that of Lhb whose proximal promoter is packaged into a nucleosome (Figure Lhb promoter (Fshb gene promoter are similarly organized and modified following GnRH exposure.This organization of the e Figure  that ence Figure , 92. Thepromoter , 93. Thepromoter . This inpromoter , 96. TheCga chromatin (The organization of the chromatin at the gonadotropin genes and its GnRH-induced modifications that facilitate transcription is crucial in understanding how these genes are activated during the reproductive lifespan, but may also have implications in non-pituitary GnRHR-expressing cancer cells. There is increasing indication, however, that much larger distal genomic regions function to determine expression of specific genes, as shown for the eRNA that regulates hromatin , 97. TheAll authors contributed to this review, and all have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past coauthorship with one of the authors PM.


Simonini et al. present an alternative auxin-sensing mechanism in which the auxin response factor ARF3/ETTIN controls gene expression through interactions with process-specific transcription factors. Tissue patterning in multicellular organisms is the output of precise spatio–temporal regulation of gene expression coupled with changes in hormone dynamics. In plants, the hormone auxin regulates growth and development at every stage of a plant's life cycle. Auxin signaling occurs through binding of the auxin molecule to a TIR1/AFB F-box ubiquitin ligase, allowing interaction with Aux/IAA transcriptional repressor proteins. These are subsequently ubiquitinated and degraded via the 26S proteasome, leading to derepression of auxin response factors (ARFs). How auxin is able to elicit such a diverse range of developmental responses through a single signaling module has not yet been resolved. Here we present an alternative auxin-sensing mechanism in which the ARF ARF3/ETTIN controls gene expression through interactions with process-specific transcription factors. This noncanonical hormone-sensing mechanism exhibits strong preference for the naturally occurring auxin indole 3-acetic acid (IAA) and is important for coordinating growth and patterning in diverse developmental contexts such as gynoecium morphogenesis, lateral root emergence, ovule development, and primary branch formation. Disrupting this IAA-sensing ability induces morphological aberrations with consequences for plant fitness. Therefore, our findings introduce a novel transcription factor-based mechanism of hormone perception in plants. TIR1/AFB complex /ARF3 gene encodes an atypical ARF lacking the PB1 domain  gene (INDEHISCENT (IND) is a basic helix–loop–helix (bHLH) transcription factor (TF) required for formation of the ispersal . In addiispersal , and, inispersal . IND cooID) gene . PID is ID) gene .Here we show that ETT and IND interact to form a complex that mediates gynoecium patterning and that auxin affects the activity of this complex in the regulation of gene expression. Our data demonstrate the existence of a novel auxin-sensing mechanism comprised of ETT and IND that exhibits a strong preference for the naturally occurring auxin indole 3-acetic acid (IAA) over synthetic auxins and is fundamental for coordinating growth and patterning during carpel development. Moreover, our findings demonstrate that the IAA-sensing function is an intrinsic property of ETT, and the disruption of this IAA-sensing ability triggers morphological aberration in other developmental contexts in addition to gynoecium patterning such as LR emergence, ovule development, and primary branch formation. Thus, our findings introduce a novel TF-based mechanism of hormone signaling in plants.Arabidopsis gynoecium is particularly well suited for such purposes due to its division into highly distinct tissues with different symmetries , the radial style deformation of the ett-3 single mutant is exacerbated in the ett-3 ind-2 double mutant, which fails to achieve complete closure at the apex , suggesting overlapping roles of these regulators. Analyses of the pETT::ETT-CFP and pIND::IND-YFP reporter lines revealed that ETT and IND expression patterns overlap in the apical region and that the fusion proteins colocalize in the nuclei in the cells of this domain . We demonstrated previously that