HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet “DNA loops”. Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates. Hox genes encode transcription factors with crucial roles during development. These genes are grouped in four different clusters names HoxA, B, C, and D. Mutations in genes of the HoxA and D clusters have been found in several human syndromes, affecting in some cases limb development. Despite their essential role and contrary to the genes of the HoxD cluster, little is known about how the HoxA genes are regulated. Here, we identified a large set of regulatory elements controlling HoxA genes during limb development. By studying spatial chromatin organization at the HoxA region, we found that the regulatory elements are spatially clustered regardless of their activity. Clustering of enhancers define tissue-specific chromatin domains that interact specifically with each other and with active genes in the limb. Our findings give support to the emerging concept that chromatin architecture defines the functional properties of genomes. Additionally, our study suggests a common constraint of the chromatin topology in the evolution of HoxA and HoxD regulation in the emergence of the hand/foot, which is one of the major morphological innovations in vertebrates. Hox gene family encodes transcription factors with central roles in patterning of the body plan and organogenesis. Hox genes are grouped into clusters in most animal species, and mammals possess 39 genes divided into four clusters named HoxA to HoxD. In mice, deletion of the HoxA cluster is embryonic lethal HoxB, HoxC, or HoxD are viable at least until birth Hox genes identified Hoxa13 as a gene required for proper placenta function and thus embryonic survival HoxA genes have been found in various human syndromes  including limb malformations. Studies of gene inactivation in mice demonstrated that genes located at the 5′ end of the HoxA cluster (Hoxa9–13) are required for proper patterning of the three limb segments: the upper arm , lower arm , and the hand/foot  The HoxA genes. This is in contrast to HoxD, which transcriptional control has been more thoroughly studied, especially in the limb where the HoxD genes play partially overlapping functions with HoxAHoxA and D clusters follows similar dynamics during limb development, and occurs in two phases Hox1) to the other (Hox13), with early activated genes expressed throughout the limb bud and those activated later (Hox10-13) gradually restricted to posterior cells HoxA and HoxD genes partly differ in the second phase (from E11.5 onwards), suggesting some differences in the regulatory mechanisms controlling the clusters in this later phase.Despite their pivotal roles during embryogenesis, little is known about the regulation of HoxD cluster is regulated long-distance by enhancers in several tissues  is controlled by several remote cis-regulatory sequences located in the gene desert upstream of the cluster Hoxa10 and Hoxa13 are also expressed in the presumptive hand/foot domain therefore raised the possibility that specific recruitment of HoxA and HoxD gene functions in developing digits stem from the implementation of similar cis-regulatory elements during the fin-to-limb transition. Whereas sequence conservation analysis of the region upstream of these clusters did not identify cognate cis-regulatory elements driving HoxA expression in limbs Hoxa13 gene, in the neighborhood of the 3-hydroxyisobutyrate dehydrogenase (Hibadh) gene Hibadh is also expressed in distal limb buds Hibadh, Hoxa10/13, or both. Thus, the enhancer sequence(s) and whether HoxA expression in limbs is regulated by long-range control mechanisms has remained unknown.Previous studies show that transcription at the HoxD genes in the presumptive digit domain is mediated by physical contacts with remote enhancers, and led to a model whereby expression of Hoxd10 to d13 associates with the formation of DNA loops between the genes and regulatory islands HoxD genes and one of its regulatory islands, specifically in digit progenitor cells β-globin locus Igh gene It was previously shown that control DNA elements could regulate the expression of remote genes by physically interacting with them HoxA genes is controlled by multiple remote enhancers located upstream of the cluster. In limb cells, these enhancers are grouped into distinct sub-megabase topological domains (sub-TADs) that contact each other and the sub-TADs containing target genes. In head tissues, the topology is drastically different, modifying both gene-enhancer and enhancer-enhancer interactions. Interestingly, enhancers located in the same sub-TAD are active in distinct subset of limb cells suggesting that spatial clustering of enhancers does not simply reflect enhancer co-activity. We also present evidence that enhancer-HoxA contacts are maintained even when enhancer activity is suppressed, suggesting that the HoxA regulatory region acquires a permissive conformation prior to gene activation. We suggest a model whereby sub-TAD formation and/or contacts between sub-TADs define the cis-regulatory network controlling gene expression. From an evolutionary perspective, this first extensive characterization of HoxA regulation in developing limbs provides new insights into the evolution of Hox regulation in the emergence of hand/foot. Our study suggests that while the DNA sequences of the distal limb enhancers for HoxA and HoxD genes are different and have likely emerged independently, chromosome partitioning into topological domains has similarly constrained the evolution of HoxA and HoxD cis-regulatory landscapes underlying the emergence of digits, one of the major morphological innovations in Vertebrates.Here, we show that during limb development, expression of HoxA expression during limb development, we used a combination of genetic and genomics approaches that probe enhancer features in mouse embryos. We first tested whether HoxA transcription in developing limbs involves cis-regulatory sequences outside of the gene cluster itself. To this end, we used two mutant lines with targeted genomic rearrangements at the HoxA cluster in situ hybridization shows that a neomycin reporter transgene located downstream of Hoxa1 is not expressed in limbs at E11.5 (left). In contrast, we found that a hygromycin transgene inserted at the opposite end of the cluster, 3.5 kb upstream of Hoxa13 is robustly transcribed in distal limbs at this stage (right). These expression patterns correlate well with the expression profile of the endogenous HoxA genes adjacent to the reporter transgenes. Upon deletion of the entire HoxA cluster, the neomycin transgene becomes activated in distal limbs suggesting that sequences upstream of the cluster are sufficient to trigger distal expression . Active enhancers are characterized by the binding of several proteins including RNA polymerase II (RNAP2), and subunits of Mediator like Med12 Hoxa13 (top).Given the results described above, we focused our analysis on the genomic region upstream of the cluster. To identify active enhancers in distal limbs, we used dissected distal forelimbs , which a2 Hoxa13 , top.HoxA was rather large, particularly compared to HoxD for which seven enhancers have been identified HoxD, the region upstream of HoxA encompasses several genes . In contrast, none of the enhancers interact with the 3′ part of the cluster containing Hoxa1 to Hoxa7 , with 3′ genes residing into one TAD and the 5′ part extending into the other HoxA and HoxD clusters.The number of candidate sequences identified upstream of al genes . Candidater top, , and thater top, , bottom.to Hoxa7 , which hHoxA genes  locate farther from the cluster, within the Jazf1 gene, and correspond to those loci most enriched in marks typical of active enhancers . e1 is located close to Evx1, within a region of high interaction frequencies with HoxA both in limb and head tissues. This is not the case for e3 so we further tested interaction frequencies between Hoxa13 and e2 to e5 using 3C .Interestingly, sequences with the highest interaction frequencies with 5′nhancers . High ind tissue , bottom.using 3C . This annhancers . Interesnhancers . As ther binding , bottom.HoxA genes and most of our candidate enhancers, we proceeded to test their in vivo activity in the mouse by transgenesis. Putative enhancer sequences were subcloned into vectors carrying the β-globin minimal promoter and lacZ reporter. Except for e1 and e2, X-Gal staining in transgenic embryos shows that all candidate enhancers tested activates transcription in developing limbs  embryos, the Gli3R domain is restricted anteriorly as a consequence of posterior Sonic hedgehog (Shh) signaling emanating from the Zone of Polarizing Activity (ZPA), which blocks processing of the full length Gli3 protein into its truncated repressor form Shh−/− limbs, the Gli3R functional domain extends posteriorly leading to the down-regulation of HoxA as well as HoxD genes While “DNA looping” is associated with long-range transcriptional control, the extent to which spatial structure exists prior to or as a consequence of enhancer activation remains elusive. This issue partly originates from the fact that most studies have compared the spatial distance of enhancers and target gene(s) in tissues expressing the genes with others where they are never expressed. To gain insight into the causative relationship between spatial proximity and long-distance enhancer regulation, we examined the outcome of enhancer silencing on long-range enhancer-gene interactions in developing limbs. During limb development, the transcriptional repressor Gli3R negatively regulates the expression of HoxA-associated limb enhancers identified, we found several that overlap with loci bound by Gli3R in the limb  while still functional in the developing genitalia (panel d). In contrast, a transgene containing the e1 enhancer, which does not overlap with a Gli-bound locus, remained expressed in a Shh−/− background (f–h) although in a smaller domain consistent with Shh−/−embryos having reduced limb size (e to f).Amongst the pression , and theenitalia , panel dckground f–h althHoxA-enhancer proximity requires enhancer activity, we measured contacts between Hoxa13 and e5 in wt and Shh−/− distal limb buds from E11.5 embryos. As e5 activity is suppressed in the absence of Shh, the enhancer should no longer interact with Hoxa13 if enhancer activity is required for the contact. 