Caenorhabditis elegans possesses a unique (with various isoforms) FOXO transcription factor DAF-16, which is notorious for its role in aging and its regulation by the insulin-PI3K-AKT pathway. In humans, five genes (including a protein-coding pseudogene) encode for FOXO transcription factors that are targeted by the PI3K-AKT axis, such as in C. elegans. This common regulation and highly conserved DNA-binding domain are the pillars of this family. In this review, I will discuss the possible meaning of possessing a group of very similar proteins and how it can generate additional functionality to more complex organisms. I frame this discussion in relation to the much larger super family of Forkhead proteins to which they belong. FOXO members are very often co-expressed in the same cell type. The overlap of function and expression creates a certain redundancy that might be a safeguard against the accidental loss of FOXO function, which could otherwise lead to disease, particularly, cancer. This is one of the points that will be examined in this “family affair” report.The nematode  Gene duplication is one of the mechanisms that generate functional novelty in evolution. One of the two paralogs created by genomic duplication escapes the pressure of having to maintain its functionality  and can therefore evolve and diversify toward a new functionality. This concept was initially formulated by Ohno in 1970 , is largIt would be intuitive to think that the degree to which the two paralogs differ will determine the degree of novelty of the new functionality and that the more important the variations are, the more functional novelty will be created. However, it could be that the creation of functional novelty is dependent on the fact that paralogs remain very similar and this because of the peculiar mode of action of the gene product involved in duplication. This assumption, which, at first may sound paradoxical, may be one of the reasons for the existence, in a given genome, of gene families that encode for very similar proteins. The FOXO family of transcription factors that is present in humans is one such family. In this review, I will discuss the functional implications of the existence of a restricted family of very similar proteins like the FOXOs, but also part of a more extended family of related FOX factors that most probably evolved from a common ancestor.Drosophila and caused by the mutation in one of the precursors of this family [s family . This dos family ,9.Subfamilies are characterized by additional similarities in the forkhead domain but mainly outside of the forkhead domain .FOX members belong to the functional group of transcription factors that are able to bind DNA in a sequence-specific way, on promoters and enhancers, to regulate gene expression. The standard view of a transcription factor binding to a specific DNA sequence and, via this process, recruiting co-factors to influence transcription is by no way wrong, but it represents only part of how transcription factors actually work. Recent studies that used new genome-wide technologies have enlarged our view about sequence-specific DNA recognition in the context of chromatin and about additional mechanisms involved ,11,12.FOXO genes are rearranged in various cancers [FOXA1 is mutated in prostate and breast tumors [FOXM1 has been shown to be a potent outcome predictor in various cancer types [FOX proteins participate in the most diverse range of biological functions ,13,14,15 cancers ,17; FOXAt tumors ,19,20,21er types .FOXO1, FOXO3, FOXO3B, FOXO4 and FOXO6. These are all regular genes, with the exception of the pseudogene FOXO3B, which has recently been shown to encode for a functional protein as well [FOXO genes originate from successive gene duplications [FOXO3/6 and FOXO1/4 lineages, and two additional duplications leading to the four current genes. C. elegans DAF-16 and the Drosophila dFOXO proteins. Besides the very structured forkhead domain, most of FOXOs consist of intrinsically disordered regions [N-terminus lies in a short conserved region that is found in all of the proteins, the middle AKT site lies at the C-terminal extremity of the DNA-binding domain in a conserved nuclear localization signal sequence (NLS) and the third AKT site lies in a relatively short conserved sequence that serves as a nuclear export signal that is not found in FOXO6. Upon phosphorylation by AKT, FOXOs translocate to the cytoplasm with the exception of FOXO6, which, in most cases, does not follow this rule, as it lacks a nuclear export signal [In humans, five genes encode for FOXO transcription factors:  as well . Bioinfoications : the fir regions . These r regions ,27. One  regions ,29,30,31t signal . Many otC. elegans and Drosophila FOXOs, we see that FOXO3 is the closest, while comparisons between Daf16 and dFOXO reveal that they have a lower identity rate, which may partly be due to differences in the generation numbers of the organisms. When all human FOXOs are compared . This process is discussed at the end of this review. I decided to examine the proteins one by one, at the level of amino-acid identity, which is the most stringent way to compare conservation. The table in lignment ), the nulignment ). FigureFOXO pseudogene, will not be treated in this review, because it is structurally quite different, with an 85-amino-acid extension at the N-terminus and a truncated non-functional forkhead domain with a stop codon in the mid-forkhead domain. The protein is permanently located in the cytoplasm.In conclusion, the average identity in human FOXOs is very high . The forkhead domain is an important part of total identity, implying that average identity is lower in the rest of the protein. Furthermore, FOXOs contain high percentages of intrinsically disordered regions that are usually less conserved . More deFOXO function is often described as and considered to be a collective family function. This is because they are often co-expressed, show co-regulation of their PMTs, have similar effects on reporter expression in vitro and have been shown, in some instances, to have additive, redundant or even synergistic effects in vivo. However, studies have often looked at one particular paralog, without studying the others in the same context. Furthermore, in some cases, double or triple conditional knockouts were compared to wild types, so that the effect of a single knockout in the exact same context remains unknown ,35.FOXOs control tissue homeostasis and are particularly involved in metabolic adaptation to nutrient intake (glucose and lipid metabolism), autophagy, protection from oxidative stress, aging, cell growth, apoptosis and cell-differentiation  and because FOXOs have a pro-apoptotic and cell-growth inhibitory role. This expectation was confirmed in the seminal publication by Paik et al., which showed that the simultaneous deletion of FOXO1, FOXO3 and FOXO4 in mice gave rise to tissue-specific tumors . The facAnother way to collectively inactivate the nuclear function of the FOXOs is by inducing the PI3K-AKT pathway ,56. ThisFOXOs also have redundant functions in glucose metabolism and, more generally, in diet response. The simultaneous deletion of FOXO1 and FOXO3 in the liver is required to observe hypoglycemia and hyperlipidemia in mice ,60,61. IFOXO genes lie on different chromosomes, and therefore have autonomous expression and expression regulation due to their individual chromatin environments, gene promoters, enhancers and regulatory regions  [Human example) . This meFOXOs have a rather broad expression in the organism, especially FOXO1 and FOXO3, which are often co-expressed, leading to functional redundancy for a common function.If FOXOs have autonomous expression, as seen above, then they are not independent, because of this. FOXOs regulate the expression of their paralogs and also their own expression ,69,70,71Many signaling pathways regulate the subcellular localization of FOXOs. In this review, I principally consider the role that FOXOs exert in the nucleus, but FOXOs have also been found to act in the cytoplasm, where they regulate, for example, autophagy ,73 and EBinding to DNA in an autonomous way. The DNA-binding capacity can be regulated by post transcriptional modification, such as phosphorylation (reviewed in ) or acetBinding to another DNA-binding protein without FOXO DNA-binding ,84,85,86Pairwise binding to DNA in which the binding of FOXO to DNA is dependent on the interaction with another protein that itself binds a nearby DNA sequence, such as for FOXO and SMAD . This moThe mode of action determines how similarity in a protein family is exploited functionally. FOXO can regulate transcription in a number of ways in regard to DNA binding, as shown in Transcription factors only bind a subset of their recognition motifs that are present in the genome. One of the reasons for this is the local chromatin structure, which may or may not permit easy access to DNA. Accessibility is epigenetically regulated through histone modification, DNA methylation and chromatin remodulation. Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) allows the genome-wide occupancy of a given factor on chromatin to be determined. ChIP-seq analyses have revealed that occupancy does not necessarily indicate the presence of high-affinity binding sites for a giving transcription factor. It is often low-affinity binding sites that are occupied, suggesting that cooperation in binding with additional factors is common. To make things even more complicated, the binding of a given transcription factor on regulatory sequences does not guarantee an effect on transcription .The Family effect can be exerted in two ways: (1) same cell effect, due mainly to the common DNA-recognition specificity, among family members, that creates functional overlap. The benefit of this effect is that a single DNA element increases its regulatory potential by being hit by more pathways and effector proteins . This maThis effect is mainly caused by the DNA-recognition specificity that is shared by FOXOs family members, but also by other FOX proteins.The crystal and solution NMR structures of several FOX protein-DNA complexes have revealed that the highly conserved residues of helix H3 bind the major groove of DNA and make the majority of the base contacts. In different FOX members, wing residues can make additional specific DNA contacts. An additional alpha-helix is present in some FOX proteins , and the FOXOs subgroup share a common 5 amino-acid insertion that changes the length of the loop between helix H2 and H3 . These fWithin each subgroup, DNA-sequence recognition is very similar if not identical. Differences in specificity can vary between subfamilies.Besides the fact that different FOX family members can recognize very similar sequences , the ideIn muscles, FOXK1 and FOXK2 repress a set of genes involved in autophagy that overlap with the one induced by FOXO3, so that, in starved conditions, FOXO3 is found on promoters were FOXK was found in non-starved conditions . In cardIn hepatocytes, 58% of DNA sites occupied by FOXO1 are also