IND promotes radial symmetry at the gynoecium apex via interaction with another bHLH protein, SPT  with the consensus sequence TGTCNN (or NNGACA on the opposite strand). Phylogenetic shadowing using mVISTA (PID genes in the Brassicaceae family and therefore are strong candidates for being recognition sites for ARF proteins (pETT::ETT-GFP (ett-3) line revealed a particularly strong interaction with a region in the PID promoter containing these two elements . Moreover, a yeast one-hybrid (Y1H) assay demonstrated that ETT specifically interacts with these sequences, while this interaction is disrupted when they are mutated to non-AuxREs .The controlled expression of lishment . Given tpression , we therg mVISTA  demonstrproteins A,B. ChroPID promoter is of relevance for PID gene expression, we performed in situ hybridization with a probe against the PID mRNA on longitudinal sections of developing wild-type gynoecia. In agreement with previous data  and together suggest that IND and ETT repress PID expression during early stages of gynoecium growth to ensure proper carpel development.To test whether the interaction of ETT and IND with the ous data , we deteynoecium C. This eynoecium D,E. Morepel body C,F. AnalETT and IND, their overlapping expression pattern, and the fact that both proteins together directly regulate PID expression, we investigated whether ETT and IND proteins interact. To this end, we used bimolecular fluorescence complementation (BiFC) . All of the techniques revealed strong interactions between the two proteins, suggesting that ETT and IND most likely heterodimerize to regulate expression of their target genes.Given the genetic interaction between n (BiFC) A–C, yeasn (BiFC) D, fluoren (BiFC) E–I, and Supplemental Fig. 3B). IND has been divided previously into three domains, including the bHLH domain in the C-terminal half and a central HEC domain with homology with the closest homologs of IND, the HECATE proteins (Arabidopsis and was therefore named the IND-specific (IS) domain. In contrast to ETT, all IND domains appear necessary to maintain the interaction with ETT . Moreover, the interaction with IND was specific to ETT, since no interaction could be detected between IND and the ETT homolog ARF4 . These results further support a synergistic role for ETT and IND in the control of gynoecium development.A deletion analysis demonstrated that the C-terminal ETTIN-specific (ES) domain of 215 amino acids is sufficient for interaction with IND , suggesting that IAA affected the ETT–IND dimerization. Performing the Y2H assay in the presence of IAA showed, in agreement with the BiFC data, that the ETT–IND interaction was sensitive and suppressed at IAA concentrations similar to those used previously for testing the activity of TIR1 and AFBs in yeast  . Although region 1 contains potential AuxREs, our phylogenetic shadowing analysis shows that none of these are conserved in PID genes of the other Brassicaceae species in the analysis. Moreover, the interaction between ETT and region 2 (containing the two conserved AuxREs) is not disrupted by the IAA treatment in both the ChIP and Y1H assays . One way to explain this could be that the increased association of ETT with region 1 in the presence of IAA may be indirect and possibly occurs via another protein bound to this region and facilitated by the modified ETT–IND interaction . The potential importance of sequences within region 1 for PID regulation is reflected in its high overall level of conservation in the phylogenetic shadowing analysis  and with or without IAA. In this analysis, 1734 genes significantly changed expression  following induction of IND. However, out of these, 796 genes required the presence of IAA for IND to have an effect .Additional support of IAA affecting IND activity emerged when we carried out a microarray experiment using a To assess the biological and functional relevance of the IAA-sensing ability in planta, we aimed to identify point mutations in IND and the ETT-ES domain that permitted the formation of the ETT–IND dimer while rendering it insensitive to IAA.IND ORF resulted in the isolation of an aspartate-to-glycine substitution at position 30 (D30G) (D30G variant under the IND promoter (pIND::INDD30G) in the strong ind-2 mutant background led to a striking