3C analysis shows that e5 interacts with Hoxa13 even in the absence of Shh  analyzed with Hi-C at the mega-base scale HoxA genes and distal limb enhancers are located in the same TAD  is one of the major morphological novelties that accompanied Vertebrates adaptation to terrestrial habitats. As autopod development requires the function of nhancers . NotablyHoxA and HoxD regulatory landscapes suggested by the absence of obvious sequence conservation of their respective enhancers is further supported by several findings. First, the recent evidence that HoxA and HoxB clusters most likely stem from the duplication of an ancestral HoxA/B cluster HoxA and HoxD should have been lost at HoxB and HoxC. This scenario however appears unlikely to account for the specific HoxA and HoxD regulation associated with hand/feet development as the tandem duplications of the ancestral Hox cluster that led to the four Vertebrate Hox clusters occurred prior to the fin-to-limb transition. Second, there is a major difference in the layout of the HoxA and HoxD regulatory landscapes controlling their expression in the developing hand/feet. While HoxD-associated enhancers are part of a gene desert HoxA-associated enhancers are embedded in genes. Notably, HoxA enhancers with the highest enrichment of RNAP2, Med12 and p300, which also show the highest frequencies of interaction with HoxA genes, are located within Hibadh and Jazf1. Moreover, the genomic domain between HoxA and Jazf1, has undergone significant expansion from fish to mice (about 50 kb in fish and 800 kb in mice), indicating that an extensive genomic reshuffling at the HoxA regulatory landscape occurred during the fin-to-limb transition, which further support an independent evolution of the HoxA and HoxD regulatory landscapes. Interestingly, this genome expansion affected both the size of Hibadh, Jazf1 and the intergenic regions. The absence of preferential localization of HoxA-associated enhancers in gene-free regions thus suggests that introns are equally amenable to sequence evolution and emergence of new regulatory elements.The independent evolution of HoxA and HoxD expression in distal limb most likely emerged independently, in both cases the distal limb regulatory landscape is located within the TAD containing the 5′ genes , and the samples were incubated 20 min at 37°C with collagenase  to obtain single-cell suspensions. The number of cells in suspension was then counted under the microscope, and each sample was diluted in 9 ml of 1×PBS containing 10% FBS (5 ml for Shh−/− embryos). The cells were then fixed with 1% formaldehyde for 10 min at room temperature. Crosslinking was stopped with glycine , and incubated 5 min at room temperature followed by 15 min on ice. Cells were centrifuged at 400 g for 10 min at 4°C. Supernatants were removed and the cell pellets were flash frozen on dry ice.Distal limb and head tissues were dissected at E12.5 for wt and at E11.5 for g for 15 min at 4°C and aliquots of the supernatant were taken for quantification and to confirm proper sonication. Remaining samples were stored at −80°C until use. Chromatin from 5 million cells was used for each immunoprecipitation. Protein G Dynal Beads (Invitrogen) were incubated 8 hours at 4°C with either 5 or 10 µg of antibodies. The chromatin was then incubated with the beads overnight. Immunoprecipitated complexes were sequentially washed in low salt ), medium salt ), LiCl ), and 1×TE buffers. The protein/DNA complexes were eluted in an SDS buffer , 10 mM EDTA) by incubation at 65°C for 15 min on a rotating platform. Crosslinks were reversed by incubating the complexes at 65°C overnight. Samples were treated one hour at 55°C with RNAseA  and then with Proteinase K for two hours. Finally, the DNA was purified on QIAquick columns (Qiagen). Specific antibodies for Med12 and RNAP2 were purchased from Bethyl (A300-774A) and Abcam (ab5131), respectively.Chromatin immunoprecipitation was performed as previously described with some modifications The ChIPed material was sequenced on a Hi-Seq 2000 high-throughput DNA sequencer. Sequencing libraries were prepared from 31 ng (RNAP2), 5 ng (Med12), and 345 ng (input) of ChIPed DNA. The libraries and flow cells were prepared by the IRCM Molecular Biology and Functional Genomics platform. The libraries were multiplexed and sequenced on one lane. The sequencing was performed by the McGill University and Génome Québec Innovation Centre following recommendations by the manufacturer .For RNAP2, Med12, and the input, we obtained a total of 151,045,509, 110,507,927, and 98,043,425 sequence reads, respectively. The first base of each sequence read was trimmed to ensure high base calling quality. The trimmed reads were mapped to the mouse mm9 genome assembly with Bowtie using the –best parameters Redundant reads were filtered out for peak finding and wiggle file generation. Thus the wiggle files enclose the total number of uniquely mapped and non-redundant reads. After processing the data, the number of sequence reads we obtained was 129,222,085 for RNAP2, 91,816,355 for Med12, and 88,141,136 for the input. The position of RNAP2 and Med12 peaks genome-wide identified in our study is provided in Shh−/− library used for the 3C experiments) were incubated for 15 min on ice in 200 µl of lysis buffer (10 mM Tris (pH 8.0), 10 mM NaCl, 0.2% NP40, supplemented with fresh protease inhibitor cocktail). Cells were then disrupted on ice with a dounce homogenizer . Cell suspensions were transferred to eppendorf tubes and centrifuged 5 min at 2000 g. Supernatants were removed and the cell pellets were washed twice with 100 µl of 1×EcoRI buffer (NEB). After the second wash, the cell pellet was resuspended in 100 µl of 1×EcoRI buffer, and divided into two eppendorf tubes containing 50 µl of cell suspension. 1×EcoRI buffer (337 µl) was added to each tube, and the mixture was incubated 10 min at 65°C with 0.1% SDS final (38 µl). Triton X-100 (44 µl of 10% Triton X-100) was added before overnight digestion with EcoRI (400 Units). The restriction enzyme was then inactivated by adding 86 µl of 10% SDS, and incubating 30 min at 65°C. Samples were then individually diluted into 7.62 ml of ligation mix . Ligation was carried out at 16°C for 2 hours.Limb and head cell pellets were treated as previously described 3C libraries were then incubated overnight at 65°C with 50 µl Proteinase K (10 mg/ml), and with an additional 50 µl Proteinase K the following day for 2 hours. The DNA was purified by one phenol and one phenol-chloroform extraction, and precipitated with 0.1 volume of 3M NaOAc pH 5.2 (800 µl) and 2.5 volumes of cold EtOH (20 ml). After at least 1 h at −80°C, the DNA was centrifuged 25 min at 20,000 g at 4°C, and the pellets were washed with cold 70% EtOH. The DNA was resuspended in 400 µl of 1×TE pH 8.0, and transferred to eppendorf tubes for another phenol-chloroform extraction and precipitation with 0.1 volume of 3M NaOAc pH 5.2 (40 µl) and 2.5 volumes of cold EtOH (1.1 ml). DNA was recovered by centrifugation (25 min at maximum speed at 4°C), and washed eight times with cold 70%EtOH. The pellets were then dissolved in 100 µl of 1×TE pH 8.0, and incubated with RNAse A (1 µl at 10 mg/ml) for 15 min at 37°C.Shh−/− distal limb, and head shown in This protocol was used to produce the 5C data for the distal limb, After the second wash, the cell pellet was resuspended in 50 µl of 1×EcoRI buffer. 1×EcoRI buffer (337 µl) was added to each tube, and the mixture was incubated 10 min at 65°C with 0.1% SDS final (38 µl). Triton X-100 (44 µl of 10% Triton X-100) was added before overnight digestion with EcoRI (400 Units). The restriction enzyme was then inactivated by incubating 30 min at 65°C. Ligation was performed in 600 µl . Ligation was carried out at 16°C for 4 hours.3C libraries were then incubated overnight at 65°C with 15 µl Proteinase K (10 mg/ml), and with an additional 15 µl Proteinase K the following day for 2 hours. The DNA was purified by one phenol and two phenol-chloroform extractions, and precipitated with 0.1 volume of 3M NaOAc pH 5.2 (64 µl) and 2.5 volumes of cold EtOH (1740 µl). After at least 1 h at −80°C, the DNA was centrifuged 25 min at maximum speed at 4°C, and the pellets were washed once with cold 70% EtOH. The DNA was resuspended in 50 µl of 1×TE pH 8.0, and incubated with RNAse A (1 µl at 10 mg/ml) for 15 min at 37°C.HoxA region , and one USP22 control region ) were mixed in equimolar ratio. Mixed BACs were digested with EcoRI and randomly ligated with T4 DNA ligase (5700 Cohesive end Units) overnight at 16°C. BAC libraries were then purified by phenol-chloroform extraction. The libraries were generated with the following BACs: RP23-420L19, RP24-359H1, RP24-242G11, RP-347D14, RP23-305I5 . These libraries were used only to correct primer pair efficiencies during 3C analysis and not in the 5C experiments.As 3C products are quantified by PCR amplification of expected ligation junctions with different primer pairs, differences in PCR primer pair efficiencies must be corrected using control 3C libraries. Control libraries were generated from bacterial artificial chromosomes (BACs) as previously described http://dostielab.biochem.mcgill.ca), and sequences are listed in USP22 region and an intergenic region within HoxA region as a reference.3C primers were designed using the ‘3CPrimer’ program  and the HoxA region  were designed using ‘my5C.primer’ 5C primers covering the CCATCTCATCCCTGCGTGTCTCCGACTCAG-(5C-specific)) and the P1-key tails ((5C-specific)-ATCACCGACTGCCCATAGAGAGG) were added to the Forward and Reverse 5C primers, respectively. Reverse 5C primers were phosphorylated at their 5′ ends. Two experimental designs were used in our study. In the “cluster