Escherichia coli (ExPEC) to colonize niches outside the intestinal tract and to establish infection is the coordinated action of numerous virulence and fitness factors. The so-called high-pathogenicity island (HPI), responsible for synthesis, secretion and uptake of the siderophore yersiniabactin, proved to be an important virulence determinant. In this study we investigated the interaction of the flagellum-mediated motility and the HPI. The impairment of yersiniabactin production by deletion of irp2 or ybtA affected significantly motility. The gain of yersiniabactin production improved motility in both pathogenic and non-pathogenic E. coli strains. The loss of flagella expression had no adverse effect on the HPI. Strikingly, external iron abundance was not able to suppress activation of the HPI during motility. The HPI activity of swarming bacteria was comparable to iron deplete conditions, and could even be maximized by supplementing excessive iron. This fact is the first description of a regulatory mechanism, which does not follow the known hierarchical regulation of siderophore systems. Transcriptional reporter fusions of the ybtA promoter demonstrated that the entire promoter region with all YbtA binding sites is necessary for complete induction in both HPI-positive and HPI-negative strains. Altogether, these results suggest that the HPI is part of a complex regulatory network, which orchestrates various virulence mechanisms to optimize the overall fitness of ExPEC.The key of success of extraintestinal pathogenic  E. coli (ExPEC) [Yersinia spp. and spread in a big bang-like moment over a variety of Enterobacteriaceae, where it plays a major role in virulence [ybtA, (ii) fyuA, (iii) irp2- irp5, (iv) irp6-irp9 [irp2, fyuA, and the divergent overlapping promoter region between irp6 and ybtA contain specific binding sites termed repeated sequences (RSs). YbtA is proposed to bind to these sites as a homodimeric complex with yersiniabactin. Full expression of Irp2, FyuA and Irp6 depend on the action of YbtA. Here the transcriptional regulator works as an activator. Regarding its own transcription, YbtA shows auto-repression.Over the last decades the growing body of evidence was helpful to elucidate the pathogenic potential of extraintestinal pathogenic  (ExPEC) –3. The o (ExPEC) –6. It is (ExPEC) . The so-irulence –10. As airulence –14: (i) irulence . The mixirulence . YbtA, arp6-irp9 –19. The fyuA additionally led to morphological changes of bacteria during biofilm maturation [in vivo expressed it fulfils all essential criteria of a potential vaccine candidate [E. coli (UPEC) strain UTI89 during growth in minimal medium revealed metabolic changes when genes of the HPI were mutated [Interestingly, the supply of ferric iron to the microbial cell is considered to be the main function, but recent studies indicate the implication of the HPI in various processes apart from just iron acquisition. So Paauw et al. reported that the high binding affinity of yersiniabactin for ferric iron not only promotes bacterial growth by supplying additional iron but also reduces the production of reactive oxygen species by activated immune cells . Furtherturation . With Fyandidate . A multiandidate ; 24. Ano mutated . An extr mutated . This is mutated ; 28. ThePseudomonas putida abolished completely bacterial surface movement [Salmonella typhimurium revealed a strong induction of iron uptake systems during motility [Vibrio parahaemolyticus, it is known that iron depletion is an essential signal for swarmer cell differentiation [flhD. Both deletion of fur and iron scarcity had a strong impact on motility. Altogether, these examples point towards an important contribution of iron acquisition systems to flagellum-mediated motility. In this work, the main objective is to examine the interaction of the HPI and motility in ExPEC. We present data showing the strong induction of the HPI in motile bacteria and the impact of iron on this interaction.The versatility of the HPI clearly shows how this acquired iron uptake system became part of a complex network that coordinates various virulence and fitness properties. This multi-functional aspect prompted us to investigate whether additional virulence mechanisms may utilize the HPI. The hostile environment of the urinary tract forces ExPEC to get access to more favourable sites to scavenge for nutrients like iron, as well as to escape the host immune response. In this regard, flagellum-mediated motility has been demonstrated to be of great importance –31. Onlymovement . A functmotility . In the ntiation . A relevntiation . The iroBacterial strains and plasmids used in the present work are listed in 600 = 1.0) was stabbed into the middle of a soft agar plate and incubated at 37°C. Four hours after inoculation motility was quantified every hour by measuring the diameter of swimming bacteria. In order to address the influence of iron excess on motility in a rich medium like LB, soft agar plates were supplemented with different concentrations of additional Fe(III)Cl3 (Fluka AG) as indicated. Samples for β-galactosidase assays, western blotting and quantitative real-time PCR