occupied by FOXA2, with carboxylic acids, lipids, steroids and vitamin A metabolism being shared functions, while FOXO1 has a specific function in DNA metabolism and FOXA2 in hepatic development . A similThe mechanisms at play when several proteins regulate transcription through identical cis-acting elements are not known with precision. The interplay of factors may follow different rules, depending on the binding site and the proteins involved: direct competition, exclusion and partial site occupancy, but also cooperation. A great deal must still be learned about binding kinetics, especially in the context of chromatin. Protein–DNA association is concentration-dependent, and when more than one factor is recognizing the sequence, the different affinities for the particular sequence, the diverse concentrations of the factors, their capacity to adapt to the local chromatin state and the presence of co-factors will decide the outcome. Moreover, the dissociation of the DNA–protein complex is relevant. Dissociation was long thought to be spontaneous and independent of concentration. However, recent data have contradicted this view. Higher concentrations of a given transcription factor increase its own off rate, which is a so-called “facilitated dissociation”, shown to occur with dimeric and also with monomeric DNA-binding proteins ,98. The Cooperation between two family members is another possible contribution to the family effect. As mentioned previously, many binding sites are not accessible to transcription factors, because the chromosomal region in which they lie in is in an unfavorable chromatin state.The peculiar characteristics of certain transcription factors enable them to bind DNA/nucleosomes that are located in unfavorable chromatin regions and to initiate a process of chromatin activation. These phenomena are often linked with changes in cell-differentiation stages. A closed chromatin state is part of a mechanism of gene silencing and needs to be relieved to permit the expression of genes that are important for the process of differentiation itself and/or for the function of the differentiated cell. The transcription factors that can perform these tasks have been therefore been called pioneer factors .A FOX member is one of the prototypes for pioneer factors. FOXA1 (HNF3) is able to bind histones and to open compacted chromatin .Transcription factors that possess pioneer factor ability start the process of chromatin remodeling, enabling further binding of transcription factors that do not have this characteristic. This collaboration may happen between FOXA members and FOXO members. FOXA members are essential for the embryonic initiation of hepatic differentiation, but not for the maintenance of nucleosome organization later on. FOXOs, and in particular, FOXO1 are important in many hepatic functions and metabolism. The abovementioned results , which dBesides the recognition of the core FOX recognition sequence by many FOX proteins, other types of sequence recognitions have been reported within FOX proteins. I will mention some here , becauseFOXN1 and FOXN4 recognize the 5bp (GACGC), which is very different from the FOX-consensus sequence ,108,109.When transcription factors bind to nucleosome, their DNA-binding specificity can change. This has been shown for the pioneer factor FOXA1 .Fox proteins bind DNA as monomers, unlike the b-ZIP or the bHLH transcription factors that require homo- or hetero-dimerization to function. However, site selection experiments have shown evidence of a dimeric mode of binding, even for proteins acting as monomers , includiDo other FOX form DNA-mediated homodimers or even FOX hetero-dimers? Some evidence that these kinds of interactions exist come from proteomic analysis, using affinity selection and mass spectrometry . FurtherC. elegans DAF-16 and mouse FOXOs give identical core binding sequence (TTGTTTAC) [Drosophila, mice and humans. They found remarkable conservation between orthologs (over 600 million years), and changes were often linked to the appearance of new functions in new cell types that were not found in Drosophila. Remarkably, human FOXO3 can functionally substitute DAF16 in C. elegans, although only partially [During evolution, the conservation of binding specificity is very strong, and TGTTTAC) . More geTGTTTAC)  have comartially . The conFOXO gene is present in the genome, there will be two possible kinds of cells , in regard to Function F: kind 1, which is negative for it, and kind 2, which expresses it. If two FOXO genes (both with Function F) are present, then we will have four situations, as shown in FOXO gene) to three (with two FOXO genes). When the FOXO gene number rises to three or four, then the number of cell kinds that are positive for Function F rises to 7 and 11, respectively.The principle is shown in FOXO genes , enables and participates in the creation of new cell types. In FOXO genes, creates seven different situations with Function F expression. With seven situations of expression (compared to one with one gene), there are many more chances for Function F expression to adapt and coevolve with the change in global expression pattern required for the establishment of new cell type phenotypes in more complex organisms.More complex organisms mean more specialized tissues and organs with many more cell types being required. In our example, the broadening of the expression of Function F, due to increase in FOXO paralog genes. An example of this is the evolutionary acquisition of functional cysteines [FOXO genes  will be redundant for Function F. With three FOXO genes, four cell kinds  will be redundant for Function F. Redundancy is discussed later, in the evolution section.Furthermore, apart from Function F that remains, new functions can be added or lost on the different ysteines . These nes as in , with boFOXO ortolog DAF-16 gene in C. elegans, which is famous for being required for lifespan extension in insulin receptor (DAF2) mutants [I started this review with a comparison between the five genes present in humans and the single  mutants ,120,121. mutants  with the mutants  in influN-terminus responsible for its different nucleo-cytoplasmic distribution compared to isoform a . The cooperation between the two DAF16 isoforms in life-span extension is dependent on both the different expression pattern and the different sensitivity levels displayed by the two isoforms toward AKT kinases [C. elegans, was confirmed by the finding that, in humans, like in C. elegans and Drosophila, FOXO might be involved in age regulation. An FOXO3 genetic variant correlated with increased longevity in humans [FOXO in C. elegans and gene variants of FOXO in humans is striking and may deserve more attention. Could it be that besides an impressive conservation in function and structure, the FOXOs from humans and DAF-16 from C. elegans also share the functional family effect described herein?Isoform d/f  is expre kinases . The remn humans ,124. TheAs discussed above, FOXOs are partly redundant in their function, with many of the characteristics of this redundancy still to be defined.Maintenance of genetic redundancy is not rare, and for some genes, it has been shown to extend over a period of billions of years of natural selection . HoweverAfter gene duplication, one of the two genes retains the initial function, meaning that there is no immediate selective pressure on the second one to be kept. Preservation theories include rapid mutations leading to new functions and changes in expression, so that gene dosage renders the expression of the two paralogs necessary . It is aOnce the paralog is established in its new function, what are the forces maintaining redundancy? Redundancy is expected to be transparent to selection pressure, and therefore, it is not maintained. The FOXOs are interesting in this regard and highlight the fact that the question of genetic redundancy should be addressed one gene or protein at a time, because the reasons for it and advantages that it represents are very much linked to the function and mechanism of action of the individual protein.One of the theories developed to explain genetic redundancy could be applied to the FOXO family: the piggyback model , which rFor multicellular organisms an additional mechanism applies. As seen in Concerning the tumor-suppressive role of FOXOs, an audacious idea comes to mind when associating The points discussed above weaken the theory that the establishment of redundancy, by co-expressing several FOXOs with a common function, was selected during evolution, to protect the organism from somatic mutations in the FOXO genes. It rather strengthens the possibility that redundancy is the result of other selective needs and is a by-product of it.More experimental data are required to answer some questions about the entangled functionality of FOXO paralog proteins, and new technologies will greatly help to resolve these questions in the future .The fate of duplication has been tested in models, such as yeast ,130,131,FOXO genes provides the possibility of the extended expression of their common functions and the segregated expression of the paralog-specific functionalities that would otherwise be expressed together if present on a single gene . The FOXOs may represent an excellent model for the study of the maintenance of functional redundancy in evolution, a phenomenon that is not easy to explain, because of an apparent lack of selection for it. One possible mechanism is that the common function is preserved on paralogs (at least two to have redundancy) during evolution because required in cells where one FOXO is expressed. When now two or more FOXOs are expressed , the redundancy is created. What are the common functions shared by FOXOs? Regulation of cell growth and apoptosis, protection from particular stresses, and cell metabolism regulation perhaps all of these ultimately influence the aging process.Although FOX proteins members, and even more, FOXO family members within their subfamily, have acquired new functions compared to their paralogs over the course of evolution, they have kept strong relations that enable mechanisms of collaboration, interference, exclusion and redundancy that enhance functionality and regulation potential. Most of these effects are conveyed through the common forkhead domain, exploiting in the paralogs the coexistence of shared and restricted DNA-binding recognition, and the coexistence of both similar and different activity regulation. The existence of several For the moment, too little is known about specific and common functions within the FOXO family. Paralogs are mostly studied individually. For example, precise protein expression and localization information, as well as the sensitivity to diverse post-translational modifications is rarely studied for all the paralogs in an identical given situation. Functional comparisons between paralogs coupled with sequence comparisons might provide a tool that can address mechanistic questions but also understand the forces that molded, through evolution, the FOXOs as we know them now.