R” design . In the “FR” design . Primer sequences are listed in The universal A-key . When 3C libraries were generated from a small cell number (106 cells), we used approximately 1 µg of DNA.5C libraries were prepared and amplified with the A-key and P1-key primers following a procedure described previously 6 cells) PCR reactions were pooled before purifying the DNA on MinElute columns (Qiagen).Before adding the 3C libraries to the reaction tubes, 5C primer stocks (20 µM) were diluted individually in water on ice, and mixed to a final concentration of 0.002 µM. Mixed diluted primers (1.7 µl) were combined with 1 µl of annealing buffer  on ice in reaction tubes. Salmon testis DNA (1.5 µg) was added to each 5C reaction, followed by the 3C libraries and water for a final volume of 10 µl. Samples were denatured at 95°C for 5 min, and annealed at 55°C for 16 hours. Ligation with Taq DNA ligase (10 U) was performed at 55°C for one hour. One tenth (3 µl) of each ligation was then PCR-amplified individually with primers against the A-key and P1-key primer tails. We used 28 cycles based on dilution series showing linear PCR amplification within that cycle range. The products from 2 (for the 3C libraries prepared from a large number of cells) to 8 (for the 3C libraries prepared from 105C libraries were quantified on agarose gel and diluted to 0.0534 ng/µl (for Xpress Template Kit v2.0) or 0.0216 ng/µl (for Ion PGM Template OT2 200 kit). One microliter of diluted 5C library was used for sequencing with an Ion PGM Sequencer. Samples were sequenced onto Ion 316 Chips following either the Ion Xpress Template Kit v2.0, and Ion Sequencing Kit v2.0 protocols, or the Ion PGM Template OT2 200 Kit, and Ion PGM Sequencing 200 Kit v2.0 protocols as recommended by the manufacturer's instructions (Life Technologies).https://main.g2.bx.psu.edu/). Briefly, the data was mapped against a customized reference file with TMAP. The reference file contained a list of all possible contacts between Forward and Reverse 5C primers covering our regions. The data was then filtered to remove low-quality reads , reads aligning more than two nucleotides away from the reference sequence start site, and reads which do not contain EcoRI restriction sites. This analysis generates an excel sheet containing interaction frequency lists (IFL) as well as a text file, which was used to visualize results using ‘my5C-heatmap’ 3. The number of total reads and of used reads is provided for each experiment in http://dostielab.biochem.mcgill.ca/Analysis of the 5C sequencing data was performed as described earlier http://www.ncbi.nlm.nih.gov/geo/. The my5C-primer and my5C-heatmap bioinformatics tools can be found at http://3dg.umassmed.edu/my5Cheatmap/heatmap.phpThe limb p300 and H3K27Ac datasets (Acc. No. GSE13845 and GSE30641) are from E11.5 embryos, and were downloaded from the Gene Expression Omnibus (GEO) website Dataset S1http://genome.ucsc.edu/) after decompression.ChIP RNAP2 chr6 wig file. A compressed wig file containing the ChIP-seq results of RNAP2 in E12.5 mouse distal limb from chromosome 6. This file can be uploaded directly onto the UCSC genome browser ((GZ)Click here for additional data file.Dataset S2http://genome.ucsc.edu/) after decompression.ChIP Med12 chr6 wig file. A compressed wig file containing the ChIP-seq results of Med12 in E12.5 mouse distal limb from chromosome 6. This file can be uploaded directly onto the UCSC genome browser ((GZ)Click here for additional data file.Dataset S3http://genome.ucsc.edu/) after decompression.ChIP input chr6 wig file. A compressed wig file containing the sequencing data of the input from E12.5 mouse distal limb on chromosome 6. This file can be uploaded directly onto the UCSC genome browser ((GZ)Click here for additional data file.Figure S1HoxA genes in the limb are reproduced in biological replicates. Physical contacts between the HoxA cluster and the upstream genomic region containing candidate enhancers were measured by 5C-seq in two biological replicates of distal limb , and a biological replicate of the head (bottom) of E12.5 embryos. The color intensity of each pixel in 5C heatmaps reflects the frequency of interaction between two genomic regions. Contact frequency is according to the respective color scales and corresponds to the number of sequence reads. Most predicted enhancers  interacted long-distance specifically with 5′ HoxA genes in the limb and contacts were weaker or absent in the head (compare top two panels with bottom). Green dotted lines link the position of enhancers along the genomic region to corresponding 5C fragments in the heatmaps. Brackets on the left hand side of each heatmap show the area containing Hoxa9, a10, a11, and a13. Green arrows indicate the chromatin fragments containing the Hibadh and Jazf1 promoters (p). Other limb-enriched interactions with HoxA genes that do not correspond to candidate enhancers are highlighted by blue stars. Restriction fragments corresponding to enhancer e6–8, 12, and 19 could not be included in the 5C design as they fell into regions that were not amenable to 5C (see Interactions between candidate enhancers and 5′ o 5C see .(EPS)Click here for additional data file.Figure S2Hoxa13 is more frequent in the limb than in the head. Physical contacts between the enhancers and the Hoxa13 promoter were detected by 3C as described in the Interaction of the e2–e5 enhancers with (EPS)Click here for additional data file.Figure S3HoxA genes in Shh−/− mutant limbs. Physical contacts between the HoxA cluster and the upstream genomic region containing the limb enhancers were measured by 5C-seq in two biological replicates of E11.5 Shh−/− distal limb , and a biological replicate of E12.5 head (bottom). The color intensity of each pixel in 5C heatmaps reflects the frequency of interaction between two genomic regions. Contact frequency is according to the respective color scales and corresponds to the number of sequence reads. The limb-specific interaction pattern between enhancers and the 5′ HoxA genes are similar in Shh−/− (middle panel) and wt distal limb buds (top panel) albeit with some interaction frequencies slightly reduced. These data are consistent with the data shown in Hoxa9, a10, a11, and a13. Green arrows indicate the chromatin fragments containing the Hibadh and Jazf1 promoters (p). Restriction fragments corresponding to enhancer e6–8, 12, and 19 could not be included in the 5C design as they fell into regions that were not amenable to 5C Click here for additional data file.Figure S4HoxA regulatory region at the megabase and sub-megabase scale in limb and head tissues. 5C interaction matrix of the HoxA cluster and its upstream regulatory region in distal limb (A) and head tissue (B). The 5C data was generated by 5C-seq using tissues from E12.5 embryos, and is presented in the form of heatmaps according to color scales as described in HoxA and Evx1 genes, and the enhancer clusters to the corresponding areas in heatmaps.The spatial organization of the (EPS)Click here for additional data file.Figure S5Hibadh promoter, and the enhancer sub-TADs. Enriched interaction between Evx1 and a subset of distal limb enhancers are shown with grey circles. Upon sub-TAD interactions in limbs, some loci get pulled away (blue) from each other compared to the head while other become closer (pink). The most enriched interactions not involving an enhancer or promoter could also represent structural contacts as those in Comparison between interaction patterns in the limb and head tissues shows major changes in chromatin architecture at the sub-megabase level. The interaction matrix of the region containing enhancer e1 to e5 with the entire distal limb regulatory landscape is shown in heatmap form. Interactions were measured by 5C-seq. The color scale represents differences in interaction frequencies in the head and limb. The robust interactions between sub-TADs in limb (black circles) modify the internal TAD architecture. Dashed circles highlight the enriched interaction between the region containing e5 and the (EPS)Click here for additional data file.Table S1(XLS)Click here for additional data file.Table S2HoxA, enhancer, and USP22 regions. The 3C primer sequences used to characterize the HoxA cluster and its regulatory landscape, and the genomic region containing housekeeping USP22 gene are listed along with their genomic position and the corresponding 3C fragment tested.Mouse 3C primers for the (XLSX)Click here for additional data file.Table S3HoxA and USP22 regions used in the anchored “R” design. This 5C primer set was used in Mouse 5C primers for (XLSX)Click here for additional data file.Table S4HoxA and USP22 regions used in the altered “FR” design. This 5C primer set was used in Mouse 5C primers for (XLSX)Click here for additional data file.Table S55C sequencing results. Summary of the number of sequence reads before and after processing from each 5C dataset presented in this study.