were prepared by scraping bacteria off the edge of a motility ring of strains swarming on a 0.5% LB swarm plate. All experiments were performed in duplicates and repeated at least three times. For statistical analysis a paired t-test or the Mann-Whitney U test were performed and results were considered statistically significant if the p-value was lower than 0.05.Swimming motility was assessed by using 0.3% LB soft agar plates. A late logarithmic phase culture  and subsequently transferred to a Protran® nitrocellulose transfer membrane (Whatman). Rabbit polyclonal antiserum to H7 flagellin (kind gift of B. Westerlund-Wikström) was used as primary antibody. For the detection of high pathogenicity island proteins bacterial cultures grown in LB medium or NBD medium were collected, adjusted to an OD600 = 1.0 and finally analysed using rabbit polyclonal antiserum to YbtA (kind gift of A. Rakin) and FyuA [For the detection of flagellar expression bacteria swarming on 0.5% LB swarm plates were scraped off carefully, resuspended in PBS and finally adjusted to an ODand FyuA . The res-∆∆ method as described by Livak and Schmittgen [Different RNA samples were prepared as described above. RNA extraction was performed using the Trizol (Invitrogen) method . Total Rhmittgen .Irp2, coding for HMWP2 (high molecular weight protein 2) [ybtA, encoding a transcriptional regulator of the AraC-like family [luc reporter assay (data not shown). Motility was first assessed performing swimming assays on LB soft agar plates . Although LB is considered to be a rich medium with excess of iron available, measuring of the diameter of swimming bacteria revealed a reduction of motility compared to the wild type strain. After 7 hours of incubation the average motility diameters of the irp2 mutant  and the ybtA mutant  were both significantly decreased compared to the wild type strain (77.50 mm ± 3.55 mm), with the deletion of irp2 resulting in a stronger impairment than the ybtA mutant (p<0.01). The complementation of both irp2 and ybtA in trans restored the motility to wild type levels, thus confirming that the phenotype was due to the deletion of irp2 and ybtA. In order to investigate this decrease in motility, we performed qPCR to determine whether the phenotype is regulated at the transcriptional level. FliC, fliD and flgK genes coding for flagellin, a filament-capping protein and a hook-filament junction protein, respectively, are class III flagellar genes, which were selected to study gene expression [Fig 1B). FliC was the most upregulated gene with a 1700-fold increase in gene expression. The amounts of fliD and flgK transcripts were increased 245-fold and 235-fold, respectively. This result confirmed the strong induction of flagellar genes at the transcriptional level. Next, we tested different HPI mutants and compared their gene expression with the wild type strain . We observed a downregulation of the studied flagellar genes in all HPI mutants. NU14 ∆irp2 demonstrated the strongest decrease in transcription of fliC, fliD and flgK, down 8.7-fold, 6.8-fold and 7.5-fold, respectively, relative to the wild type strain. Transcriptional analysis indicated that the abolishment of yersiniabactin production, as represented by the irp2 mutant, resulted in decreased flagellin expression. Therefore, we investigated the production of flagella in different HPI mutants using H7 flagellin antiserum . The reduced gene expression of fliC in the irp2 mutant correlated with a decrease in flagellin expression. These data clearly demonstrated that the ability to produce yersiniabactin promotes flagella expression and motility in ExPEC. This finding prompted us to investigate whether the gain of yersiniabactin biosynthesis in a yersiniabactin deficient strain would also affect motility. UPEC strain CFT073 was isolated from a patient with acute pyelonephritis [Y. enterocolitica [. After 9 hours of incubation on a LB soft agar plate the diameter of CFT073 Ybt+ was 42.5 mm (± 3.5 mm) compared to 16 mm (± 2 mm) (p<0.01). Even the equipment of K12 E. coli strain DH5α, which is an HPI-negative strain, with a functional HPI promotes motility . After 12 hours of incubation the motility diameter of DH5α increased from 26 mm (± 1 mm) to 43 mm (± 0.87 mm) due to additional production of yersiniabactin (p<0.001). These results support the contribution of yersiniabactin production to motility.In a first attempt to determine the role of the HPI in flagellum-mediated motility, defined mutations affecting genes involved in the biosynthesis of yersiniabactin were generated in the prototypic UPEC strain NU14. otein 2) , and ybte family , were dee family . The funpression . Performephritis  and reprcolitica  creatingfliI, a class II gene, which codes for an ATPase involved in type III flagellar export [ nor detectable expression of flagellin , and consequently, the mutant was non-motile on LB soft agar plates . The cultivation of the flagellar mutant under iron deplete conditions . Also the detection of FyuA expression by western blot analysis of the fliI mutant under iron limited cultivation in NBD medium showed no difference compared to the wild