HoxD cluster to study how these regulatory architectures are formed and deconstructed once their function achieved. We describe this TAD as a functional unit, with several regulatory sequences acting together to elicit a transcriptional response. With one exception, deletion of these sequences didn’t modify the transcriptional outcome, a result at odds with a conventional view of enhancer function. The deletion and inversion of a CTCF site located near these regulatory sequences did not affect transcription of the target gene. Slight modifications were nevertheless observed, in agreement with the loop extrusion model. We discuss these unexpected results considering both conventional and alternative explanations relying on the accumulation of poorly specific factors within the TAD backbone.Developmental genes are often controlled by large regulatory landscapes matching topologically associating domains (TADs). In various contexts, the associated chromatin backbone is modified by specific enhancer–enhancer and enhancer–promoter interactions. We used a TAD flanking the mouse  Hox gene family of transcription factors. These proteins instruct progenitor cells at different levels along the main anterior to posterior axis, about their developmental fates. In addition to this ancient role in trunk patterning, subsets of the four Hox gene clusters were co-opted during evolution to promote the development of secondary body axes such as the limbs and the external genitalia   .HoxD cluster, the control of gene transcription in the emerging GT involves cis-regulatory sequences located in a 700 kb large regulatory landscape positioned 5’ to the cluster, referred to as C-DOM (centromeric domain). This rebolished  and subsbolished . Geneticbolished . Overallbolished .HoxD cluster (T-DOM), which includes a large variety of enhancers with distinct specificities regulating ‘anterior’ Hoxd genes, the C-DOM appears to be devoted to the control of the most posterior and distal terminal body structures by regulating mostly Hoxd13 either in digit cells or in the GT. The tropism of C-DOM enhancers for Hoxd13 results from the presence of a strong chromatin boundary between this target gene and the rest of the cluster, which concentrates the action of centromeric enhancer on this precise target  effect on transcription in the GT. Moreover, the deletion of the only bound CTCF site detected in the central part of the regulatory landscape, did not impact the transcriptional outcome, even though its inversion reallocated contacts in a manner compatible with the loop extrusion model  and analyzed datasets from three different stages of GT embryonic development starting from embryonic day 12.5 (E12.5), E16.5 and E18.5. We observed that genes positioned in the 5’ portion of both HoxA (Hoxa7 to Hoxa13) and HoxD (Hoxd8 to Hoxd13) clusters were expressed at all developmental stages  at E10.5, the major contributing embryonic tissue to the emergence of the GT , GT at E13.5  and GT at E17.5  and performed ATAC-seq and ChIP-seq for both H3K27ac and H3K27me3 chromatin marks. At E10.5, prior to GT formation, all Hoxd genes and Evx2 were accessible as defined by ATAC-seq  dataset    became vHoxd13 continued to increase when compared to the CR sample , contacts between various enhancer regions and R sample . As devex or GT2 .Hoxd13 during GT development, and the island V region which showed more constitutive contacts, as viewpoints in 4C-seq experiments. We used 4C-seq libraries for E12.5, E13.5, E15.5, and E17.5 GT cells and E12.5 forebrain cells as the negative control. We confirmed that the interactions between GT2 and the Hoxd13 region substantially decreased from E13.5 to E17.5, whereas contact frequencies between island V and Hoxd13 was essentially stable, regardless of the stage and tissue analyzed , a protein described to facilitate enhancer-promoter contacts by DNA-looping .We next explored the functional dynamics of C-DOM during GT development. A detailed analysis of our CR ATAC-seq dataset revealed several accessible chromatin sites, some of which match previously identified GT enhancers such as GT2 . In E17.Hoxd genes during GT development by using a series of partial deletions, in particular the Del(rel1-rel5), Del(rel5-SB) and the Del(SB-Atf2) alleles (Del(IV-SB) allele corresponding to a deletion between island IV and SB . We analyzed the effect of each of these four deletions on Hoxd genes transcription by RT-qPCR at E12.5.We evaluated the functional importance of C-DOM for the transcriptional control of  alleles , bottom,V and SB , bottom.Del(rel1-rel5) allele, one-third of C-DOM is removed, including two digit and/or GT enhancers (GCR and Prox) (Hoxd13 mRNA levels was scored in the GT (p=0.0005), whereas Hoxd12, Hoxd11 and Hoxd10 were less affected (Del(rel5-SB) allele is a 300 kb large deletion of C-DOM including the GT2, and the island III, IV and V regulatory sequences. Mice carrying this deletion displayed a greater effect on the steady-state level Hoxd13 mRNAs, which was reduced by 76% (p<0.0001). Again, Hoxd12, Hoxd11 and Hoxd10 were affected to a lower extent (Del(IV-SB) allele and noticed a 38% decrease in the amount of Hoxd13 mRNAs (p=0.0066), yet no significant effect was detected for any other genes (Del(SB-Atf2) allele where the most centromeric part of the TAD had been deleted. In these mutant mice, we observed a slight but significant upregulation of Hoxd13 mRNA levels (p=0.003) in the GT, whereas other genes were not affected (Hoxd13 in the developing GT.In the nd Prox) , bottom.affected . The Delr extent . We nexter genes . Finallyaffected . Taken tHoxd13 transcription. We applied CRISPR/Cas9 genome editing to fertilized eggs and generated a series of alleles where these elements were either deleted or inverted. We initially focused on the region between the rel1 and rel5 breakpoints (Hoxd13 and was accessible in the GT at E13.5 as seen by ATAC-seq (Del(Prox) allele, a 13 kb large deletion including the Prox sequence (Hoxd13 by RT-qPCR in E12.5 GTs (p=0.006) (Hoxd13 expression pattern detected by whole mount in situ hybridization (WISH) remained spatially unchanged  . This senchanged . This reDel(Prox) mutant E13.5 GTs  (3 kb large deletion), Del(IV) (11 kb) or Del(IIIE) (39 kb) allele. In addition, to assess the importance of bound CTCF proteins within island V, we both deleted and inverted this 14 kb large region (Del(V) and Inv(V), respectively; Hoxd13 mRNA levels by RT-qPCR and examined the transcript distribution by WISH. Unexpectedly, we did not detect any significant difference, either in transcript levels or in their spatial patterns, in any of the Del(GT2), Del(IV), Del(IIIE), Del(V) and Inv(V) alleles  allele was particularly surprising since this sequence displayed a strong, highly specific and continuous staining in the GT in transgenic embryos and also because of the robust transcriptional down-regulation obtained when using a larger deletion including it. Consequently, we performed both 4C-seq and ATAC-seq in Del(GT2) homozygous GT at E13.5 to assess whether this deletion would at least impact the functional organization of the regulatory landscape . Amongst them, the possibility that the functional contribution of GT2 is required at a particular stage of GT development, which was not considered in our analyses. To explore this possibility, we used RT-qPCR to measure the cription . At this  and Inv(V) alleles to evaluate the importance of this element in ensuring proper 3D-chromatin organization at the HoxD locus. We first defined the CTCF binding profile in wildtype and mutant E13.5 GTs by using both ChIP-seq and CUT and RUN. In the wildtype locus, our ChIP-seq results showed several CTCF binding sites in the centromeric part of C-DOM, primarily between island II and Atf2 and matching with other islands and 4C-seq peaks, close to the centromeric TAD boundary (Evx2 and island II (approximately 550 kb) where a clear binding of this protein was detected  allele showed a loss of the associated CTCF binding (Inv(V) allele in GT at E13.5, indicating that the inversion did not affect its binding capacity (Del(V), and Inv(V) homozygous GT at E13.5. In mutant Del(V) GT cells, with the exception of the deleted region, we did not observe any change in the ATAC-seq profile (Inv(V) GT cells, we observed the loss of one ATAC-seq peak located between the GT2 and CsB sequences (Del(GT2) allele. Therefore, neither the deletion nor the inversion of this centrally-located CTCF site had any substantial effect on the accessibility of the remaining regulatory elements, corroborating the RT-qPCR results where expression of Hoxd13 was unchanged in these two alleles  and Inv(V) mutant alleles on GT cells at E13.5 to investigate whether either the absence or the inversion of the CTCF site would affect the interaction landscape within C-DOM. When Hoxd13 was taken as a viewpoint for the Del(V) allele, the global interaction profile between Hoxd13 and C-DOM was virtually identical to control  allele modified the orientation of this centrally-positioned CTCF site, we analyzed the impact of this inversion upon chromatin conformation. Qualitative analysis of the interaction profile generated using Hoxd13 as a viewpoint revealed a slight disruption in the contacts between Hoxd13 and island V (Hoxd13 thus confirming the previous result (Hoxd13 (The majority of CTCF mediated chromatin loops are established between sites displaying opposite and convergent orientations (i.e. with CTCF motifs pointing toward each other) . Becauses result . Notewors result . This ins result . When weHoxd13 and neighboring Hoxd genes during distal limb bud development, a structure that resembles in many respects the developing genitals . Among them is the possibility that the transcriptional outcome of the C-DOM regulation may rely upon an unspecific and global effect of accumulating various factors within the landscape architecture, thus licensing the TAD for activation of the target genes. The same C-DOM TAD was previously shown to regulate genitals . In thiscificity .HoxD regulatory landscapes , rather than a field containing a range of disparate enhancers with specific features and acting at different times. Also, the chromatin architecture associated with this specific developmental context was already observed in the E10.5 CR, that is before the emergence of the GT. Therefore, this internal-TAD micro-organization predates the outgrowth of the GT structure, which suggests -but does not demonstrate- a causal relationship or at least a necessity for the TAD to be fully primed for the structure to develop.We looked at chromatin dynamics at the Hoxd13. The highest frequency of interactions with C-DOM was scored in E12.5 and E13.5 GTs, which correlated with an increase in Hoxd13 transcription, an enrichment of H3K27ac marks and increase in binding of HOX13 proteins at discrete enhancer elements. After this time-point, a decrease in Hoxd transcript levels were scored in parallel with a reduction of all contacts associated with the active regulatory regions within the C-DOM. By E17.5 the C-DOM structure within the GT was reduced to a framework of constitutive interactions associated with CTCF binding sites, similar to the one observed in ES cells and fetal forebrain cells.Our time-series sampling gave us the unique opportunity to follow the C-DOM TAD dynamics in a developing system where most of the cells at E17.5 derive from a homogenous population of mesodermal cells in the nascent genital bud at E12.5, all expressing Hoxd13 expression was scored in the anlagen of the corpus cavernosum while other cells of the tubercle became negative. Of note, this concentration of positive cells in the blastema and subsequent restriction to the periphery  differentiates from this region in the mouse as in many other mammals.This global decrease, observed at a cell population level, can be explained either through a general decrease in transcription or through the selective transcriptional switch-off in some cell types along with their progressive differentiation thus leading to a dilution effect. Detailed ISH analyses see Figures 2 and 3 in eriphery  resembleHoxd13. HOX13 products are indeed potent dominant negative proteins  of Hoxd13. Upon deletion of this region, Hoxd13 contacts with functional enhancers would be re-enforced. Also, this effect may be a consequence of a global reorganization of the genomic landscape, for this specific deletion removes a TAD boundary. Because of the observed weak transcriptional effect and the difficulty to discriminate between many parameters, the analysis of this allele was not pursued further.The deletion of the most centromeric part of C-DOM unexpectedly resulted in a slight but significant upregulation of rel5 to SB DNA fragment was deleted, a substantial decrease in Hoxd13 transcription was observed. However, the deletion of any single candidate sequence in isolation identified within this segment did not elicit any detectable decrease in transcription. This systematic analysis echoes previous studies where deleting a single and well-characterized enhancer did not have the expected effect upon its target gene  CR at E10.5.In contrast, the deletion of the Prox enhancer resulted in a decrease in nditions . Our tranditions . FinallyBesides these potential explanations, the binding of HOX13 proteins to most -if not all- of these C-DOM regulatory sequences raise yet another potential explanation related to recent work showing that phase-separation-induced condensates of RNA Pol II, transcription factors (TF) and the Mediator complex are present at particular enhancers leading to transcriptional activation . In thisRel5-SB sub-region of C-DOM contains the largest series of defined GT regulatory sequences involved in Hoxd13 