(XLSX)Click here for additional data file.Table S6top) presented in matrix format. Predicted EcoRI restriction fragments and corresponding genomic regions are named at the top of each column and on the left of each row. Intersecting rows and columns identify the pair-wise contacts corresponding to interaction frequencies. The 5C data was filtered as described in Mouse 5C results for the distal limb anchored “cluster R” design. Distal limb 5C dataset from (XLSX)Click here for additional data file.Table S7top). Predicted EcoRI restriction fragments and corresponding genomic regions are named at the top of each column and on the left of each row. Intersecting rows and columns identify the pair-wise contacts corresponding to interaction frequencies. The 5C data was filtered as described in Mouse 5C results for the distal limb replicate 1 anchored “cluster R” design. First biological replicate of the distal limb 5C dataset presented in matrix format. This data is shown in (XLSX)Click here for additional data file.Table S8middle). Predicted EcoRI restriction fragments and corresponding genomic regions are named at the top of each column and on the left of each row. Intersecting rows and columns identify the pair-wise contacts corresponding to interaction frequencies. The 5C data was filtered as described in Mouse 5C results for the distal limb replicate 2 anchored “cluster R” design. Second biological replicate of the distal limb 5C dataset presented in matrix format. This data is shown in (XLSX)Click here for additional data file.Table S9middle) presented in matrix format. Predicted EcoRI restriction fragments and corresponding genomic regions are named at the top of each column and on the left of each row. Intersecting rows and columns identify the pair-wise contacts corresponding to interaction frequencies. The 5C data was filtered as described in Mouse 5C results for the head anchored “cluster R” design. Head 5C dataset from (XLSX)Click here for additional data file.Table S10bottom). Predicted EcoRI restriction fragments and corresponding genomic regions are named at the top of each column and on the left of each row. Intersecting rows and columns identify the pair-wise contacts corresponding to interaction frequencies. The 5C data was filtered as described in Mouse 5C results for the head replicate 1 anchored “cluster R” design. Biological replicate of the Head 5C dataset presented in matrix format. This data is shown in (XLSX)Click here for additional data file.Table S11Mouse 5C results for the distal limb alternating “FR” design. Distal limb 5C dataset from (XLSX)Click here for additional data file.Table S12Mouse 5C results for the distal limb replicate alternating “FR” design. Biological replicate of the distal limb 5C dataset presented in matrix format. This data is shown in (XLSX)Click here for additional data file.Table S13Mouse 5C results for the head alternating “FR” design. Head 5C dataset from (XLSX)Click here for additional data file.Table S14Mouse 5C results for the head replicate alternating “FR” design. Biological replicate of the head 5C dataset presented in matrix format. This data is shown in (XLSX)Click here for additional data file.Table S15top) presented in matrix format. This 5C data was generated from wt distal limbs 3C libraries produced with a 3C protocol for a small number of cells . Distal limb 5C dataset from ells see . Predict(XLSX)Click here for additional data file.Table S16Shh−/− anchored “cluster R” design . Shh−/− distal limb 5C dataset from middle) presented in matrix format. This 5C data was generated from Shh−/− distal limbs 3C libraries produced with a 3C protocol for a small number of cells Click here for additional data file.Table S17Shh−/− replicate 1 anchored “cluster R” design . First biological replicate of the Shh−/− distal limb 5C dataset presented in matrix format. This 5C data is shown in top), and was generated from Shh−/− distal limbs 3C libraries produced with a 3C protocol for a small number of cells Click here for additional data file.Table S18Shh−/− replicate 2 anchored “cluster R” design . Second biological replicate of the Shh−/− distal limb 5C dataset presented in matrix format. This 5C data is shown in middle), and was generated from Shh−/− distal limbs 3C libraries produced with a 3C protocol for a small number of cells Click here for additional data file.Table S19bottom) presented in matrix format. This 5C data was generated from wt head tissue 3C libraries produced with a 3C protocol for a small number of cells . Head 5C dataset from ells see . Predict(XLSX)Click here for additional data file.Table S20bottom), and was generated from head tissue 3C libraries produced with a 3C protocol for a small number of cells . Biological replicate of the head 5C dataset presented in matrix format. This 5C data is shown in ells see . Predict(XLSX)Click here for additional data file.Table S21RNAP2 peaks in E12.5 mouse distal limb. The start and end position of the RNAP2 peaks identified in mouse distal limbs at E12.5 is listed genome-wide.(XLSX)Click here for additional data file.Table S22Med12 peaks in E12.5 mouse distal limb. The start and end position of the Med12 peaks identified in mouse distal limbs at E12.5 is listed genome-wide.(XLSX)Click here for additional data file.


Bacillus coagulans, an industrial relevant organism that is recalcitrant for transformation. B. coagulans comK gene under its own promoter region into Bacillus subtilis comK strain results in low transcriptional induction of the late competence gene comGA, but lacking bistable expression. The promoter regions of B. coagulans comK and the comGA genes are recognized in B. subtilis and expression from these promoters is activated by B. subtilis ComK. Purified ComK protein of B. coagulans showed DNA-binding ability in gel retardation assays with B. subtilis- and B. coagulans-derived probes. These experiments suggest that the function of B. coagulans ComK is similar to that of ComK of B. subtilis. When its own comK is overexpressed in B. coagulans the comGA gene expression increases 40-fold, while the expression of another late competence gene, comC is not elevated and no reproducible DNA-uptake could be observed under these conditions. Our results demonstrate that B. coagulans ComK can recognize several B.subtilis comK-responsive elements, and vice versa, but indicate that the activation of the transcription of complete sets of genes coding for a putative DNA uptake apparatus in B. coagulans might differ from that of B. subtilis.The genes for DNA uptake and recombination in Bacilli are commonly regulated by the transcriptional factor ComK. We have identified a ComK homologue in  The ability to take-up DNA from the environment is widely spread among eubacteria, including Gram-positive and Gram-negative species B. subtilisBacilli, like B. licheniformisB. amyloliquefaciensB. cereusB. subtiliscomG operon encodes a type IV pilus that facilitates DNA to pass the cell wall and reach the cell membrane The genes coding for DNA uptake and recombination are conserved among Bacilli B. subtilis chromosome. To coordinate the expression of these genes and operons, B subtilis utilizes the global transcription factor ComK. If the protein level of ComK increases in the cells, ComK directly or indirectly activates more than 100 genes 5-TTTT) separated by a spacer of a discrete number of helical turns comK gene and the protein level of ComK are tightly regulated. Transcription of comK is repressed by AbrB, CodY, and Rok and activated by the DegU protein comS gene, embedded in the srfA operon, is activated in a quorum sensing dependent manner B. subtilis, like sporulation, motility, biofilm formation, and protease production The late competence genes are scattered around the Bacillus coagulans ComK homologue in B.coagulans and in B. subtilis. B. coagulans is a spore forming, microaerophilic, lactic acid producing species of the Bacillus genus. It is frequently isolated as food spoilage organism B. coagulans, our aim was to better characterize the ComK homologue from B. coagulans DSM 1. First, we assayed the B. coagulans comK gene (denoted as BcocomK) and promoter regions of BcocomK and BcocomG in the heterologous host, B. subtilis. In vitro studies further supported the conserved role of ComKBco as a DNA binding protein. Finally, we assayed the effect of BcocomK overexpression in B. coagulans.In this study we characterized the function of the B. coagulans strains, including DSM 1 (unpublished data), 36D1 and 2–6 showed that several genes and operons can be identified with high sequence similarity to Bacillus genes that code for the late competence genes in B.subtilis and their homologues in other Bacillus species . The B. coagulans ComK homologues are somewhat shorter than the ComK protein of B.subtilis (13 aminoacids shorter compared to ComK of B. subtilis), but most regions are conserved  that directly or indirectly control oagulans . InteresBcocomK gene of B. coagulans DSM1 appears to code for another member of the ComK family. Therefore, we wanted to test if the product of the comK gene can activate transcription. For this we first introduced the BcocomK gene (cloned in pATK4) into B. subtilis harboring a PBsucomGA-gfp reporter that enables us to monitor the activation of gene expression. The expression of BcocomK is driven by its own promoter region. Subsequently, we deleted the endogenous BsucomK gene in this strain so we can solely monitor the effect of BcocomK. In this synthetic background, the reporter activity observed depends on the presence of ComKBco. As depicted in BsucomGA promoter when the BcocomK gene was introduced, but not when the empty plasmid was present. The gene expression was detected using both flow cytometry .On the basis of its protein sequence analysis, the ytometry  and fluoytometry . The expBco can activate gene expression in the heterologous host B. subtilis. The transcription activation by ComK proteins depends on the promoter sequences they bind and their relative amount, and they either activate gene expression  into the ΔBsucomK strain containing the pATK6 plasmid showed that ComKBco can activate gene expression from the promoter of BcocomGA (BsucomK with pATK4) similarly to that observed for the BsucomGA promoter .ComKsubtilis . This exBco affects expression from the BsucomGA promoter and that the BcocomGA and BcocomK promoters are also recognized by ComKBsu. To test this in more details, we examined the in vitro DNA binding ability of ComKBco. We overexpressed a malE-BcocomK fusion construct in Escherichia coli and purified the ComKBco protein with the aid of the maltose binding protein (MBP) tag  tag . MBP fus protein . A smallon assay . We alsoromoters . The MBPeriments .B. subtilis suggest that the ComK proteins can activate gene expression on the heterologous comGA promoters (see above). To examine if this effect of ComK proteins is achieved by direct binding and transcription activation, we examined the in vitro binding of various ComK proteins on the B.coagulans and B. subtilis promoters of the comGA and comK genes. Results depicted in B. coagulans and B. subtilis to the heterologous promoter fragments is in agreement with the in vivo