overproliferation of stigmatic tissue accompanied by reduced pollen tube density and decreased fertility, which was not seen when ind-2 was transformed with a wild-type pIND::IND construct or in the untransformed ind-2 mutant . These results strongly suggest that the IAA sensitivity of the ETT–IND complex is important for proper gynoecium development and plant reproduction.A screening for random point mutations introduced in the 0 (D30G) A, which 0 (D30G) B. ExprespIND::INDD30G in the ind-2 mutant background was perfectly able to rescue its indehiscent phenotype . This observation shows that the INDD30G protein is functional and that the IAA sensitivity of the IND and ETT interaction is not required for specification of the valve margin. This is in agreement with the lack of ETT expression in this tissue .In contrast to the gynoecium defect, expressing D30G complex affected the regulation of downstream target genes. In situ hybridization showed that PID expression was reduced in gynoecia of the pIND::INDD30G line as compared with wild type , thus suggesting that IAA regulates the activity of the ETT–IND complex toward its downstream targets. In the case of PID expression, it is conceivable that ETT–IND acts as a repressor in the absence of IAA, while accumulation of IAA leads to an ETT–IND-dependent induction of PID expression. Additional evidence of an ETT–IND-mediated effect on auxin dynamics in the gynoecium comes from a persisting DR5::GFP signal at the late developmental stages in the pIND::INDD30G line . This emphasizes a defect in auxin dynamics when disrupting the IAA-sensitive ETT–IND activity.Since both ETT and IND are TFs, we tested whether the IAA insensitivity of the ETT–INDSupplemental Fig. 7A,B). In contrast, combining C-to-S mutations in positions 452 and 506 led to growth of yeast also in the presence of IAA (pETT::ETT-C452S-C506S IAA-insensitive variant (referred to here as pETT::ETT2C-S) in the ett-3 mutant background led to the same overproliferated stigmatic tissue phenotype observed in the pIND::INDD30G line . These data therefore strongly support the hypothesis that gynoecium development requires the ETT–IND-mediated IAA-sensing pathway.As opposed to the random mutagenesis approach with IND, we took a site-directed approach with ETT due to the presence of four cysteine residues in the ES domain. Cysteines have been shown previously to modulate transactivational activity of TFs  and coule of IAA A,B. Rema30G line F–H, whilett mutants; however, ETT has been reported to function in a diverse range of developmental contexts in addition to the gynoecium; namely, LR initiation, ovule integument development, and stem–pedicel fusion (pETT(8Kb)::GUS reporter line (pETT::ETT-GFP complementation line confirmed the presence of ETT expression in domains relevant to these three processes. ETT expression was detected (1) in the pericycle (the tissue that will give rise to LRs) and during all stages of LR formation ; (2) during ovule development, coinciding with the region from which the integuments will later emerge ; and (3) at the abaxial side of the new developing primary branches, marking the region that corresponds to the point of conjunction between the stem and the branch .Abnormal carpel development is the most obvious and severe defect in l fusion . Analyseter line  and the Arabidopsis) library of TFs  belonging to different TF families, including HOMEOBOX , AP2 domain (BABYBOOM [BBM]), and TCP . Since ETT already has been reported to interact with ABERRANT TESTA SHAPE  during ovule development  despite the lack of conserved AuxREs .In contrast, IND function appears to be very specific to gynoecium and fruit development . It is ty of TFs . This anelopment , ATS waselopment A. Similaelopment A, thus selopment , ovules elopment , stem–peelopment , and carelopment . Remarkart codon . This reett-3, pETT::ETT-GFP, and pETT::ETT2C-S genotypes with respect to these three different developmental contexts.We next wondered whether the IAA-sensing activity of ETT is a generally conserved mechanism for modulating organ and tissue morphogenesis in response to high IAA concentrations. Indeed, LR initiation, ovule integument development, and primary branch fusion are strictly connected to and dependent on auxin accumulation . TherefoETT