regulation. Within this region lies island V, which contains the only occupied CTCF site in the central part of C-DOM. We initially assumed that this site could help increasing the spatial proximity between these enhancers and Hoxd13 through looping due to the presence of several CTCF sites around Hoxd13 with convergent orientations , Del(rel5-SB) and Del(SB-Atf2) alleles can be found in Genotyping of all alleles was done by PCR. Mouse tissue biopsies were lysed for 15’ at 95°C, 800 rpm, in lysis buffer . For all genotyping reactions PCR was performed with a standardized cycling protocol (1x (94°3’), 2x , 30x , 1x (72°10’)). The primers used to genotype the Del(rel1-rel5), Del(rel5-SB) and Del(SB-Atf2) alleles (crispr.mit.edu web tool (the Zang laboratory) for the Del(V), Inv(V) and Del(GT2) alleles, or CCTop (Del(IV), Del(IIIE), Del(Prox) and Del(IV-SB) alleles (Del(V), Inv(V) and Del(GT2) strains were produced by pronuclear injection (Del(IV-SB), Del(IV), Del(IIIE) and Del(Prox) strains were produced by electroporation   . Their ices.org)  and targHoxd13 WISH probe was previously described in Whole-mount in situ hybridization (WISH) was performed according to later stabilization reagent (Invitrogen). RNA was extracted from single micro-dissected GT (E12.5) or single cloaca region (CR) (E10.5), using Qiagen Tissue Lyser and RNeasy Plus kit (Qiagen), according to the manufacturer’s instructions. RNA was reverse transcribed using Superscript III (Invitrogen) or Superscript IV (Invitrogen) and random hexamers. qPCR was performed on a CFX96 real-time system (BioRad) using GoTaq qPCR Master Mix (Promega). Primers were previously described in −ΔCt method using a reference gene. Tubβ was chosen as internal control and the mean of wildtype control samples was set as reference to calculate the ratio between the different samples. Graphical representation and statistical analysis were performed with GraphPad Prism 7.Before processing, all tissues were stored at −80°C in RNACircular chromosome conformation capture (4C-seq) was performed as described in http://htsstation.epfl.ch) (HoxD cluster and smoothened using a window size of 11 fragments. Quantifications of the interactions established with the cis-regulatory elements in www.r-project.org).4C-seq reads were demultiplexed, mapped on GRCm38/mm10 mouse assembly, and analyzed using the 4C-seq pipeline of the Bioinformatics and Biostatistics Core Facility (BBCF) HTSstation (epfl.ch)  or usingMicro-dissected GT (35 to 40) or CR (70) were crosslinked in 1% formaldehyde/PBS for 20 min and stored at −80°C until further processing. Chromatin was sheared using a water-bath sonicator (Covaris E220 evolution ultra-sonicator). Immunoprecipitation was done using the following antibodies, anti-CTCF , anti- H3K27ac , and anti-H3K27me3 . Libraries were prepared using the TruSeq protocol, and sequenced on the Illumina HiSeq system (100 bp single-end reads) according to manufactures instructions.GATCGGAAGAGCACACGTCTGAACTCCAGTCAC). Reads were mapped to the mouse genome (mm10) using Bowtie2 (v2.3.4.1) (gsize 1870000000) using the corresponding input data as control BAM (-c). CTCF motif orientation was assessed using the CTCFBSDB 2.0 database  , with st2.3.4.1) . Peak cadatabase , with EM2 and 0.1% IGEPAL CA-630), nuclei were carefully resuspended in 50 μl transposition reaction mix  and incubated at 37°C for 30 min. DNA was isolated with a MinElute DNA Purification Kit (Qiagen). Library amplification was performed by PCR (10 to 12 cycles) using NEBNext High-Fidelity 2x PCR Master Mix . Library quality was checked on a fragment analyzer, and paired-end sequencing was performed on an Illumina NextSeq 500 instrument (read length 2 × 37 base pairs).ATAC–seq was performed as described in fr --dovetail --very-sensitive-local). Reads with mapping quality below 30, mapping to mitochondria, or not properly paired were removed from the analysis. PCR duplicates were filtered using Picard (v1.56.0). Peak calling was done using MACS2 (v2.1.0.20151222) callpeak . The coverage was done using the center of the Tn5 insertion and extended on both sides by 20 bp (script developed by L. Lopez-Delisle). When indicated, coverage profiles represent an average of the replicates, this was done by dividing each replicate by the number of million reads that fall within peaks in each sample  and calculating the average coverage.ATAC-seq reads processing was done on the Duboule local Galaxy server . Reads wMicro-dissected GT from different embryonic stages were stored individually at −80°C in RNAlater stabilization reagent (Ambion) before further sample processing. Total RNA was extracted from tissues using Qiagen RNeasy Plus Micro Kit (Qiagen) after disruption and homogenization. RNA quality was assessed using an Agilent 2100 Bioanalyser. Sequencing libraries were prepared according to TruSeq Stranded mRNA Illumina protocol, with polyA selection. RNA-seq libraries were sequenced on an Illumina HiSeq 2500 sequencer, as single-end reads (read length 100 base pairs).Raw RNA-seq reads were aligned on the mouse mm10 genome assembly using TopHat 2.0.9 . Gene ex2, 0.05% Digitonin) was added and samples were incubated on an ice-cold block for 30 min. STOP buffer  was added and samples were incubated at 37°C for 30 min, replaced on a magnet stand and the liquid was removed to a fresh tube. DNA was extracted by Phenol-Chloroform extraction and ethanol precipitation. Libraries were prepared as described in CUT and RUN  was perffr --dovetail --very-sensitive). Reads with mapping quality below 30 or not properly paired were removed from the analysis. The output BAM file was converted to BED using bamtobed bedtools v2.18.2 (format BED --keep-dup 1 --bdg --nomodel --extsize 200 --shift −100).CUT and RUN reads processing was done on the Duboule local Galaxy server . Adapter v2.18.2 . The cov v2.18.2  , under license No GE 81/14 (to DD). In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by three peer reviewers, including Job Dekker as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: José Luis Gómez-Skármeta (Reviewer #3).Thank you for submitting your article "A complex regulatory landscape involved in the development of external genitals" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.HoxD locus and external genitalia as paradigm, the authors perform timecourse experiments to understand the correlation between structure and function and, importantly, generate a series of transgenic mouse lines to functionally disentangle the role of individual regulatory elements. Based on these observation they make the following conclusions: 1) the TAD regulatory environment of the HoxD locus is mostly unchanged during development, however specific regulatory elements become accessible and differentially interact with Hoxd13 promoter upon gene activation; 2) while large deletions have strong effect on transcription, single enhancer deletions have surprisingly little effect on gene expression (with one notable exception) and 3) Inversion or deletion of CTCF contacts alters chromatin architecture landscape in agreement with predictions of the loop-extrusion model but has no effect on gene expression or enhancer-promoter contacts.In this manuscript, Duboule and colleagues examine not only how complex regulatory architectures are established to achieve a specific transcriptional outcome, but also how they are rewired after. Using the eLife provided that the following important issues are addressed:In general, the work is well done; the findings are interesting and novel and provide evidence that enhancer-promoter contacts are dynamically regulated within the general architecture of the encompassing TAD. The extensive transgenic mouse lines provide evidence that functional outcome of genetic manipulations cannot be readily predicted based on chromatin accessibility and 3D proximity and further confirmation that enhancer regulation can also be CTCF-independent. Overall, I believe that the manuscript could be potentially suitable for publication in Essential revisions:1) Replicates and biological reproducibilityIn many figures the 4C signal is quantified  and conclusions are made based on differences across conditions without showing what is the biological variability of such quantifications. Based on the Transparent Reporting File 2 biological replicates exist for these experiment – they should be analyzed separately to determine whether the differences are meaningful or due to biological variability.2) Data presentation consistencyHoxd13 has been downregulated (at E17.5). The authors should display consistently the ATAC and H3K27ac tracks across all three timepoints for a fair comparison. Similar issue is in Figure 2B when we compare the 4C profiles between mES and CRE10.5 with gene expression in Figure 1B, which doesn't have the mES timepoint. Does expression of Hoxd13 increase between mES and CR E10.5?In many cases only selected timepoints are shown in the figure panels which sometimes makes the interpretation difficult. For example, in Figure 3A ATAC tracks from CR E10.5 and GT E13.5 are compared with H3K27ac tracks from GT E13.5 and GT E17.5. This makes it difficult to see if the accessibility at the Prox enhancer persists also after Hoxd13 expression3) Contribution of the GT1 region to Hoxd13 promoter in a dynamic manner, is open at E13.5 but has no H3K27ac. Furthermore, in the Del(SB-Atf2) line, where the GT1 region is also removed, Hox13 expression is upregulated. One hypothesis would be that this region represents a transcription repressor , but the authors never discuss this. Such hypothesis could also potentially explain why the long deletion in the Del(rel5-SB) line has a major effect but there is little contribution of individual enhancer elements – it could be that the big deletion reduces the genomic distance between the Hoxd13 promoter and GT1, therefore facilitating repression. It would be of great interest to see: 1) Does the interaction between Hoxd13 and the GT1 region increase in the Del(rel5-SB) line? 2) Are there any motifs present in GT1, suggesting binding of repressors, is it marked by Polycomb? and 3) would the precise deletion/mutation of GT1 also lead to upregulation of Hoxd13.The region labeled as GT1 in Figure 2 appears to interact strongly with Lnpk, Evx2 and Atf2 (in the C-Dom) affected upon Prox deletion? This will address if Prox is regulating only HoxD13 or has a general enhancer activity within the TAD. It would also help discriminated between additional layer of E-P specificity versus a global activation within the TAD. What is the expression of Hoxd13 in this line at CR 10.5 and E17.5? Analogous for Figure 6 – does the deletion of GT2 affects the expression of neighboring genes? Lnpk is of particular interest, given the potential interaction based on Figure 2—figure supplement 1. Related to this point: Does inversion of the CTCF site in element lead to leakage of GT2 or Prox activity towards genes on the centromeric side of HoxD? It may not be surprising that deletion of element sC, II, III, IV by themselves do nothing, as they are "behind" the CTCF site in element V . When element V is deleted: does deletion of these centromeric distal elements now alter Hoxd13?4) In Figure 4: Is the expression of neighboring genes such as 5) A major conclusion is that the deletions do not affect the overall architecture of the locus. The question is whether 4C-seq, with just one or two anchors has enough power to conclude that there is no effect on the global architecture of the C-DOM landscape.Hoxd13 now interact with other elements ? If so, are these elements contained within the larger deletion that did have an effect on Hoxd13 expression?6) When GT2 or Prox are deleted: does eLife. Your article has been reviewed by three peer reviewers, including Job Dekker at the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: José Luis Gómez-Skármeta (Reviewer #3).Thank you for submitting your article "A complex regulatory landscape involved in the development of mammalian external genitals" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Essential revisions:eLife.The current 4C data represent only 1 replicate. Two reviewers indicated that it was critical to demonstrate reproducibility. They also understood the technical challenges. Therefore, we ask for adding biological replicates to confirm the most important and key findings. This would include replicates of some of the 4C analyses. When such data can be included the work would be suitable for  Essential revisions:1) Replicates and biological reproducibilityIn many figures the 4C signal is quantified  and conclusions are made based on differences across conditions without showing what is the biological variability of such quantifications. Based on the Transparent Reporting File 2 biological replicates exist for these experiment – they should be analyzed separately to determine whether the differences are meaningful or due to biological variability.We apologize for not being very clear regarding the number of replicates. We did produce replicates for all the deletion alleles and the corresponding controls (GT E13.5). The number of biological replicates for each condition  are indicated in the corresponding figure legends. However, we did not produce replicates for the samples used in the “developmental time series” . We determined it was not necessary in this specific case, mainly for two reasons. First because we made the converse experiment (i.e. using viewpoints on the islands) to confirm the changes. We understand that this is not the same as a biological replicate, yet the same result obtained by two (or more) different viewpoints is certainly as valid as running one duplicate for a single viewpoint. In the case where the material is