type strain , indicating the strong activation of the HPI during motility. This strong induction of transcription prompted us to study whether additional iron may suppress this activation during motility or stimulate even stronger. For that purpose, we supplemented LB soft agar plates with different concentrations of Fe(III)Cl3. We applied 10 μM, 100 μM and 1 mM of ferric iron. Motility assays indicated a significant increase only for 10 μM Fe(III)Cl3 compared to standard LB soft agar plates without any additional iron (p<0.05) . Higher concentrations appeared to mediate no relevant benefit for motility. Real-time PCR was further carried out to analyse the gene expression of both the HPI and the flagellar system . In the case of supplementation with 10 μM of ferric iron we observed a stronger induction of genes of the HPI. YbtA increased from 25-fold to 84-fold induction (p<0.05), irp2 showed a rise from 1800-fold to 2800-fold (p<0.05), and fyuA was upregulated from 28-fold to 70-fold (p<0.001). Using higher concentrations of iron we observed a suppression of transcript levels, which finally resulted in the inactivation of the HPI at 1 mM of FeCl3. The transcription of fliC could be stimulated with additional iron, but displayed no difference for the various concentrations of iron used. Corresponding results could be obtained by western blotting . The expression of both YbtA and FyuA increased when 10 μM of ferric iron were added, and diminished at higher concentrations until no expression was detectable. The investigation of flagellin production revealed a rise in expression with additional iron, but according to the qPCR data we could not observe significant changes for different concentrations of FeCl3. At this point, our data clearly prove that the HPI is active during motility under iron rich conditions, and the HPI is even stronger activated when 10 μM of ferric iron is added, instead of being repressed by this excess of iron.Iron uptake systems are essential to the overall fitness of pathogens and they are supposed to be activated only by scarcity of iron . Strikinirp2, irp6 and fyuA has been elucidated in previous studies [ybtA transcription, representing the starting point for activation of the HPI, our first step was to construct several transcriptional lacZ reporter fusions with different fragments of the ybtA promoter region . We fused the entire promoter including all three YbtA binding sites, termed repeated sequences (RS), and the Fur box to the lacZ gene to confirm the published data concerning ybtA regulation. YbtA is supposed to activate the transcription of irp2, irp6 and fyuA and represses its own transcription [ybtA we introduced both plasmid p3RS and plasmid pWKS30-ybtA into UPEC strain NU14. PWKS30-ybtA contains ybtA under the control of a lac promoter, so iron independent expression of YbtA is achieved. As depicted in  NU14 with additional YbtA expression showed a reduced reporter activity of 330 miller units compared to 540 miller units (p<0.01) in the strain carrying the empty vector control. We wanted to know whether this expected phenotype could be reproduced in an HPI-negative background, like in K12 E. coli DH5α. This setup actually allows the exclusive analysis of the interaction of YbtA with its own promoter, because the influence of all known and still unknown cis regulatory elements on the HPI can be excluded. Interestingly, in DH5α the additional expression of YbtA led to an increase in reporter activity of 41% relative to the strain without YbtA expression (p<0.001). This surprising observation prompted us to study this effect in the UPEC strain NU14 ∆hpi, where the entire HPI has been deleted. Measuring of miller units in the mutant NU14 ∆hpi revealed that the extra expression of YbtA indeed induced the reporter activity. We observed an almost 40% increase of miller units when additional YbtA was expressed (p<0.001). After all, these promoter studies suggest that YbtA itself activates its own transcription, at least in an HPI-negative genomic background.The YbtA-mediated transcriptional regulation of genes like  studies ; 18, whicription –19. In oybtA promoter region in flagellum-mediated motility. The activation of the HPI starts always with the induction of ybtA transcription. So we fused different parts of the ybtA promoter region to the lacZ gene and analyzed the reporter activities of motile bacteria . The constructs comprised a selection of reporter fusions including the whole promoter region (p3RS), two YbtA binding sites plus the Fur box (p2RS+), two YbtA binding sites without the Fur box (p2RS), only one YbtA binding site (p1RS) and finally a short fragment of the promoter region without any known binding site. Initially, we compared promoter activities in UPEC strain NU14 using reporter plasmid p3RS under different conditions, i.e. cultivation in NBD medium, cultivation in LB medium and swarming NU14 . As expected, iron depletion activated the ybtA promoter compared to the cultivation under iron excess. Consistent with the data presented above, the reporter activity was highly elevated during swarming. Approximately 420 miller units (± 20.1) for