experiments done in B. subtilis. Taken together the in vivo and in vitro experiments all suggest that B. coagulans possesses a functional ComK homologue that is presumably able to activate the transcription of several late competence genes in B. coagulans .Experiments performed in Bsu activates transcription by binding K-boxes that are composed of two AT-boxes with a consensus sequence AAAA-N5-TTTT. The boxes are separated of a discrete number of helical turns , which places them on the same side of the DNA-helix B.coagulans showed the presence of several AT-boxes , however, K-boxes could be only found in the promoter regions of BcocomK and BcocomC . This motif is not found within the promoter regions of the BcocomGA and BcocomK genes. However, due to the low number of analyzed promoters, we cannot conclude whether the K-boxes or this latter overrepresented motif are functional in B. coagulans and their role requires additional functional characterization.ComK comCBco . InteresBcocomK overexpression in B. coagulans DSM 1. For this, we cloned the BcocomK gene under control of the IPTG  inducible spac promoter, resulting in plasmid pATK10. We introduced this construct into B. coagulans DSM 1 by electroporation and assayed whether the level of ComK protein is enhanced in B. coagulans containing pATK10 upon induction. An increased level of ComK protein was detected in Western blot analysis using antibodies against ComKBsu (BcocomGA gene was increased (ratio of 30.5±3.7) in the strain where BcocomK expression was induced with 1 mM IPTG compared to the wild type strain that lacks the plasmid. However, the expression level of another late competence gene (i.e. BcocomC) showed no significant change (ratio of 0.85±0.3) in the BcocomK-overexpression strain compared to the plasmid-free strain under this given condition. Other late competence genes (BcocomEA-C and BcocomFAC) also lacked the increased expression in the BcocomK overexpression strain (data not shown). Overexpression of the BsucomK gene using the previously published pNWBsucomK plasmid BcocomGA expression (ratio of 3.2±1.2) and unaltered BcocomC transcription (ratio of 1.3±0.5) compared to the plasmid free wild type strain. These experiments demonstrate that ComKBco can activate gene expression in B.coagulans in line with previous observations presented above.In our final experiments, we assayed the effect of  ComKBsu . Next, wBcocomGA by BcocomK overexpression is sufficient to observe a functional DNA uptake in B. coagulans, we tested the uptake of genomic DNA  after induction. Cells were plated on medium containing chloramphenicol. We could not observe reproducible DNA uptake under the above presented BcocomK overexpressing conditions in B. coagulans, suggesting the lack of a fully functional DNA uptake machinery under these specific conditions. Similarly, DNA uptake was not detected in B. coagulans when BsucomK was overexpressed, in contrast to the experiments with B. cereusBco is detected by Western blot analysis when BcocomK is overexpressed in B.coagulans . Previous studies on the binding site of ComKBsu described K-boxes, where the distance between the two AT-boxes is important for its function BcocomK and BcocomC genes. In contrast with these in silico observations, purified ComKBsu protein binds in vitro to the promoter regions of late competence genes of B. coagulans . However, our study clearly shows that ComKBco is a DNA-binding protein that is capable of activating gene expression. Therefore, it presents an important element of future research for better understanding of late competence gene induction in B. coagulans.While overexpression of B. coagulans strains were grown in BC medium at 50°C, 120 rpm 4)2HPO4, 3.5 g (NH4)2SO4, 10 g Bis-Tris (bis[2-hydroxymethyl]iminotris[hydroxymethyl]-methane), 5 mg MgCl2 · 6 H2O, 3 mg CaCl2 · 2 H2O, 1 ml of filter sterilized trace elements , pH 6.7. B. subtilis strains were grown in minimal medium Escherichia coli DH5α and Lactococcus lactis MG1363 were grown in TY and GM17 (37.5 g M17 broth (Difco) per liter supplemented with 0.5% glucose) medium, respectively, grown at 30°C or 37°C. Antibiotics were used at a concentration of 5 µg ml−1 for chloramphenicol, 6 µg ml−1 for tetracycline, and 100 µg ml−1 for ampicillin. Transformation of L. lactis and B.coagulans was performed by electroporation as previously described E. coli was performed by heat-shock B. subtilis strains using natural competence The strains and plasmids used in this study are listed in Bco, the BcocomK gene was PCR amplified from the genome of B. coagulans DSM 1 using oligos oATK26 and oATK14  into the BamHI-ScaI digested pATK8 vector, resulting pATK10. The resulting vectors were validated using restriction analysis and inserts were verified by sequencing.To facilitate the purification of MBP-ComKleotides , digestegfp gene was first obtained from pSG1151 using KpnI-XbaI restriction enzymes and ligated into the corresponding sites of the broad host range pNW33N vector, resulting pATK2. pATK2 was digested with KpnI and ApaI and used to ligate the promoter fragments of BcocomK and BcocomGA obtained with PCR reaction using oligonucleotides oATK5 and oATK6 (for BcocomK) and oATK7 and oATK8 (for BcocomG) and digested with the same restriction enzyme pairs. The integrity of cloned fragments was verified by sequencing.The Bco or pMALcomK −1 of IPTG at 0.8 of OD600. Cells were harvested by centrifugation . Pellets were washed with a buffer containing 1.17% NaCl, 25 mmol l−1 EDTA, 10 mmol l−1 NaN3, 0.15% DTT, 50 mmol l−1 Tris-HCl pH 7.4. Cells were lysed by sonification , and the sonicated fractions were centrifuged  to obtain a supernatant that contains the MBP-ComK. The fraction with the MBP-ComK has been loaded on an amylose column which had been equilibrated with a buffer containing 0.5 mM DTT, 20 mM Tris-HCl pH 8.0. Elution was performed with the same buffer, now containing 10 mM maltose. The fractions were stored immediately at 4°C . The purity of MBP-ComKBco and MBP-ComKBsu was verified on SDS-PAGE and the purified proteins were also validated using Western hybridization with antibody raised against ComKBsu as described in B. coagulans samples were performed as described previously for B. cereusB. coagulans wild-type and BcocomK overexpression strains were grown in BC medium until 0.8 of OD600 and induced with 0.1 mmol l−1 IPTG. Three hours after induction, samples were harvested by centrifugation , disrupted using lysozyme treatment. The ComKBco protein level was detected after SDS-PAGE using Western hybridization with ComKBsu– specific antibody 1 liter culture of cells containing the pMALcomKB. coagulans putative competence genes comK, comG, comC and comFA were obtained by PCR using oligos oATK5-oATK6, oATK7-oATK8, oATK48-oATK49, and oATK50-oATK51, respectively . Purified MBP-ComKBco and MBP-ComKBsu proteins and probes were premixed on ice in binding buffer. Reaction mixtures contained poly (dI-dC) that is known to eliminate non-specific DNA binding of ComK −1 Tris, 0,089 mmol l−1 Boric Acid, 0,022 mmol l−1 EDTA) at 90 V for 60 minutes, dried in a vacuum dryer and autoradiographed using phosphoscreens and a Cyclone PhosphorImager .Gel retardation assays were carried out essentially as described by Susanna et al. ectively . The B. B. coagulans wild type and BcocomK overexpression strains were grown in BC medium until 0.8 of OD600 and induced with 0.1 mmol l−1 IPTG. Two hours after induction, samples were harvested by centrifugation . A total of three independent biological replicates were included. RNA preparation of quantitative PCR was performed as described before −1 Tris·HCl (pH 7.5), 2.5 mmol l−1 MgCl2, 0.1 mmol l−1 CaCl2), and re-purified with the Roche RNA isolation Kit. RNA concentration and purity was assessed using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed with 50 pmol random nonamers on 4 µg of total RNA using RevertAid™ H Minus M-MuLV Reverse Transcriptase . Quantification of cDNA was performed on an CFX96 Real-Time PCR System  using Maxima SYBR Green qPCR Master Mix . The following oligos were used: for comGA, oATK87 and oATK88, for comC, oATK89 and oATK90 and for rpiA gene of B. coagulans, oATK83 and oATK84 . For the flow cytometric analyses, cultures were inoculated into fresh minimal medium. Samples were taken after transition to stationer phase every hour. Cells were diluted 10 fold in minimal salts and analyzed on a Coulter Epics XL-MCL flow cytometer  operating an argon laser at 488 nm. Green fluorescent protein (GFP) signals were collected through an FITC filter with the photomultiplier voltage set between 700 and 800 V. Date were obtained using EXPO32 software (Beckman Coulter) and further analyzed using WinMDI 2.8 (The Scripps Research Institute). Figures were prepared using WinMDI 2.8 and Adobe CS4 Illustrator.BsucomGA-gfp in individual cells using fluorescence microscopy was performed as described by Smits et al.The fluorescence of the GFP reporter protein was visualized with a Zeiss Axiophot microscope, using filter set 09 . Imaging of PBcocomK), JX518620 (BcocomGA), JX518621 (BcocomC), JX518622 (BcocomFA), JX518623 (BcorpsD), JX518624 (BcorpiA).Sequences used in this study have been deposited in GenBank under accession numbers JX518619 , open boxes indicate upstream open reading frames and com genes, numbers denote spacing between AT-boxes resulting in a so called K-box . B. Sequences of B. coagulans DSM1 promoter regions related to competence. Bold letters indicate putative AT-boxes. The putative open reading frames, com genes are indicated below the sequence.(PDF)Click here for additional data file.Figure S2Bco protein.Detection of the ComK Equal amounts of proteins were loaded in each lane, Samples were taken from induced (+) or non-induced (−) cultures. Cells were centrifuged, lysed and analysed by Western blotting using ComKBsu-specific antibodies. The arrows indicate ComK specific signal (K) and non-specific signal (NP). Positions of molecular weight marker bands are indicated on the right side of the gel.(TIF)Click here for additional data file.