rare and necessitates too many embryos (e.g. CRE10.5), we generally prefer using various viewpoints. Secondly, the time course of the GT in vivo (unique thus far in the literature) is self-controlled, i.e. the interactions that are not “specific” for one particular stage are used as internal comparative controls to identify those contacts that evolve along with the time course. This is particularly visible in Figure 2, where some peaks initially increase and subsequently decrease, when compared both vertically  and horizontally (control interactions at the same time-point).The Transparent Reporting File was corrected accordingly.2) Data presentation consistencyIn many cases only selected timepoints are shown in the figure panels which sometimes makes the interpretation difficult. For example, in Figure 3A ATAC tracks from CR E10.5 and GT E13.5 are compared with H3K27ac tracks from GT E13.5 and GT E17.5. This makes it difficult to see if the accessibility at the Prox enhancer persists also after Hoxd13 has been downregulated (at E17.5). The authors should display consistently the ATAC and H3K27ac tracks across all three timepoints for a fair comparison. Similar issue is in Figure 2B when we compare the 4C profiles between mES and CRE10.5 with gene expression in Figure 1B, which doesn't have the mES timepoint. Does expression of Hoxd13 increase between mES and CR E10.5?We tried to show the most significant datasets for the message we like to convey and thus we did not overpack the figures with confirmatory results. Nevertheless, we agree with this comment and we have re-organized the datasets to increase consistency. Concerning the first part of the question, we have now added to Figures 1 and 3 the ATAC-seq dataset for E17.5 for better comparison. We also removed the H3K27ac ChIP-seq data from Figure 3. Concerning the H3K27ac ChIP-seq for the cloaca, while we are confident in the results in the gene cluster , we would need to do the experiment once again to determine that the C-DOM is virtually depleted of this mark. This experiment would require dissecting hundreds of young fetuses, which we do not think is necessary, nor desirable, considering the small amount of information that this experiment will deliver. As a consequence, we have removed the data from the figure.Hoxd genes increases from mES to the CRE10.5 sample, for there is essentially no expression of thesegenes in murine ES cells . Likewise, Hoxd genes are not transcribed in forebrain cells at any developmental stage considered and, consequently, there is little basis to quantifying Hoxd13 either in mES cells, or in FB cells. On lines 188 and 189, we write: “These two profiles likely reflected the 3D chromatin state of C-DOM in the complete absence of transcription”, which we think is clear enough.To the second aspect of the question, the transcription of Hoxd13 in Figure 2. Also, we slightly amended Figure2A to make it clear that Atf2 is not part of the C-TAD.To clarify this issue, we added a schematic of the expression of 3) Contribution of the GT1 region to Hoxd13 expressionThe region labeled as GT1 in Figure 2 appears to interact strongly with Hoxd13 promoter in a dynamic manner, is open at E13.5 but has no H3K27ac. Furthermore, in the Del(SB-Atf2) line, where the GT1 region is also removed, Hox13 expression is upregulated. One hypothesis would be that this region represents a transcription repressor , but the authors never discuss this. Such hypothesis could also potentially explain why the long deletion in the Del(rel5-SB) line has a major effect but there is little contribution of individual enhancer elements – it could be that the big deletion reduces the genomic distance between the Hoxd13 promoter and GT1, therefore facilitating repression. It would be of great interest to see: 1) Does the interaction between Hoxd13 and the GT1 region increase in the Del(rel5-SB) line? 2) Are there any motifs present in GT1, suggesting binding of repressors, is it marked by Polycomb? and 3) would the precise deletion/mutation of GT1 also lead to upregulation of Hoxd13.Prox) had the strongest impact on Hoxd13 transcription. The situation turned out to be complex enough, with potential activating enhancers, such that we decided to ignore the up-regulation of Hoxd genes upon removal of the most centromeric deletion. The main reason for this is that the GT1 sequence was isolated because it displayed transgenic activity in the GT (LacZ staining). Therefore, should this sequence have a repressive activity, it would need to be a context-specific repression, whereas the sequence would be an activator outside its genomic context. While of potential interest, this investigation would require years of work using embryonic material and is in fact not on the agenda.We certainly understand this interest for the GT1 sequence. We nevertheless decided to focus this work on the central and telomeric parts of C-DOM, rather than on the centromeric part containing GT1, mostly because the central deletion (and that of We nevertheless introduced a paragraph in the Discussion section “Mechanism(s) of action of long-range enhancers”, which now proposes some potential explanations related to the GT1-containing deletion.Also, GT1 does not contain any salient DNA motive that would be known either to bind a repressor, or to induce formation of a chromatin structure that may be associated with transcriptional down-regulation (such as a TE). Along this line, the GT1 sequence does not display any histone modifications associated with polycomb binding.4) In Figure 4: Is the expression of neighboring genes such as Lnpk, Evx2 and Atf2 (in the C-Dom) affected upon Prox deletion? This will address if Prox is regulating only HoxD13 or has a general enhancer activity within the TAD. It would also help discriminated between additional layer of E-P specificity versus a global activation within the TAD. What is the expression of Hoxd13 in this line at CR 10.5 and E17.5? Analogous for Figure 6 – does the deletion of GT2 affects the expression of neighboring genes? Lnpk is of particular interest, given the potential interaction based on Figure 2—figure supplement 1. Related to this point: Does inversion of the CTCF site in element lead to leakage of GT2 or Prox activity towards genes on the centromeric side of HoxD? It may not be surprising that deletion of element sC, II, III, IV by themselves do nothing, as they are "behind" the CTCF site in element V . When element V is deleted: does deletion of these centromeric distal elements now alter Hoxd13?Hoxd13 and Hoxa13 alone completely abrogates GT formation, leading to an open cloaca (see Introduction). To our knowledge, none of the genes located nearby (i.e. Lnpk and Evx2) has any function during either limb or GT development (the Atf2 gene is not addressed here as it stands outside -and not inside as suggested- the C-DOM TAD). It is possible that these non-Hox genes nevertheless play a role by being part of the general enhancer-promoter equilibrium necessary for Hoxd13 to be optimally regulated. The genetic stocks we use in this paper were not produced to address this question and only inferences can be obtained from our deleted mice. It would be necessary to produce mice lacking promoters (or the part responsible for the contact), a set of experiments largely outside the scope of this paper.Several issues are raised here. First, one has to remember that the combined inactivation of As it stands, we can propose the following answers:del(prox) mice, the steady state mRNA levels of Evx2 and Hoxd12 are significantly decreased. This is not surprising as we showed previously  that every transcription unit present in this landscape would fall under the control of this set of enhancers . In this manuscript, we did not look at Lnpk, whose transcription is also controlled by the limb and genital enhancers present in C-DOM.In the Hoxd13 in the CR of del(Prox) embryos at E10.5, because this element is not accessible at this stage , we considered that Prox was not active in these cells and hence we did not include this stage  when analyzing this allele. Regarding the E17.5 stage, it would be difficult to interpret the results of this experiment (we expect only a moderate quantitative effect) as we will not be able to discriminate between a direct transcriptional effect, on the one hand, and a cellular dilution effect induced by the huge growth of the structure, with only some cells remaining positive for Hoxd13 (see the text), on the other hand.Concerning the expression of Evx2 but not that of Hox genes .Also, the deletion of GT2 alone affects the transcription of Does inversion of the CTCF site in element lead to leakage of GT2 or Prox activity towards genes on the centromeric side of HoxD?Evx2 and Hoxd genes in this CTCF inverted allele.There isn’t any detectable change in the expression of the It may not be surprising that deletion of elements C, II, III, IV by themselves do nothing, as they are "behind" the CTCF site in element V .III and IV) at the same time while keeping the CTCF site associated to island V intact.Well, this does not seem to be the case, for we see an effect when deleting two elements  and a long-time frame. Also, the number of possibilities of combined deletions in-cis is very large and there is no a priori one or a few particular combinations that should be done in priority, leaving us with the necessity either to have an unbiased approach or to have no approach at all. We selected the latter.This is of course one of the valid questions one may ask in experiments combining micro-deletions in-del(IV-SB) and the del(rel5-SB) alleles affect Hoxd13 expression, although the first one keeps the island V intact and the second allele also removes GT2.However, our educated guess is probably yes, because both the 5) A major conclusion is that the deletions do not affect the overall architecture of the locus. The question is whether 4C-seq, with just one or two anchors has enough power to conclude that there is no effect on the global architecture of the C-DOM landscape.region V), which induces a clear (and expected) modification in the distribution of interactions, does not globally change the contact profile to an extent similar form example to the inversion or the deletion of a strong TAD boundary region.We think that indeed our approach allows us to draw such a conclusion, provided the word “global” is used. It is certainly possible that minor changes occur that cannot be detected using our 4C approach, and the same question can be asked at any level of resolution. For example, a slight difference in the representativity of one particular spatial configuration amongst others would not even be detected by using a high resolution Hi-C approach. By global architecture we mean the presence of the TADs and of the main contact points within these TADs, suggesting that these various regulatory sequences do not contribute strongly to the building of the structure. Even the inversion of the CTCF site (6) When GT2 or Prox are deleted: does Hoxd13 now interact with other elements ? If so, are these elements contained within the larger deletion that did have an effect on Hoxd13 expression?Unless we do not fully understand this comment, this point is addressed in the paper. For Prox in Figure 4E and F and in the text. For GT2, in Figure 6 and figure supplement 3A and 3B as well as in the text too.Essential revisions:The current 4C data represent only 1 replicate. Two reviewers indicated that it was critical to demonstrate reproducibility. They also understood the technical challenges. Therefore, we ask for adding biological replicates to confirm the most important and key findings. This would include replicates of some of the 4C analyses. When such data can be included the work would be suitable for eLife.To the first point, there are two issues.1) the appearance and disappearance, along with time, of particular peaks . This is highly significant and we have tried to make it clearer in this version. For this time course, we had made one replicate at E13.5 to have an estimation of the variability. This variability is now indicated in the new Figure 2C (open circles in the E13.5 points). You can see that the data for GT2 are very clear. For Prox, you can see that if one applies this variability to all the points, only the time points 12.5, 13,5 and 15.5 are clearly significant, whereas the 10.5 and 17.5 points are borderline. This simply means that the Prox element peaks in its interactions within a shorter time window, which is in full agreement with the fact that it is opened (ATAC seq data) later than the GT2 sequence.2) The general increase and decrease of contacts throughout the TAD (C-DOM), following the same dynamics. This was not the main issue of this Figure 2 and we agree that these exact percentages did not have much sense, except to indicate that there is a clear tendency. In this revised text, we removed the numbers from Figure 2 and simply mention in the text, after the description of GT2 and Prox dynamics that, in addition, the overall number of reads over the entire landscape showed a tendency… etc. A clear description of how we calculated this global increase/decrease in the number of reads has been added in the Materials and methodsTo the second point, we have added a table including the 66 datasets described in this work. They are accompanied by the numbers of the figure(s) where they appear, the number of replicates (if any) and an explanation as to why replicates were not processed in some cases. This table is now included as supplementary file 6 and referred to at the end of the Materials and methods section as: “A complete list of datasets and replicate status of all experiments is described in Supplementary file 6”.