bacteria in NBD medium and 530 miller units (± 70.2) for swarming bacteria confirmed, that the HPI is almost equally activated. To determine the role of single promoter fragments containing different regulatory elements, we introduced various reporter constructs into the NU14 wild type strain and compared the reporter signals of bacteria grown under iron starvation with motile bacteria . The resulting data demonstrated that the entire promoter region with all three YbtA binding sites is necessary to fully activate the ybtA promoter. Only the reporter plasmid p3RS displayed a strong signal of 417 miller units (± 20.9). When the swarming UPEC NU14 was examined, we were able to observe the same pattern. Only the wild type strain carrying the reporter construct p3RS including the entire promoter region showed an elevated reporter activity of 528 miller units (± 73.5). To investigate if the observed pattern in UPEC strain NU14 also accounted for a HPI negative strain, reporter activities were detected in K12 E. coli strain DH5α carrying also the same reporter constructs  for reporter plasmid p3RS. Interestingly, we observed a slight reporter activity for constructs p2RS+ and p2RS, with both containing two YbtA binding sites. Once again, the pattern of reporter activity was identical to the results acquired under iron depletion. Plasmid p3RS displayed the highest rate with 918 miller units (± 100.6), whereas p2RS+ and p2RS produced weak signals. Overall, the promoter studies clearly show that the entire promoter region including all YbtA binding sites is mandatory for complete promoter activity. Furthermore, the lacZ reporter activity patterns during motility were similar in the HPI-positive strain NU14 and the HPI-negative strain DH5α suggesting a common mechanism of induction for the HPI, which is independent of YbtA and yersiniabactin production.We kept on investigating the role of the ybtA and irp2 resulting in the impairment of yersiniabactin production affected flagellar motility. The most interesting aspect of this observation is that motility assays were performed on LB soft agar plates, with LB representing an iron rich condition. External iron excess is supposed to repress transcription of genes involved in iron acquisition through the action of the ferric uptake regulator Fur [E. coli strains. These results prove that a functional HPI promotes flagellum-mediated motility. We investigated whether this interaction between HPI and flagellar system is mutual. The disruption of flagella expression had no effect on the activity of the HPI under iron deplete cultivation. It appears that induction of flagellum-mediated motility constitutes a stimulus that activates the HPI regardless of the external content of iron. This unexpected phenomenon prompted us to investigate how different concentrations of iron might affect this interaction. Most strikingly, during motility additional iron induced even more both motility and the HPI. Beside the activation of the HPI under iron excess, the further stimulation of a siderophore system with higher amounts of iron under iron excess is quite astonishing. To our knowledge, this is the first report of a higher regulatory mechanism of iron homeostasis that extends beyond the regulation by Fur and external iron content. The cultivation in an iron rich medium like LB shows no induction of iron uptake systems due to the binding of Fur to Fur boxes upstream of iron regulated genes [lacZ transcriptional reporter fusions we analysed the ybtA promoter activity under different conditions to identify elements of the promoter region necessary for targeting the HPI during motility. We confirm previously published data [ybtA regulation was the unexpected stimulation of the ybtA promoter by YbtA itself. Published reports provided evidence for auto-repression [cis-regulatory elements located on the HPI can be excluded. In an HPI-free background we were able to detect activation of the ybtA promoter lacZ fusion with the additional expression of YbtA for both pathogenic and non-pathogenic strains. This is not the only interesting observation. It is important to stress, that the binding of YbtA to its promoter can occur in the absence of yersiniabactin. The results for K12 E. coli DH5α carrying the YbtA expressing plasmid demonstrated the yersiniabactin-independent interaction of YbtA with its promoter. These new insights complement the current understanding of YbtA-mediated regulation of the HPI. The initial induction of the HPI starts with transcription and translation of YbtA. Small amounts of this regulator will stimulate stronger the expression of YbtA, so that all the other YbtA-dependent operons can be transcribed. This will start the production of yersiniabactin until the HPI is fully activated. At this point, further expression of YbtA may be damped. The molecular mechanism of this inhibition cannot be explained with our experimental set up. But DNA bending mechanisms stabilized through YbtA with or without yersiniabactin, as it is known for the AraC-mediated regulation, may be possible [ybtA promoter fragments fused to the lacZ reporter gene allowed to