expression pattern, the ett-3 mutant displays defects in all the three tissues: a significantly higher number of developing LRs , aberrant ovule integument morphology . In contrast, analyses of the pETT::ETT2C-S line revealed morphological aberrations in all three developmental contexts that could not be explained by complementation of the ett-3 phenotypes.In accordance with the Supplemental Fig. 8C–F). Second, during ovule development, we observed an extensive overgrowth of the outer integument that brings it particularly close to the funiculus , thus placing the micropile aperture, the point of entrance of the pollen tube, in an unfavorable position for fertilization. As a consequence, pETT::ETT2C-S plants displayed very high ovule abortion frequency and a drastically reduced seed set . These results demonstrate that ETT-mediated IAA perception plays multiple diverse roles throughout plant development.First, during the LR emergence process, we detected a significant overproduction of LRs along the root length and in proximity to the root tip . The IAA-dependent modulation of ETT protein interactions implicates that, in a precise spatio–temporal window that coincides with ETT expression, a specific set of ETT partners must be available to achieve a tissue-specific transcriptional response. The identification here of a number of proteins from diverse TF families that interact with ETT in an IAA-sensitive manner supports this hypothesis.Our data suggest that IAA affects the activity of ETT toward downstream targets by directly interfering with ETT–TF interactions O. SpecifThe genetic results as well as the protein–protein interaction data indicate that IAA directly affects the ETT–TF dimer, opening the possibility that the IAA perception occurs via direct contact between the IAA molecule and the ETT protein. If so, this mechanism may be reminiscent of the way by which the thyroid hormone receptor (TR) regulates its targets in animals ::GUS was kindly provided by Toshiro Ito (Plants were grown on soil in long-day conditions (16 h light/8 h dark). Mutant line ckground ; ett-3,  ecotype , was inthiro Ito . SelectiSEMs and sectioning were performed as described previously . GUS stapETT::ETT-GFP in the ett-3 line, and wild-type plants were used as a negative control. Data were analyzed as in pETT::ETT-GFP and wild-type plants with a solution containing 0.1 mM IAA, 10 µM NPA, and 0.03% silwet and collecting material 6 h after treatment. Primers sequences are in Supplemental Table 2.ChIP was performed in triplicate according to PID promoter fragments were cloned in the pHISi vector (Clontech). Interaction was tested on selective yeast synthetic dropout (YSD) medium lacking Leu (L) and His (H) supplemented with different concentrations  of 3-aminotriazole (3-AT). IAA (Sigma) was dissolved in ethanol and added at the desired concentration directly to the cooling medium.The Y1H assay was performed in strains Y187 and PJ64a following the protocol described in The Y2H assays were performed at 28°C in the yeast strain AH109 (Clontech) using the cotransformation technique . Coding Screening of the REGIA library was performed with the mating technique; the ETT coding sequence was cloned in the pDEST32 plasmid (Invitrogen) passing through the pDONR207 (Invitrogen) and transformed in the PJ69a strain. The REGIA library was in the PJ69alfa mating type. After mating, cells were resuspended in sterile water and plated on YSD − W-L-H-A supplemented with different concentration of 3-AT. Plasmids were extracted from positive clones and sequenced.http://www.ibmcp.upv.es/ferrandolabvectors.php) passing through pDONR207 (Life Technologies). The pEAQ-HT vector  using cDNA as template. Developing inflorescences were fixed and embedded in Paraplast Plus embedding medium, cut in 8-μm sections, and then hybridized as described previously  optics.The eviously . For in Arabidopsis (Columbia wild-type) mesophyll protoplasts (n = 28) were analyzed, and average FLIMs of different combinations were exported for generating a box plot. The statistical significance of differences between samples was determined using a two-tailed Student's t-test.FRET/FLIM was performed as described previously  using trtoplasts . Images toplasts . Severalhttp://genome.lbl.gov/vista/index.shtml). PID