Carassius auratus). The structural domains, three-dimensional structures, and amino acid sequences of gfLyz-C1 and gfLyz-C2 were highly comparable, as the two proteins shared 89.7% sequence identity. The gfLyz-C1 and gfLyz-C2 recombinant proteins were generated in the insoluble fractions of an Escherichia coli system. Based on the results of lysoplate and turbidimetric assays, gfLyz-C1 and gfLyz-C2 showed broad-spectrum antimicrobial properties with high levels of activity against Micrococcus lysodeikticus, Vibrio parahemolyticus, and Edwardsiella tarda, and relatively low activity against E. coli. Both gfLyz-C1 and gfLyz-C2 mRNAs were mainly expressed in the trunk kidney and head kidney, and gfLyz-C1 was expressed at much higher levels than gfLyz-C2 in the corresponding tissues. The expression of the gfLyz-C1 and gfLyz-C2 transcripts in the trunk kidney and head kidney was induced in these tissues by challenge with heat-inactivated E. coli and lipopolysaccharides (LPS), and the transcriptional responses of gfLyz-C1 were more intense. In goldfish primary trunk kidney cells, the levels of the gfLyz-C1 and gfLyz-C2 transcripts were upregulated by heat-inactivated E. coli, V. parahemolyticus, and E. tarda, as well as LPS, and downregulated by treatment with dexamethasone and leptins. Overall, this study may provide new insights that will improve our understanding of the roles of c-type lysozymes in the innate immunity of cyprinid fish, including the structural and phylogenetic characteristics, antimicrobial effects, and regulatory mechanism.Lysozymes are key antimicrobial peptides in the host innate immune system that protect against pathogen infection. In this study, the full-length cDNAs of two c-type lysozymes (gfLyz-C1 and gfLyz-C2) were cloned from goldfish ( The prie system . The ante system . Lysozyme system . Althouge system .The c-, g-, and i-type lysozymes are presumed to have originated from a common ancestor ; howeverCarassius auratus) is a domesticated cyprinid teleost that is closely related to crucian carp and common carp [gfLyz-C1 and gfLyz-C2) cDNAs from goldfish and investigated their expression patterns in normal individuals and in specific tissues from the individuals challenged with pathogenic bacteria and immunostimulants. Recombinant gfLyz-C1 and gfLyz-C2 proteins were generated in a prokaryotic system, and their antimicrobial activities against different bacterial species were characterized by lysoplate and turbidimetric assays. Furthermore, the changes in the expression of the gfLyz-C1 and gfLyz-C2 transcripts in response to immune challenge and hormonal treatment were further examined in goldfish primary trunk kidney cells.Goldfish , a 102-bp 3′-UTR region, and a 438-bp open reading frame (ORF) encoding a 145-amino acid (a.a.) protein precursor that is composed of an 18-a.a. signal peptide followed by a 127-a.a. mature protein with a deduced molecular weight of 14.5 kDa . A SDS-PAGE analysis confirmed the expression of the recombinant gfLyz-C1 (~16.3 kDa) and gfLyz-C2 (~16.4 kDa) proteins after IPTG induction, and high-purity protein products were ultimately obtained  leptins, but not dexamethasone, suppressed the basal levels of gfLyz-C1. Dexamethasone is an exogenous glucocorticoid and cortisone derivative, and glucocorticoids are involved in the metabolism of carbohydrates, protein, and fat, and possess anti-inflammatory activity. When goldfish are exposed to a potential predator, the plasma cortisol (an endogenous glucocorticoid) levels increase [HSP70 mRNA expression in the brain [gfLyz-C1 and gfLyz-C2 mRNA expression induced by immune challenge. On the other hand, leptin is a peripheral satiety factor that is mainly produced in the adipose tissue of mammals or the liver of fish [leptin-AI and leptin-AII transcripts in the goldfish liver is controlled by a combination of insulin and glucagon [The expression of the imulants A, but alimulants B and lepimulants C. Some dincrease , leadinghe brain . In addihe brain . Furtherhe brain . Our pre of fish ,42. In g of fish . Leptins of fish  and pitu of fish  in goldfglucagon ,46. In hglucagon . HowevergfLyz-C1 and gfLyz-C2 mRNAs were predominantly expressed in the trunk kidney and head kidney and were induced by challenge with heat-inactivated E. coli and LPS in these tissues. Notably, gfLyz-C1 is considered the major form of c-type lysozymes in goldfish based on its absolutely higher expression levels and greater responses to challenge with immunostimulants. The bacteriolytic and bactericidal activities of the recombinant gfLyz-C1 and gfLyz-C2 proteins against the standard species M. lysodeikticus and E. coli and pathogenic species V. parahemolyticus and E. tarda were comparable. In goldfish primary trunk kidney cells, the expression of the gfLyz-C1 and gfLyz-C2 transcripts was upregulated by heat-inactivated E. coli, V. parahemolyticus, and E. tarda, as well as LPS, and downregulated by treatment with dexamethasone and leptin. Our current findings may provide new insights that will improve our understanding of the potential mechanisms of action and regulation of c-type lysozymes, as well as the host/pathogen interactions, in innate immunity in cyprinid fish.In conclusion, the full-length cDNAs of two c-type lysozymes were cloned and characterized in cyprinid goldfish. The structural and phylogenetic features of gfLyz-C1 and gfLyz-C2 were analyzed and compared. Both Carassius auratus) with body lengths of ~12 cm and body weights of ~25 g were acquired from local suppliers and maintained individually in 100-L tanks at 25 °C under a 12:12 h dark–light photoperiod with a regular feeding schedule. Before tissue sampling, the goldfish were sacrificed by spinosectomy after anesthesia with 0.05% tricaine methanesulfonate . Given that the sexual dimorphism was not appeared in sexually immature fish, goldfish of mixed sexes were used for the in vivo intraperitoneal (i.p.) injection and in vitro cell cultural experiments. All animal experiments were conducted in accordance with the guidelines of the Lingnan Normal University.Goldfish (gfLyz-C1 and gfLyz-C2). The corresponding full-length sequences of gfLyz-C1 and gfLyz-C2 were obtained by performing 3′- and 5′-rapid amplification of cDNA ends (RACE).Total RNA was extracted from the goldfish trunk kidney using TRIzol reagent , digested with DNase I (Invitrogen), and reverse transcribed into first-strand cDNAs with a PrimeScript™ RT Kit . By performing a BLAST analysis of a previously constructed goldfish transcriptome, two unigenes of 645 bp and 713 bp, respectively , were foSignal peptides were predicted with the SignalP 3.0 program. Structural domains were illustrated by using the SMART and ScanProsite programs. Three-dimensional (3-D) models were deduced with the knowledge-based modeling program ProMod II provided by the SWISS-MODEL server. A phylogenetic analysis was constructed based on nucleotide differences (p-distance) by the neighbor-joining method with 1000 bootstrap replicates with Mega 6.0 software. The a.a. sequence alignment of c-type lysozymes from different vertebrate species was constructed using Clustalx1.8 and presented with GeneDoc.E. coli (BL21). The bacterial cells were grown at 37 °C until the OD600 reached 0.6, and the expression of the recombinant gfLyz-C1 and gfLyz-C2 proteins was induced by adding IPTG to a final concentration of 0.1 mM for an additional 24 h at 28 °C. The cell pellets were homogenized by sonication and the insoluble fraction was separated by centrifugation. The recombinant gfLyz-C1 and gfLyz-C2 proteins that appeared in the inclusion body were dissolved in 8 M urea, purified by using His-Bind Kits (Novagen) under a denaturing condition and renatured by dialysis as described in a previous study [Fragments of the mature gfLyz-C1 and gfLyz-C2 peptides were respectively subcloned into the PET28a vector  and then transformed into us study .M. lysodeikticus  or E. coli (JM109), V. parahemolyticus, and E. tarda at an OD600 of 0.4 were prepared. Then, 50 µL of recombinant gfLyz-C1 or gfLyz-C2 proteins (20 µg/µL) diluted in PBS or PBS alone was applied to the wells with diameters of 6 mm in individual plates and then incubated at 30 °C. After 24 h of treatment, the diameters of the cleared zones were measured.The lysoplate assay was performed using a previously described protocol  with sliM. lysodeikticus, E. coli, V. parahemolyticus, and E. tarda in PBS  at an OD450 of 0.6 and was mixed with the recombinant gfLyz-C1 or gfLyz-C2 protein (20 µg) in a final volume of 2 mL. The reactions were incubated at 28 °C, and the absorbance at OD450 was measured from 0 to 30 min at 1-min intervals with a microplate reader .The antimicrobial activities of the recombinant gfLyz-C1 and gfLyz-C2 proteins against different bacteria were examined with the turbidimetric assay as described previously  with a sgfLyz-C1 and gfLyz-C2 mRNAs in different tissues of goldfish, including the brain, pituitary, gill, heart, stomach, intestine, liver, trunk kidney, head kidney, spleen, muscle, fat, and blood cells from sexually immature fish, and the testis and ovary from sexually mature fish, were examined in three individuals. The expression of the gfLyz-C1 and gfLyz-C2 mRNAs was assessed by quantitative PCR. The quantitative PCR procedure is described in detail below, and elongation factor-1α  was used as an internal control for normalizing mRNA levels.Tissue expression profiles of the gfLyz-C1 and gfLyz-C2 mRNA expression in different tissues of goldfish were analyzed by i.p. injection as described previously [Escherichia coli  or lipogfLyz-C1 or gfLyz-C2 transcripts in response to immune challenge and hormonal treatment were further examined in goldfish primary trunk kidney cells. Goldfish trunk kidney cells were isolated as described previously [n = 5) were excised aseptically and washed three times with ice-cold Ca2+/Mg2+-free HBSS . The trunk kidney fragments were diced into small, 1-mm3 pieces by a McIlwain tissue chopper (Ted Pella) and dispersed into single cells by incubating the fragments with 0.25% trypsin (Sigma) in Ca2+/Mg2+-free HBSS at 28 °C for 30 min. The trunk kidney cells were then separated from the remaining fragments by filtration through a sterile 70-μm mesh and harvested by centrifugation at 100× g for 15 min at 4 °C. The trunk kidney cells were resuspended in culture medium 199 . The viability of the cells was assessed using a Trypan blue exclusion assay, and only preparations with more than 95% viability were used in subsequent experiments. The cells were diluted to a density of 0.5 × 106 cells/mL/well in M199, seeded in 24 well-plates, and then incubated in an atmosphere with 5% CO2 and saturated humidity at 28 °C overnight for recovery. On the second day after cell preparation, test substances prepared in DMEM/F12 were gently overlaid after the removal of the old culture medium. Cells were incubated with the test substances for another 24 h. In this study, the tested substrates included heat-killed E. coli, V. parahemolyticus, and E. tarda, as well as LPS, dexamethasone (Sigma) and recombinant goldfish leptin-AI and leptin-AII proteins generated in the eukaryotic methylotrophic yeast system [The changes in the expression of the eviously ,49, witht system . FinallygfLyz-C1 or gfLyz-C2 mRNAs was detected using SYBR Premix Ex Taq™ II (TaKaRa) in the RotorGene RG-3000 Real-Time PCR System . Specific primers for gfLyz-C1 or gfLyz-C2  test.Total RNA from the tissue and cell samples was isolated by using TRIzol reagent, digested with DNase I, and reverse transcribed into the first-strand cDNAs with a PrimeScript™ RT Kit (TaKaRa). The expression of the gfLyz-C2  were desgfLyz-C2 . EF-1α s