Foxa1-only sites are enriched for p53 binding sites and are frequently found near genes important to cell cycle regulation, while Foxa2-restricted sites show only a limited match to the forkhead consensus and are found in genes involved in steroid and lipid metabolism. Thus, Foxa1 and Foxa2, while redundant during development, have evolved divergent roles in the adult liver, ensuring the maintenance of both genes during evolution.Gene duplication is a powerful driver of evolution. Newly duplicated genes acquire new roles that are relevant to fitness, or they will be lost over time. A potential path to functional relevance is mutation of the coding sequence leading to the acquisition of novel biochemical properties, as analyzed here for the highly homologous paralogs Foxa1 and Foxa2 transcriptional regulators. We determine by genome-wide location analysis (ChIP-Seq) that, although Foxa1 and Foxa2 share a large fraction of binding sites in the liver, each protein also occupies distinct regulatory elements  The duplication of a gene from a common ancestor, resulting in two copies known as paralogs, plays an important role in evolution. Newly duplicated genes must acquire new functions in order to remain relevant, otherwise they are lost via mutation over time. We have performed genome-wide location analysis (ChIP–Seq) in adult liver to examine the differences between two paralogous DNA binding proteins, Foxa1 and Foxa2. While Foxa1 and Foxa2 bind a number of common genomic locations, each protein also localizes to distinct regulatory regions. Sites specific for Foxa1 also contain a DNA motif bound by tumor suppressor p53 and are found near genes important to cell cycle regulation, while Foxa2-only sites are found near genes essential to steroid and lipid metabolism. Hence, Foxa1 and Foxa2 have developed unique functions in adult liver, contributing to the maintenance of both genes during evolution. While their main function during development is in directing correct pattern formation The developmental regulators Foxa1 and Foxa2, members of the winged helix transcription factor family, share a highly conserved 100 amino acid DNA binding domain, and have been shown to cooperate and direct early liver and pancreas development in vivo, contribute to their evolutionary diversification. We find that Foxa1 and Foxa2, while redundant during development, are functionally diversified in the adult liver through target occupancy, ensuring their evolutionary fitness as distinct regulators of transcription.Here we investigated whether alterations in the biochemical properties of close paralogs, as shown by genome-wide target preferences Gene duplication followed by functional diversification of the duplicates is a primary driver of evolution. The developmental regulators Foxa1 and Foxa2, the closest paralogs in the Foxa subfamily of winged-helix transcription factors , have beFoxa1−/−;loxP/loxPFoxa2;Alfp.Cre), by analyzing RNA isolated on embryonic day 18. The number of genes dependent on each single factor was quite limited, compared to the number of genes that were differentially expressed in the double mutant  for Foxa1 and Foxa2. We identified 5,682 binding sites for Foxa1 and 11,097 for Foxa2 using the GLITR algorithm Chromatin-association of both Foxa1 and Foxa2 was previously correlated with the methylation status of histone H3 lysine 4 de novo motif analysis of these sequences, we found that all target sequences contain at least one perfect match to the previously known Foxa consensus binding site , and frequently also a second Foxa-like motif, containing one or two degenerate nucleotides  and those with alleles associated with diabetes risk identified by genome-wide association studies (GWAS) (Fto (fat mass and obesity associated gene), where Foxa1 and Foxa2 bind four distinct intronic regions  . An exam regions . These fAmong the Foxa1/Foxa2 targets are twelve nuclear hormone receptors and multiple liver-enriched DNA binding proteins . In addition, Foxa1 and Foxa2 bind regulatory regions of three members of the CTF/nuclear factor I family  and the transcriptional repressor CTCF. Interestingly, the recognition motifs for Hnf1a, Hnf4a, Hnf6, and the nuclear factor I family members are also enriched in the target sequences bound by both Foxa paralogs , thus sude novo motif analysis using the sequences bound by Foxa1 only, as defined above. The top motif in the Foxa1-only set (yellow circle) was a weak forkhead motif, with three-hundred ninety-eight (or twenty-two percent) sequences containing a perfect Foxa consensus (T[A/G]TT[G/T]AC)  . Results[G/T]AC) .forkhead consenus, several other motifs appeared in the Foxa1-only set. The first  and TNF receptor superfamily with p53 .Afp gene, where the two transcription factors regulate expression of Afp in opposite directions, and Foxa1 binding is enhanced in p53-null livers For most sites examined, there was a trend toward increased enrichment of p53 binding in Foxa1-deficient livers. Foxa1 and p53 have been shown to have an antagonistic relationship at the distal promoter element of Functional categories over-represented in the genes bound by only Foxa1 are associated with cell cycle regulation, which is not surprising as many of them are p53 targets . ‘CyclinOur analysis also confirms a number of motifs found previously in regions bound by Foxa1, such as those of the androgen receptor (AR) and the upstream stimulatory factor (Usf) cis-regulatory elements of additional transcription factors, their co-activators and targets genes, in regulatory feed-forward loops. Foxa1 has been shown to cooperate with the estrogen receptor (ER) in gene activation in breast cancer cell lines Foxa1 also binds to the de novo analysis for both Foxa2-only set resemble a stronger forkhead binding site , which targets serine 283 Recently, Kohler and Cirillo have reported that acetylation of Foxa1 by p300 attenuates binding of Foxa1 to DNA in vivoforkhead consensus, as well as motifs for liver-enriched transcription factors and nuclear receptors, and AT-rich motifs, including homeodomain transcription factors, Nfil3, and Hmga1. Hence, specialization of Foxa paralogs in the adult liver has resulted in Foxa2 acquiring a specific role in coordinating the transcriptional regulatory network that controls bile acid and lipid metabolism.Binding of Foxa2 to its targets in the adult liver has been studied previously Afp) gene The function of Foxa1 has been studied primarily in a variety of cancer cell lines A study by Gao and Matusik  to detect potential DNA binding complexes occupying the TS2 regulatory element of the probasin gene, bound by Foxa1 in the prostate cis-regulatory elements that contain a high-affinity forkhead binding site and regulate genes essential in development and those implicated in etiology of diabetes. It is possible that gene regulatory networks of important disease susceptibility genes have redundant modules and closely related paralogs that compensate for each other, as occurs in developmental regulation. However, while Foxa1 retains the more ancient role of regulating proliferation and growth by influencing DNA binding of p53, Foxa2 has acquired mutations in its DNA binding domain and a new role in the hepatocyte, regulating genes involved in lipid metabolism. In this instance, it is more advantageous for duplicated paralogs to perform different functions and transduce the many different physiological signals propagated through differentiated tissues. We propose that this functional diversification of the Foxa paralogs contributed to the maintenance of both genes during evolution.In summary, the transcriptional regulators Foxa1 and Foxa2 share a significant fraction of Foxa1 null and loxP/loxPFoxa1 mice, the Alfp.Cre transgenic line to achieve hepatocyte-specific deletion of Cre, and the loxP/loxP;Alfp.CreFoxa2 mouse model has described previously The derivation of Foxa1 null, loxP/loxP;Alfp.CreFoxa2, Foxa1−/−;loxP/loxPFoxa2;Alfp.Cre and control embryos (e18.5) and loxP/loxP;Alfp.CreFoxa1 and control mice (2–3 months), and quantitative reverse transcription-PCR performed as described Liver RNA was isolated from ChIP and the following real time PCR reactions were performed as described Protein analysis by immunoblot was performed as reported previously Foxa1 ChIP libraries for five biological replicates and two input libraries were sequenced in a total of seven lanes to 36 nucleotides. The reads were aligned to the mouse genome  using the ELAND aligner (Illumina). Reads with a unique best alignment were retained for further processing. We have pooled reads from previously published Foxa2 ChIP-Seq  and 2 biological replicates sequenced on GAII, resulting in 28,625,810 total reads for Foxa1 ChIP and 32,852,268 total reads for Foxa2 ChIP. The GLITR algorithm was run 10 times on a random sample of 28 million reads each from Foxa1 or Foxa2 ChIPseq and input samples which were compared to a large pool of input reads from multiple mouse tissues. Regions were defined as bound and subjected to further analysis if identified by GLITR in 3 or more runs.The set of all Foxa1 or Foxa2 sites was created by taking the union of regions from each GLITR analysis and merging regions that overlapped. The set of common sites was defined as the set of merged regions. To identify truly unique binding sites for each factor for subsequent analyses, we defined stringent criteria for Foxa1-only and Foxa2-only binding sites. The set of unique targets contains peaks for the first factor that include at most one tag per million per kilobase (Kb) for the other. All region manipulations were performed using Perl and R scripts in the TESS Location Analysis package.de novo motif finding. Sequence alignment of Foxa proteins was performed by ClustalW2 algorithm CisFinder The Asap software was used for positional weight matrix enrichment analysis  http://www.ebi.ac.uk/arrayexpress/) under accession nos. E-MEXP-2106, E-MEXP-3426, and E-MEXP-3428. The ChIP-seq data from this study can be accessed at GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession nos. GSE25836, GSE26729, and GSE33666.The microarray data from this study can be accessed at ArrayExpress , ‘:’ (highly conserved column), ‘.’ (weakly conserved column).(PDF)Click here for additional data file.Table S1Overrepresented Forkhead Positional Weight Matrices in Foxa1-specific, Foxa2-specific, and Dual ChIP-Seq Targets.(XLS)Click here for additional data file.Table S2Cis-regulatory Elements of Diabetes Susceptibility Genes Bound by Both Foxa1 and Foxa2.(DOC)Click here for additional data file.Table S3Overrepresented Other (non-Forkhead) Positional Weight Matrices in Foxa1-specific, Foxa2-specific, and Dual ChIP-Seq Targets.(XLS)Click here for additional data file.