genomic sequences from Brassicaceae species were obtained from public databases via BLAST search on http://www.ncbi.nlm.nih.gov using the Arabidopsis PID gene as the query sequence.Phylogenetic shadowing was performed using the mVISTA program  glucose, 0.5× Murashige, and Skoog medium with constant shaking and constant light. Seedlings were treated for 6 h with either DMSO mock or 10 μM IAA ± 10 μM DEX. RNA was extracted from three biological replicates . The Robust Multiarray Average (RMA) algorithm was used to normalize the microarray data using Affymetrix expression console. Affymetrix transcriptome analysis console (TAC) software was used for differential gene expression analysis. Genes with a >1.5-fold differential expression and a Pistils were emasculated and pollinated after 24 h with wild-type pollen. After 16–18 h, pistils were carefully isolated from the plants and fixed in a solution of acetic acid and absolute ethanol (1:3), cleared with 8 M NaOH, and labeled with aniline blue (Sigma). Images were taken using a Leica DM6000.pIND (3.2kb)::IND-YFP and pETT(3.7kb)::ETT-CFP constructs were assembled using the Golden Gate method and appropriate plasmids as described previously  was cloned in the pPZP222 plasmid already harboring the GFP-coding region and the tNOS terminator and transformed in ett-3 (Col-0 background). Thirteen independent T1 lines were analyzed for GFP expression pattern. Quantitative real-time PCR analysis was used to estimate the numbers of transgene copies in these individual lines, similar to the approach reported previously  was cloned in the pPZP222 plasmid already harboring a tNOS terminator and transformed in ett-3 (Col-0 background). Cysteine-to-serine mutations were introduced by site-specific mutagenesis. Primer sequences are in Supplemental Table 2.For construction of ETT versions with cysteines mutated to serines, the 2, 0.15 mM MnCl2, and unbalanced dNTP mix . The pool of PCR fragments was incubated with the pGADT7-rec plasmid (Clontech) following the cotransformation protocol described in the Matchmaker library construction and screening user manual (Clontech). Cells were plated on YSD-selective medium lacking Trp (W), Leu (L), Ade (A), and His (H) and supplemented with 100 μM IAA. Plasmids were recovered from those colonies able to grow in the presence of IAA and sequenced . One clone carried five mismatches, including the D30G mutation, and three had a deletion of 12 amino acids, including the D30 residue. The remaining 23 clones contained several errors that affected the correct frame and for this reason were discarded. The IND promoter (3.2 kb upstream of the ATG) plus the IND/INDD30G-coding region were cloned into the pCGN1547 vector already harboring the tNOS terminator. The Agrobacterium tumefaciens strain GV3101 containing the final vector was transformed into ind-2 plants using the floral dip method.Error-prone PCR mix was composed of G2 polymerase buffer (Promega) and G2 polymerase, 5 mM MgCl


GT‐1 cis‐element widely exists in many plant gene promoters. However, the molecular mechanism that underlies the response of the GT‐1 cis‐element to abiotic and biotic stresses remains elusive in rice. We previously isolated a rice short‐chain peptide‐encoding gene, Os2H16, and demonstrated that it plays important roles in both disease resistance and drought tolerance. Here, we conducted a promoter assay of Os2H16 and identified GT‐1 as an important cis‐element that mediates Os2H16 expression in response to pathogen attack and osmotic stress. Using the repeated GT‐1 as bait, we characterized an abscisic acid, stress and ripening 2 (ASR2) protein from yeast‐one hybridization screening. Sequence alignments showed that the carboxy‐terminal domain of OsASR2 containing residues 80–138 was the DNA‐binding domain. Furthermore, we identified that OsASR2 was specifically bound to GT‐1 and activated the expression of the target gene Os2H16, as well as GFP driven by the chimeric promoter of 2 × GT‐1‐35S mini construct. Additionally, the expression of ASR2Os was elevated by pathogens and osmotic stress challenges. Overexpression of ASR2Os enhanced the resistance against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani, and tolerance to drought in rice. These results suggest that the interaction between