The temporal and spatial expression of genes is controlled by promoters and enhancers. Findings obtained over the last decade that not only promoters but also enhancers are characterized by bidirectional, divergent transcription have challenged the traditional notion that promoters and enhancers represent distinct classes of regulatory elements. Over half of human promoters are associated with CpG islands (CGIs), relatively CpG-rich stretches of generally several hundred nucleotides that are often associated with housekeeping genes. Only about 6% of transcribed enhancers defined by CAGE-tag analysis are associated with CGIs. Here, we present an analysis of enhancer and promoter characteristics and relate them to the presence or absence of CGIs. We show that transcribed enhancers share a number of CGI-dependent characteristics with promoters, including statistically significant local overrepresentation of core promoter elements. CGI-associated enhancers are longer, display higher directionality of transcription, greater expression, a lesser degree of tissue specificity, and a higher frequency of transcription-factor binding events than non-CGI-associated enhancers. Genes putatively regulated by CGI-associated enhancers are enriched for transcription regulator activity. Our findings show that CGI-associated transcribed enhancers display a series of characteristics related to sequence, expression and function that distinguish them from enhancers not associated with CGIs. Enhancers control gene regulation in a way that is essential for cell- and developmental-specific gene expression  upstream and 50 bp downstream of the transcription start site (TSS) and serves as a binding site for RNA polymerase II (RNAPII) and its associated general transcription factors (GTFs). Core promoters initiate the transcription of protein-coding and many non-coding genes, but usually have a low basal activity that can be modulated by the proximal promoter and by enhancers . Enhancepression . Similarpression .Many promoters produce antisense RNAPII divergent transcripts ,5. ReceneRNAs are typically 0.5–2 kb in length, and their expression levels tend to correlate with the cis-regulatory activity of their template enhancers . The funIn this work, we investigate correlations of core promoter elements (CPEs), CGIs, and transcription-factor binding events with functional characteristics of transcribed enhancers. CPEs are binding sites for general transcription factors , which recruit RNAPII ,22–24. CCAGE  sequencing was used by the FANTOM consortium to profile the transcriptomes of a large panel of human tissues and cell types, demonstrating the existence of over 60 000 bidirectionally transcribed enhancers that gave rise to mainly nuclear and non-polyadenylated RNAs . TranscrIn this work, we show that CPEs demonstrate statistically significant localized overexpression in transcribed enhancers. Furthermore, we demonstrate that promoters and transcribed enhancers share a number of characteristics whose magnitude in both cases correlates with the presence or absence of a CGI.Hs_EPDnew_006_hg38.bed). This dataset represents a compilation of 29 598 promoter sequences for which the TSSs have been determined experimentally.The work presented in this manuscript is based on promoter definitions taken from the Eukaryotic Promoter Database New (EPDnew) dataset, version 006   were computed for twelve CPEs. A PWM of length ℓ assigns each oligonucleotide of length ℓ a matching score ng sites   sequence in which the observed frequency of CpG dinucleotides is close to the frequency that would be expected based on the individual frequency of G and C bases are termed CpG islands (CGIs). CGIs are associated with the upstream region of many genes generally covering all or part of the promoter and displaying an average size of ∼1 kb ,39.To identify CGIs in this study, a 100-nucleotide window was shifted in 1 bp intervals across the promoter sequences from position [−200, −100) relative to the TSS to . Enhancers were classified into two groups depending on whether a CGI overlapped at least one of the two TSSs. Non-parametric analysis was performed with a Mann–Whitney U test.We extracted the length of bidirectionally transcribed enhancers from the FANTOM5 file , which ranges between 0.0 for housekeeping genes and 1.0 for tissue-specific genes ,44. Let n = 15 tissues and top n = 15 primary cells by total log-transformed expression over all promoters.To compute τ in this study, expression per CAGE library was normalized and converted to expression per 29 distinct tissues and 36 distinct primary cells. For each promoter and tissue/primary cell, we then added up expression between positions −100 and +100 relative to and on the same strand as the TSS, scaled the result by a factor 1000, took the binary logarithm, and computed τ separately for the top R) and within +200 bp of the mid position on the forward strand (F). Directionality is defined as (F − R)/(F + R), with a value close to 0.0 indicating balanced bidirectional transcription and a value close to −1.0 or 1.0 indicating unidirectional transcription   relative to the TSS to  Figure . CPE− co+ (CPE2+) contained promoters in which the first (second) CPE was present in its functional window. Correspondingly, CPE1− (CPE2−) contained promoters in which the first (second) CPE was not present in its functional window.Fisher’s exact test was used to assess the statistical significance of co-occurrence of pairs of CPEs in promoters and enhancers. To carry out the test, we partitioned promoter sequences twice into two sets for each pair of distinct CPEs. CPE1P-values for the overrepresentation of promoters showing co-occurrence of both CPEsN is the count of promoters and−4.We then computed BED files representing the results of H3K27ac ChIP-seq analysis were downloaded from the ENCODE data portal . The BEDhg38_cismotifs), which contain overlapping peaks detected in two or more experimental datasets and by at least two peak-calling tools, corresponding to a total of 124 unique transcription factors.We used a dataset comprised of statistically significant ChIP-seq peaks for 599 human transcription factors . For ourThe promoter region of protein-coding genes was defined as comprising 500 nt upstream and 200 nt downstream of the TSS.In this work, we analyzed bidirectionally transcribed enhancers from the FANTOM5 project . 3587 ofCPEs can be defined computationally based on overrepresentation of a sequence motif in a specified location with respect to the TSS . We reasoned that transcribed enhancers might demonstrate a comparable overrepresentation of CPEs because these enhancers are transcribed by RNAPII. In addition to the classic CPEs , we investigated eight other CPEs that have been proposed in the literature Figure .Of the twelve CPEs tested, eight displayed statistically significant local overrepresentation in core promoters of protein-coding genes . Seven oCertain pairs of CPEs such as DPE/Inr have been shown to function cooperatively in some promoters . It was Drosophila that BREu suppresses the ability of the transcription factor Caudal to activate TATA-dependent promoters, indicating that BREu contributes to CPE-mediated transcriptional regulation in TATA-containing promoters  binds cooperatively to the Inr and DPE motifs . The obsromoters . SpeculaCAGE tag analysis of promoters showed that CGI-associated promoters tend to initiate transcription from a broad region, while non-CGI-associated promoters tend to have sharp peaks of transcription initiation . We confP = 1.63 × 10−150 by the Mann–Whitney U test). 93 CGI-present enhancers were over 1000 bp in length , while only 269 CGI-absent enhancers were over 1000 bp .We compared the lengths of the bidirectionally transcribed enhancers according to whether an enhancer overlaps with a CGI on one or both of its TSSs (present) or not (absent). While the overall length distribution was similar Figure , the meaThe molecular mechanisms controlling tissue specificity remain incompletely understood, but measures including Shannon entropy and τ (tau) have been used to characterize the overall tissue specificity of a gene. These measures characterize the extent to which a gene tends to be tissue-specific or broadly expressed (housekeeping), irrespective of the specific tissue or tissue in which it is expressed . Some feIn accordance with previous findings , we founP < 10−300, Mann–Whitney U test; CGIs colocalize with the promoters of constitutively expressed genes and ∼40% of genes with a tissue-restricted expression profile . TherefoH3K27ac is a histone mark that can be associated with active promoters and enhancers ,53. 