Retrotransposons' high capacity for mutagenesis is a threat that genomes need to control tightly. Transcriptional gene silencing is a general and highly effective control of retrotransposon expression. Yet, some retrotransposons manage to transpose and proliferate in plant genomes, suggesting that, as shown for plant viruses, retrotransposons can escape silencing. However no evidence of retrotransposon silencing escape has been reported. Here we analyze the silencing control of the tobacco Tnt1 retrotransposon and report that even though constructs driven by the Tnt1 promoter become silenced when stably integrated in tobacco, the endogenous Tnt1 elements remain active. Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction. Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host. Arabidopsis thalianaMobile genetic elements are present in virtually all genomes where they occupy a variable but very high proportion. In plants they can account from 21% of the genome as in LORE1 from Lotus japonicusTos17Arabidopsis they can transpose, but become silenced and heavily methylated when their copy number increases Most retrotransposons are highly silenced in plants and are only reactivated in certain mutant backgrounds or under severe stress conditions. Cell culture stresses can reactivate epigenetically-silenced retrotransposons such as While epigenetic silencing of transposons is generally very efficient, a handful of elements remain active even when present in high copy numbers in plant genomes. One of the best studied plant retrotransposons, the tobacco Tnt1 element, is present in hundreds of copies and still an important fraction of them remain transcriptionally active locus, the expression of the transgene was completely silenced. Upon treatment with R10, which induced the endogenous Tnt1 expression, the expression of GUS was not recovered  corresponding to the 35S promoter in order to transiently silence a 35S-GUS construct stably introduced in tobacco. Agroinfiltration of a hairpin of the 35S promoter sequences driven by the promoter of the AtBH2 gene, highly active in leaf tissue In summary we have not detected any recovery of the silenced 35S promoter in R10-treated leaves that would indicate a global decrease of TGS and explain Tnt1 expression.http://smallrna.udel.edu/) confirmed that tobacco produces siRNAs matching the Tnt1 sequence. The size distribution of the siRNAs complementary to the Tnt1 sequence shows two different peaks at 21–22 and 24 nt  in which a GFP reporter gene was flanked by two Tnt1 LTRs, providing the promoter and terminator sequences, in an arrangement that resembles that of the endogenous Tnt1 elements . While tThe analysis of other constructs containing fragments of the Tnt1 LTR together with fragments of the 35S promoter gave a similar result. While these constructs were expressed as expected in transient expression assays (not shown), they were silenced when stably introduced in tobacco . As expeIn plants TGS is usually accompanied by DNA methylation of the silenced promoters, in particular in the case of transposable elements The results presented here clearly show that Tnt1 promoter sequences are a target of TGS in tobacco and that they can be highly methylated and efficiently silenced when present in reporter constructs stably introduced in tobacco.Tnt1 is present in hundreds of copies in tobacco, an important fraction of which is transcriptionally competent Altogether these results show that the endogenous Tnt1 elements are significantly less methylated at asymmetrical positions than the silenced LTR-GFP-LTR transgenes , and sugIn addition to DNA methylation, TGS is often correlated with the deposition of chromatin marks characteristic of inactive chromatin. We have therefore analyzed the histone modifications associated with the chromatin of the silenced LTR-GFP-LTR transgene as well as the endogenous Tnt1 elements. Both transgene and endogenous Tnt1 elements are associated with active chromatin epigenetic marks  both in normal and inductive stress conditions . In factIn conclusion, although methylation at asymmetrical positions is higher at the silenced transgenes than at the active endogenous elements, this does not correlate with the presence of inactive chromatin epigenetic marks on the silenced transgene sequences.It has been recently shown that the incorporation of the histone variant H2A.Z into promoters correlates with their capacity to be activated The results presented here show that the promoter of Tnt1 is a target of silencing in tobacco and that, outside of the context of the entire Tnt1 element, silencing correlates with a higher level of methylation at asymmetric positions and the inability to incorporate the histone variant H2A.Z into the promoter region upon inductive treatments. Our results also show that endogenous Tnt1 elements, which remain transcriptionally active, are only partially methylated and incorporate H2A.Z into their promoters upon induction.The main difference between the endogenous Tnt1 elements and the constructs containing LTR sequences that may explain their different sensitivity to silencing is that the transgenes lack most of the internal Tnt1 sequences. Indeed, although the LTR-GFP-LTR constructs have a structure resembling a Tnt1 element, with a central coding region flanked by two LTRs, they do not contain any of the Tnt1 coding regions. This strongly suggests that the central Tnt1 coding region is needed to confer methylation and silencing resistance. We have tried to precisely define the sequences that may provide silencing protection by analyzing the expression of LTR-driven transgenes containing also internal Tnt1 sequences. We analyzed first a construct driven by an extended Tnt1 LTR containing the region coding for the first 25 amino acids of the GAG protein (named Hut-2) that has already been shown to be transcriptionally active in vitro culture While accounting for a major part of plant genomes, retrotransposons are very efficiently controlled by the silencing machinery of the cell and they are only expressed in very special situations. Apart from a transient de-repression in gametophytes and seeds, meant to reinforce their silencing in subsequent generations As a general release of silencing cannot be invoked to explain the expression of Tnt1, only two other possible explanations remain. Either Tnt1 is not a target of silencing in tobacco or, alternatively, it is but specifically escapes this control. Tobacco produces siRNAs of 24 nt against Tnt1, and we show here that they preferentially target the LTRs of the element suggesting that Tnt1 is regulated by siRNA-related silencing. Moreover, the results presented here show that constructs containing Tnt1 LTR sequences are strongly silenced and the Tnt1-related sequences are specifically methylated when stably introduced in tobacco, showing that tobacco is able to silence the Tnt1 promoter. The silencing of Tnt1 is highly efficient, as a single Tnt1 LTR (or fragments of it) added to the pool of hundreds of endogenous LTRs is detected, methylated and silenced. Interestingly, while Tnt1 sequences that are part of a transgene construct are methylated and silenced, endogenous Tnt1 elements escape this control remaining inducible and less methylated at asymmetrical positions.Arabidopsis only the targets of the RdDM located in heterochromatic regions present also high levels of H3K9me2, whereas targets located in euchromatin are often associated with active histone epigenetic marks, such as histone H3 acetylation and H3K4me3, and lack H3K9me2, and it has been proposed that their silencing could be more easily reverted The silencing of Tnt1 LTRs present within the transgene is not accompanied by repressive histone epigenetic marks. Indeed, the silenced LTR transgenes show high histone H3 acetylation, are associated to H3K4me2 and H3K4me3, and lack H3K9me2 and H3K27me3. It has been shown that although transposons are known to be one of the main targets of RNA-directed DNA methylation (RdDM) in plants The main difference between the endogenous Tnt1 elements and the constructs containing LTR sequences that may explain their different sensitivity to silencing is that the transgenes lack most of the internal Tnt1 sequences, which suggests that the central Tnt1 coding region may be needed to confer methylation and silencing resistance. For this reason we have tried to precisely define the sequences that may provide silencing protection but failed. Although some of the constructs containing Tnt1 internal regions showed some expression, it was not consistently maintained. Our results suggest that whereas internal Tnt1 sequences probably play a role in protecting Tnt1 from being fully methylated and silenced, other factors influence their expression.per se does not explain the ability for a particular Tnt1 to be expressed, we do have seen a high variability of expression of the –END construct or the constructs driven by chimerical Tnt1-35S promoters. It is interesting to note that, while Tnt1 does not have strict target site specificity, it integrates in genic regions and most Tnt1 copies are located near genes The chromosomal environment could be one of these factors. Although, Tnt1 expression analyses have shown that multiple Tnt1 elements (sitting at different genomic locations) are concomitantly expressed in tobacco ddm1) mutants in Arabidopsis is highly stochastic, with different elements proliferating in different plants Another factor that may influence the expression and explain the difference between endogenous Tnt1 elements and Tnt1-containing constructs could be related to the way they are integrated into the genome. Whereas transgenes integrate at the level of double strand breaks, usually repaired by non-homologous end joining (NHEJ), retrotransposons use their own integrase to catalyze their insertion. This may influence their different sensitivity to silencing mechanisms and make Tnt1 sequences introduced as a part of a transgene more prone to silencing than their endogenous counterparts. Finally, retrotransposon activation is probably a highly stochastic event, and not all potentially active Tnt1 promoters may be activated at once. Indeed, it has recently been shown that the retrotransposon release from silencing in decreased in DNA methylation  and harvested 3 hours after infiltration for RNA isolation.Agrobacterium tumefaciens strain LBA4404 for