OsASR2 and GT‐1 plays an important role in the crosstalk of the response of rice to biotic and abiotic stresses.The  To date, over 41 major resistance genes have been identified to against various strains of Xoo and some have been characterized  and R. solani, as well as abiotic salt or drought stress rapidly induced the expression of this gene. Overexpressing Os2H16 significantly enhanced rice resistance to bacterial blight disease, sheath blight disease and drought, suggesting that the Os2H16 promoter is a multipathogen‐inducible and drought‐inducible promoter that contains one or more pathogen‐inducible and drought‐inducible cis‐elements  plays an important role in the Os2H16 promoter response to pathogen infection and osmotic stress. This finding suggested a novel function for GT‐1 in addition to its salt‐ and pathogen‐inducible activities . According to the PLACE database , and this fluorescence was maximized at 12 h p.i. compared with the control. Treatment with Xoc strain RS105 resulted in similar GFP fluorescence patterns. R. solani strain YWK196 induced GFP expression at 12 h p.i., and this expression continued to increase until 24 h p.i. , −1259 (pCXGFP D2) and −720 (pCXGFP D3) showed GFP induction almost equal to that of the pC1381 D0 construct. In contrast, GFP inducible activity was nearly lost in the pCXGFP D4 construct containing a deletion up to −309 , which was reported to be responsive to Pseudomonas syringae in Arabidopsis  and transformed it into the rice cultivar Zhonghua 11. Transgenic rice plants were then inoculated with PXO99 and YWK196 for 24 h, and no infection was used as control. The GFP gene was strongly induced by PXO99 or YWK196 in pC1381 D0 construct, but this induction was significantly reduced in the pCXGFP‐ΔGT‐1 construct . The empty vector (35S mini‐GFP) was used as a control. First, by transient assay in N. benthamiana leaves, a large amount of GFP accumulation was observed in leaves expressing the 2 × GT‐1‐35S mini‐GFP construct, while weak GFP fluorescence was observed in leaves expressing the empty vector ‐treated leaves of Os2H16 promoter transgenic rice, while approximately three quarters of reduction in GFP induction was detected in the pCXGFP‐ΔGT‐1 transgenic rice. This result indicated that the GT‐1 cis‐element is partly responsible for the Os2H16 promoter response to osmotic stress. Moreover, relatively high (threefold) induction of 2 × GT‐1 suggested that the individual GT‐1 cis‐element could also respond to osmotic stress  could induce the expression of the s Figure b.cis‐element and regulate gene expression in response to pathogen and salt treatment in Arabidopsis  analysis to screen the rice cDNA library with GT‐1 as bait. Interestingly, no trihelix DNA‐binding factors were identified from the library, while an abscisic acid, stress and ripening 2 (ASR2) protein was identified and temporarily named OsASR2. OsASR2 contains 138 amino acid residues with a calculated molecular mass of 15.5 kD. Structural analysis showed that it contains an abscisic acid/water deficit stress (ABA/WDS) domain from residues 78 to 128 . In contrast, yeast cells carrying empty vector did not grow on medium lacking Ura and Leu in the presence of 150 ng/mL AbA  was validated by EMSA. As shown in Figure cis‐element. Lanes 3, 4 and 5, which contained fivefold to 50‐fold molar excesses of the unlabelled GT‐1 cis‐element, showed that the intensities of the combined bands weakened. To demonstrate whether the GT‐1 cis‐element was essential for the sequence‐specific binding activity, we tested a labelled mutant probe in which the TTTTTC sequence was changed to CCCCCA. No binding band was detected  and Western blotting  and three RNA interference (RNAi) Tg Figure . As showcis‐element was validated in transgenic rice through ChIP‐qPCR. Chromatin was harvested, and immunoprecipitations were performed using c‐Myc Tag antibody. In addition, no‐Myc immunoprecipitation was used as a negative control. The primers were designed to amplify the promoter sequences with or without the GT‐1 cis‐element . Nine motifs including the GT‐1 cis‐element were identified  and −0.942 (P = 0.009), respectively.To assess the function of OsASR2 in rice disease resistance, we first examined the responses of WT and s Figure a,b. Thres Figure c,d. The