89.8CGIs tend to lack sequence conservation over long evolutionary distances, and it is thought that their GC richness may increase the probability of binding of ubiquitous transcription factors such as SP1 . DNA fooP < 10−300, Mann–Whitney U test). If we instead compare the body of enhancers, there were 3.26 ChIP-seq binding events for enhancers associated with a CGI, again compared to 1.15 per 1000 nt for enhancers not associated with CGIs . There was also a total higher number of ChIP-seq binding events at enhancers associated with CGIs  as compared to promoters not associated with CGIs (1.89 per 1000 nt). For the enhancers, the density of ChIP-seq binding events at enhancer-associated CGIs was 2.46 per 1000 nt compared to 1.15 per 1000 nt for enhancers not associated with CGIs , chromosome (GO:0005694), and regulatory region nucleic acid binding (GO:0001067), indicating that many of the genes likely to be regulated by CGI-associated enhancers are themselves involved in transcriptional regulation or encode gene products that have a chromosomal location.Out of a total of 13 881 genes that were putatively regulated by at least one enhancer in that dataset, 2743 were regulated by CGI-associated enhancers. Seventeen Gene Ontology (GO) terms were significantly overrepresented with a e <0.001 . Among tA major goal of biology is to understand the molecular mechanisms that mediate tissue- and developmental stage-specific gene regulation patterns that underlie development, cell identity and function, and whose malfunction contributes to disease. The last decade has seen a paradigm shift in our understanding of enhancers, which were traditionally thought to be strictly distinct from promoters. As noted in the introduction, many enhancers are bidirectionally transcribed and in many cases their function depends on the transcription products (eRNAs) or perhaps on the transcription process itself. In the last decade, numerous studies have shown that enhancers and promoters share many features, including similar sequence motifs, transcription machinery, chromatin environment, and changes in activity upon binding of activators or repressors . HoweverThe contribution of the current study centers around a detailed analysis of the relation between enhancer and promoter characteristics and the presence or absence of CGIs. The function of CGIs is not completely understood but is thought to involve the establishment of transcriptionally permissive chromatin states by destabilizing nucleosomes and attracting DNA-binding proteins ,54. CGIsEnhancers are defined as DNA sequences that modulate the expression of target genes in a space and time-dependent manner, whereby the relative orientation of the enhancer to the target genes is irrelevant and the enhancers can be located kilobases or even megabases distant from their target promoters. This does not easily lead to an operational definition of an enhancer that can be used for a specific and sensitive enhancer assay. Correspondingly, we still do not have a comprehensive and accurate catalog of mammalian enhancers. The classic and still generally accepted definition of an enhancer focuses on the functional capacity of DNA to enhance transcription of a reporter gene in an orientation and position-independent manner ,60. The The enhancer candidates defined by these methods often do not show a high degree of overlap, even if one only considers methods with a functional readout such as STARR-seq, CAGE-tag analysis, and reporter gene assays of target enhancer sequences. This could be due to technical limitations of the assays, differences in the biological systems being analyzed, or other factors. Table The FANTOM5 dataset is unique in that it allows the precise boundaries of enhancers to be defined, which is a prerequisite for some of the analysis approaches presented here such as the localized overrepresentation of CPEs.Transcription initiation at promoters requires the stepwise assembly of GTFs  and RNAPII. The TATA-binding protein (TBP) subunit of TFIID can bind the TATA box found in some core promoters, and other subunits of TFIID (the TBP-associated factors or TAFs) appear to interact with Inr and DPEs . HoweverDrosophila , and a higher overall expression than enhancers lacking a CGI, which is comparable in direction if not in amplitude to the analogous findings in promoters. Our finding of higher overall expression in CGI-associated enhancers may be related to a recent finding that GC dinucleotide repeat motifs are enriched in broadly active enhancers compared to both the genomic background and context-specific enhancers .Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) is a powerful technology to identify the genome-wide locations of transcription factors and other DNA-binding proteins. ChIP-seq can identify both sharp peaks typically associated with sequence-specific transcription factors, as well as broad histone-modification signals, and involves formaldehyde-mediated cross-linking of chromatin followed by fragmentation of protein-DNA complexes into short fragments, which are then subjected to immunoprecipitation using an antibody directed against a protein of interest ,69. We lIn this work, we have investigated associations of a number of characteristics of transcribed enhancers and their relations with the presence or absence of CGIs. Although transcribed enhancers are likely to represent a heterogeneous set of genomic elements with different regulatory mechanisms, we have shown that the subset of CGI-associated enhancers display a number of distinguishing characteristics that differentiate them from non-CGI-associated enhancers. CGI-associated enhancers are longer, display a higher degree of directionality and strength of expression, show a higher frequency of transcription factor binding events, more H3K27ac signal, and putatively regulate a set of genes enriched for functions including transcriptional regulation.gkaa223_Supplemental_FileClick here for additional data file.


Ocm consists of two active Ca2+-specific domains of the EF-hand type (“helix-loop-helix” motif), covered by an EF-hand domain with inactive EF-hand loop, which contains a highly conservative cysteine with unknown function. In this study, we have explored peculiarities of the microenvironment of the conservative Cys18 of recombinant rat Ocm (rWT Ocm), redox properties of this residue, and structural/functional sensitivity of rWT Ocm to the homologous C18S substitution. We have found that apK of the Cys18 thiol lays beyond the physiological pH range. The measurement of redox dependence of rWT Ocm thiol–disulfide equilibrium (glutathione redox pair) showed that redox potential of Cys18 for the metal-free and Ca2+-loaded protein is of −168 mV and −176 mV, respectively. Therefore, the conservative thiol of rWT Ocm is prone to disulfide dimerization under physiological redox conditions. The C18S substitution drastically reduces α-helices content of the metal-free and Mg2+-bound Ocm, increases solvent accessibility of its hydrophobic residues, eliminates the cooperative thermal transition in the apo-protein, suppresses Ca2+/Mg2+ affinity of the EF site, and accelerates Ca2+ dissociation from Ocm. The distinct structural and functional consequences of the minor structural modification of Cys18 indicate its possible redox sensory function. Since some other EF-hand proteins also contain a conservative redox-sensitive cysteine located in an inactive EF-hand loop, it is reasonable to suggest that in the course of evolution, some of the EF-hands attained redox sensitivity at the expense of the loss of their Ca2+ affinity.Oncomodulin (Ocm), or parvalbumin β, is an 11–12 kDa Ca Combining the equation for K with the material balance equation 2[M–M] + [M] = M0  it is easy to obtain weight fraction of the dimeric protein, Df, which is expressed as follows,x equals to log([GSH]2/[GSSG]).The redox equilibrium in the system of a protein with one thiol group and GSH/GSSG couple can be described by the scheme [I]:Df on x for dOcm sample was fitted by Equation (1) using OriginPro v.9.0  software and K as a fitting parameter (the changes in [GSH] and [GSSG] due to reaction [I] were neglected).The experimental dependence of M–M and M (scheme [I]) their weight fractions are equal, the x value at this point can be expressed as follows.Considering that at the middle of the transition between apK value of GSH , the eqof 0.1 M .GSSG + hE, is defined by Nernst equation (activity coefficients of GSSG and GSH are commonly assumed to be close to 1)  ) was estimated for the each well as a difference between the total Mg2+ concentrations measured for both halves of the well, assuming that free Mg2+ concentrations ([fM]) do not differ between the two halves of the well. The total protein concentration (P0) was determined spectrophotometrically for the each well after the equilibrium dialysis. Since at Ocm concentration of 220 μM the Donnan coefficient is close to 1, the error arising due to the Gibbs–Donnan effect is negligible compared to the experimental uncertainties in Mg2+ and Ocm concentrations. The experimental data were analyzed using the sequential metal binding scheme [IV]. The number of Mg2+ ions bound per Ocm molecule was described by the following equation.Mgis, LLC) ,55. Eachis, LLC) . One halK1) and log(K2) were used as fitting parameters in OriginPro v.9.0 software . The weighting factor Log(Rattus norvegicus) oncomodulin  and its mutant form with homologous substitution of the highly conservative Cys18 in the inactive AB-loop (Recombinant wild type rat ( AB-loop  for Ser 2+-bound and/or oxidized states of the abovementioned forms. The major fraction (~81%) of C18S Ocm had a mass of 12,041 Da, which corresponds to the mass expected for the protein without Met1 and N-terminal acetyl group. The remaining C18S Ocm (~19%) had a mass consistent with the presence of Met1.Mass spectrometry measurements revealed two major fractions of the reduced rWT Ocm sample. One of them (~45%) had a mass of 12,057 Da, which is consistent with mass of the reduced Swiss-Prot entry P02631 without Met1 and N-terminal acetyl group. The other rWT Ocm fraction (~46%) was 131 Da heavier, in line with the presence of Met1. The minor fractions (~9%) of this sample corresponded to the CaapK value, we tried to estimate the apK value of Cys18 of rWT Ocm. Deprotonated alkyl thiol group displays an absorption band around 240 nm (ε ≈ 4000 M−1cm−1) in the UV absorption spectrum, where absorption of the protonated thiol is negligible ) at 35 °C has been studied mainly as described in , of 30.7 °C–2+/Ca2+-loaded states of C18S Ocm demonstrate well-defined heat sorption peaks, shifted relative to the corresponding peaks of rWT Ocm towards lower temperatures by 5 °C and 3.7 °C, respectively , while the Ca2+ removal causes the opposite effect. Analysis of the data according to the sequential metal-binding scheme [IV] shows that our estimates of Ca2+ affinity of rWT Ocm . Ca2or, EDTA a. The bi rWT Ocm  exceed taffinity a. Indeed rWT Ocm . As the 2+-binding to rWT/C18S Ocm was studied by equilibrium dialysis method with determination of total Mg2+ concentration using electrothermal atomization atomic absorption spectrometry ) equals to the same ratio calculated for rWT Ocm . In contrast to the dissociation stage, Ca2+ association with the first site of the C18S mutant is 20-fold slower in comparison to rWT Ocm (2+ affinity of C18S Ocm is decreased mostly due to the drop in Ca2+ association rate. Mg2+ dissociation from rWT/C18S Ocm occurs within the dead time of the instrument (data not shown), in accordance with their relatively low Mg2+ affinities  significantly affects structural and functional status of Ocm, its thiol derivatives are expected to possess altered functional activity. Thus, oxidizing conditions are likely to favor accumulation of the oxidized forms of Ocm with modified activity, thereby supporting the redox sensory function of its C18 thiol. The effects described in the present work closely resemble those reported for the conservative among neuronal calcium sensor proteins C39 residue of recoverin, located in the loop of its inactive EF-hand 1. Light-induced oxidation of this thiol results in appearance under in vivo conditions of disulfide dimer of recoverin and its oxidized monomer, which demonstrate altered structural and functional properties ,63. Ocm