agrobacterium-mediated transformation of Nicotiana tabacum (cv Xanthi) plants. Plants with a single copy of the transgene were selected for further analysis.The LTR-GFP-LTR construct has been previously described Arabidopsis thaliana ATHB2 gene Xho1 and Kpn1 or BamH1 and Cla1 restriction sites respectively. Both PCR fragments were cloned into the respective sites of the modified pHannibal to create a 35S hairpin construct. The hairpin cassette was then released from pHannibal by digestion with Not1 and cloned into pEnt3C (Invitrogen) and subsequently incorporated into the gateway binary vector pMDC99. Binary construct was transformed into Agrobacterium tumefaciens strain LBA4404. Overnight cultures grown in presence of 20 µM acetosyringone were pelleted by centrifugation and cells resuspended in 10 µM MgCl2, 10 µM MES (pH 5.7) and 150 µM acetosyringone to an OD600 of 0.3. Upon 4 hr incubation at room temperature, Agrobacterium suspension was infiltrated into expanded tobacco leaves using a needleless syringe. Three days after infiltration, non-treated and R10 cellulase-treated leaves were harvested for RNA isolation.The sequence of the 35S promoter was assembled in sense and antisense orientation into a pHannibal suppression vector Agrobacterium tumefaciens C58C1 strain. Agroinfiltration was carried out as described above. Ttobacco leaves were treated with R10 three days after infiltration.For transient expression of the silencing suppressor, a 35S promoter-based construct expressing the helper component protease (Hc-Pro) of Tobacco Etch Virus (TEV) was used 32P-labelled by PCR. The Tnt1 probe was generated by PCR amplification of a 624 bp fragment (RT3-RT6 oligos) from the Tnt1 region coding for the reverse transcriptase. Membranes were hybridized overnight at 65°C in Church buffer Total RNA was isolated from tobacco leaves using guanidine hydrochloride as described The DNA methylation status was analyzed by Southern hybridization and bisulfite sequencing. For Southern hybridization, 25 µg of genomic DNA was digested with HindIII in combination with the methylation sensitive restriction enzymes AluI, DdeI or HpaII. The digested DNA was loaded onto a 1% agarose gel, blotted to Hybond-N membranes by standard capillary transfer and UV cross-linked. Membranes were hybridized overnight at 65°C in Church buffer 2, 1% Triton-X100, 1 mM DTT), resuspended in lysis buffer  containing 0.1% SDS and sonicated in a Bioruptor. The DNA/protein complexes were immunoprecipitated with α-H3Ac (Upstate 06-599), α-dimethyl H3K4 (Upstate 07-030), α-trimethyl H3K4 (Abcam 8580), α-dimethyl H3K9 (Upstate 07-441) or α-H2AZ (Abcam 18263) as previously described Transgenic tobacco leaves were cross-linked with 1% formaldehyde in 10 mM Tris-HCl pH 8.0, 0.4 M sucrose, 1 mM EDTA and 0.5% Triton-X100. Nuclei were isolated in extraction buffer  Northern blot analysis of GUS expression in leaves of a 35S-GUS transgenic plant, after infiltration with a 35S hairpin expression construct. Different lanes correspond to different number of days after infiltration (DAI). The expression of an endogenous ubiquitin gene is shown beneath as control. B) Southern analysis of DNA from the same tobacco leaves analyzed in (A) digested with methylation sensitive enzymes to assess DNA methylation. The enzymes used are indicated on the left of each panel. A schema showing the position of the restriction enzymes sites, the probe used (black box) and the expected fragments is shown below. The 35S promoter is shown as a grey box and the GUS coding sequence is shown as a blue box. C) Northern blot analysis of GUS expression in 35S-GUS transgenic plants non silenced (−), silenced by crossing with the 271 locus (×271) or by infiltration with a 35S hairpin expression construct (ds35S). D) Southern blot analysis of DNA from the same tobacco leaves analyzed in (C) digested with methylation sensitive enzymes to assess DNA methylation. The expected bands are the same as in (B).(PDF)Click here for additional data file.Figure S2Tobacco siRNAs targeting the Tnt1 sequence. A) size distribution of tobacco siRNAs from leaves  which target the Tnt1 sequence. B) Distribution of the siRNAs of 24 nt directed against Tnt1 along the Tnt1 sequence.(PDF)Click here for additional data file.Figure S3Analysis of the potential post-transcriptional silencing of the LTR-GFP-LTR constructs. Northern analysis of tobacco leaves of two different transgenic lines containing a single copy of the LTR-GFP-LTR transgene treated (+) or non treated (−) with R10. Half of the leaves were infiltrated with a construct expressing the viral silencing suppressor HcPro (H) three days prior to R10 treatment. A transgenic line expressing a 35S-GFP was included as an hybridization control. An image of the EtBr staining of the RNA gel used as loading control is shown underneath.(PDF)Click here for additional data file.Figure S4Silencing of chimeric promoters containing part of the Tnt1 LTR. Qualitative expression of 5 different constructs driven by promoters containing Tnt1 LTR fragments, stably integrated in tobacco. The expression of a control 35S driven construct is shown as control. A schema of the analyzed construct and the name of the construct is given above each set of results. Tnt1 LTR sequences are shown as red boxes and 35S sequences are shown as yellow boxes. Constructs contain: a complete 35S promoter (35SG); a complete U3 sequence of a Tnt1 element (U3AG); the upstream U3 sequence, up to the TATA box, of Tnt1 plus the −90 region of a 35S, including the TATA box and the transcriptional start (A90G); the enhancer region of the 35S promoter and the TATA box and transcriptional start region of a Tnt1 U3 (ETAG); the enhancer region of the 35S promoter upstream of a complete U3 region of Tnt1 (EAG), and the enhancer region of the 35S promoter upstream of a A90G construct (EE90G). For each construct the expression in untreated leaves and leaves treated with R10 (R10) in different lines containing a single copy of the transgene was analyzed. The expected expression  of each construct based on the knowledge of the 35S and Tnt1 promoter is shown in red. The actual expression of the different lines for each construct is shown in black. Very low expression is shown by a small + sign. For some of the constructs different individual plants of the same line were analyzed to assess individual variability of expression.(PDF)Click here for additional data file.Figure S5DNA Methylation status of the 3′LTR of the LTR-GFP-LTR silenced transgene. The 3′ region of the LTR-GFP-LTR transgene, including the 3′ LTR, was amplified and sequenced from bisulfite converted DNA from R10-treated leaves of the LTR-GFP-LTR 6–11 transgenic line. Ten clones were sequenced from each transgene . The different regions of the transgene are shown under the sequence.The methylation state of each cytosine is shown as in (PDF)Click here for additional data file.Figure S6Comparison of the LTR sequences of endogenous Tnt1 elements together with Tnt1 RNA LTR sequences. Phylogenetic analysis of 157 Tnt1 LTRs present in public databases (shown as red dots) with 25 Tnt1 mRNA sequences obtained from R10 induced leaves (shown as green dots). LTRs selected for further analysis are shown by a red arrow.(PDF)Click here for additional data file.Figure S7DNA Methylation status of the 3′LTR of an endogenous Tnt1 element. The 3′ region of the s231f endogenous Tnt1 element, including the 3′ LTR, was amplified and sequenced from bisulfite converted DNA from R10-treated leaves of the LTR-GFP-LTR 6-11 transgenic line. Ten clones were sequenced from each transgene . The methylation state of each cytosine is shown as in (PDF)Click here for additional data file.Figure S8Global methylation analysis of the endogenous Tnt1 population. Southern blot analysis of tobacco DNA obtained from leaves untreated (0) or treated with R10 for different periods of time  digested with enzymes sensitive to asymmetrical  and symmetrical  methylation. A schema of the position of the probe used for hybridization and the expected band sizes are shown below. The approximate sizes of the hybridizing bands is shown.(PDF)Click here for additional data file.Figure S9Histone epigenetic marks associated with the LTR-GFP-LTR transgene and endogenous Tnt1 elements. ChIP analyses were performed with the LTR-GFP-LTR transgenic line 6–11 non treated (Control) or treated with R10 (R10) using antibodies recognizing H3 acetylation (Ac), H3K4me2 (2K4), and H3K9me2 (2K9), or no antibody (−). Different endogenous Tnt1 elements tested for, as well as the LTR-GFP-LTR transgene and an actin gene fragment used as control. A quantification of the band intensity relative to the second input dilution is shown below each panel.(PDF)Click here for additional data file.Figure S10The Hut-2 transgene is not silenced when stably introduced in tobacco. (A) Schema of the Hut-2 transgene. A schema of the Tnt1 element is given for comparison. B) Northen blot analysis of the expression of the Hut-2 transgene in leaves of two independent transgenic lines, non-treated (−) or treated (+) with R10. The name of the line is given on top. The hybridization with a Tnt1 probe and an image of the EtBr staining of the RNA gel are shown underneath as controls. C) Methylation analysis of the Hut-2 transgene. The 5′ region of the Hut-2 transgene, including the 5′ LTR, was amplified and sequenced from bisulfite converted DNA from R10-treated leaves. At least 10 clones were sequenced from each transgene . The methylation state of each cytosine is shown as in (PDF)Click here for additional data file.Figure S11Constructs containing most of the internal sequence of Tnt1 can be expressed in tobacco transgenic plants. (A) Schema of the – END transgene. A schema of the Tnt1 element is given for comparison. B) Northern analysis of GFP expression in wild type plants agroinfiltrated with the –END construct and expressing it transiently (trans.), as well as in plants stably transformed with the same construct (the number of the transgenic line is indicated on the top). RNA was extracted from leaves treated (+) and non-treated (−) with R10, and probed with a GFP probe. An image of the EtBr staining of the RNA gel is shown underneath as loading control. The figure presents the analysis of 7 lines representative of the 36 obtained.(PDF)Click here for additional data file.Table S1Primer pairs.(PDF)Click here for additional data file.