R. solani could induce OsASR2 expression, we investigated the role of OsASR2 in the resistance to R. solani. Three T1 OE lines were inoculated with YWK196 and investigated further. After 7 days of inoculation, the OE lines showed reductions in lesion length of 4.0–4.7 cm, compared with 6.2 cm for WT plants  was located in the −411 to −309 region. Moreover, deletion of the GT‐1 cis‐element in −411 to −309 region significantly affected the induction levels of the Os2H16 promoter by pathogens , did not affect the pathogen‐inducible activity of the Os2H16 promoter  small subunit gene and identified as the box II elemenτ ‐A‐A‐(T/A)  is involved in cell‐type‐specific transcriptional regulation  responds to P. syringae treatment  Zhou, . The higi Figure b, suggest Figure b, suggeset al., et al., et al., cis‐element was determined both in vitro and in vivo . Roots were harvested at 3, 6, 12 and 24 h after treatment for total RNA isolation.Rice plants (IRBB13 and Zhonghua 11) and OsActin were used to normalize the expression of each gene. Expression changes were calculated using the ΔΔ Ct method. The primers used are listed in Table Total RNA was extracted using TRI reagent  according to the manufacturer's instructions. First‐strand cDNA synthesis was conducted with SuperQuickRT MasterMix Kit . Quantitative real‐time PCR was performed in an UltraSYBR Mixture (CWBIO) with an ABI QuantStudio™ 6 Flex real‐time PCR detection system. et al., Xoo, lesion length was measured on T0 transgenic lines and WT plants. Lesion length was measured on infected sheaths from transgenic and WT plants at 7 dpi with R. solani YWK196. Three independent T1 transgenic lines and over ten plants for each line were assessed.As previously reported . Illumina sequencing libraries were constructed with the OsASR2‐Myc DNA samples according to the manufacturer's instructions. The end of DNA fragments were repaired and ligated to an adaptor. Then, DNA fragment of ~200 bp was selected using SPRIselect beads and amplified by PCR for 18–20 cycles. The amplified DNA products were collected and sequenced with Hiseq2000.Yeast one‐hybrid assay was performed with Matchmaker Gold Yeast One‐Hybrid (Y1H) Library Screening System  according to the manufacturer's instructions. The probe containing GT‐1 element was synthesized artificially and labelled with dUTP using terminal deoxynucleotidyl transferase  and incubated with the purified OsASR2 protein (1 μg per reaction). Subsequently, EMSA was conducted using a LightShift Chemiluminescent EMSA kit according to the manufacturer's instructions . Chromatin immunoprecipitation (ChIP) was performed with anti‐myc tag antibody‐ChIP grade . Library construction and sequencing was performed by Wuhan IGENEBOOK Biotechnology Co., Ltd (t‐test, and P values <0.05 were considered significant.All data analyses were repeated three times with three replicate experiments independently. Standard deviations were indicated by error bars, and the statistical significances were determined by one‐way variance analysis. The mean differences were compared using Student's PBJ online.Details of the methods for plasmid construction and plant transformation, subcellular localization, yeast one‐hybrid, prokaryotic expression, ChIP and transactivation assays are available in supplementary methods Appendix  at PBJ oThe authors declare no conflict of interest.Figure S1 Schematic map of the Os2H16 promoter with putative promoter elements.Figure S2 Tissue expression patterns of the Os2H16 promoter.Figure S3 Pathogen‐inducible expression patterns of the Os2H16 promoter.Figure S4 Analysis of amino acid sequence of OsASR2.Figure S5 Binding of OsASR2 to the GT‐1 cis‐element in a ChIP assay.Figure S6 Verification of OsASR2 transgenic lines.Figure S7 Overview of ChIP‐seq data.Figure S8 Binding motifs identified in OsASR2 binding peaks in the ChIP‐seq assay.Figure S9 Time causes of OsASR2 expression in response to pathogen and drought treatments.Figure S10 Sequence alignments of OsASR2 with the tomato ASR1.Click here for additional data file.Table S1 PCR primers used.Click here for additional data file.Table S2 GO analysis of genes containing the GT‐1 cis‐element in the ChIP‐seq data.Click here for additional data file.Appendix S1 Supplementary methods.Click here for additional data file.