examine whether the promoter activity of ybtA under iron deplete conditions was comparable to the process of motility. Strikingly, maximum promoter activity was only detected for the entire promoter region of the reporter construct p3RS. This was the case for both UPEC strain NU14 and laboratory strain DH5α. The full activity under iron restriction might be explained by the inactivation of Fur, but the results of motility assays under iron rich conditions point towards a different mechanism. Fur is supposed to inhibit transcription because of iron abundance. However, a strong signal was measured in both strains. Reporter constructs p2RS+ and p2RS, with a Fur box only included in p2RS+, indicated that Fur might not be the missing link of the flagella-HPI interaction since there is no difference in promoter activity between both reporter plasmids. It is remarkable for the laboratory strain DH5α that neither YbtA nor yersiniabactin can be involved in ybtA promoter activity, but there is still a strong signal detectable. Due to the presence of a Fur box in p3RS and p2RS+, the role of Fur for this phenomenon seems to be negligible, otherwise plasmid p2RS+ is supposed to display the same activity. The possibility of an unknown factor, maybe another regulator of the AraC-like family, binding to all YbtA binding sites in YbtA-like fashion may be a theory to explain this observation. This hypothesis might also account for the induction of YbtA expression during motility. In both strains NU14 and DH5α, an unknown factor acting alone or in complex with additional elements seems to occupy the entire promoter region in order to activate YbtA expression. It is unclear, whether this missing link is encoded by the E. coli core genome, so the same factor is responsible, or whether the mediating factors differ in the strains NU14 and DH5α.Numerous studies have been published over the last decades highlighting the fundamental role of iron uptake systems for the overall fitness of pathogenic bacteria ; 50–53. ator Fur . Howevered genes . But as hed data  that allpression ; 19, butpossible . FurtherybtA promoter region. The evasion of important regulatory mechanisms of iron homeostasis seems to be counterbalanced to optimize motility. This work highlights the multi-functional role of the HPI and promotes the concept of a complex but orchestrated network of virulence and fitness factors supporting ExPEC to develop into this successful pathogen.Overall, the data presented in this study clearly showed how the flagellar system activates the HPI by addressing the


We identified sequence motifs, which enhance or reduce the ability of the Arabidopsis phage-type RNA polymerases RPOTm , RPOTp , and RPOTmp (active in both organelles) to recognize their promoters in vitro with help of a ‘specificity loop’. The importance of this data for the evolution and function of the organellar RNA polymerases is discussed.Arabidopsis RPOTm and RPOTp enzymes. By comparing different truncated and mutagenized promoter constructs, we observed the same minimal promoter sequence supposed to be needed in vivo for transcription initiation. Moreover, we identified elements of core and flanking sequences, which are of critical importance for promoter recognition and activity in vitro. We further intended to reveal why RPOTmp does not efficiently recognize promoters in vitro and if promoter recognition is based on a structurally defined specificity loop of the plant enzymes as described for the yeast and T7 RNAPs. Interestingly, the exchange of only three amino acids within the putative specificity loop of RPOTmp enabled the enzyme for specific promoter transcription in vitro. Thus, also in plant phage-type RNAPs the specificity loop is engaged in promoter recognition. The results are discussed with respect to their relevance for transcription in organello and to the evolution of RPOT enzymes including the divergence of their functions.The single-subunit RNA polymerase (RNAP) of bacteriophage T7 is able to perform all steps of transcription without additional transcription factors. Dicotyledonous plants possess three phage-type RNAPs, RPOTm—the mitochondrial RNAP, RPOTp—the plastidial RNAP, and RPOTmp—an RNAP active in both organelles. RPOTm and RPOTp, like the T7 polymerase, are able to recognize promoters, while RPOTmp displays no significant promoter specificity in vitro. To find out which promoter motifs are crucial for recognition by the polymerases we performed in vitro transcription assays with recombinant The online version of this article (doi:10.1007/s11103-016-0518-z) contains supplementary material, which is available to authorized users. Escherichia coli phage, N4, needs for efficient transcription the E. coli single-stranded DNA binding protein (SSB). SSB presents the hairpin-form promoter to the N4 RNAP for binding and participates in the elongation of transcription by separating the DNA/RNA hybrid from the RNAP elongation complex to enable multiple rounds of transcription  is imported into plastids. The third RPOT gene (RPOTmp) encodes a protein that is dually targeted into both mitochondria and plastids. Consequently, dicotyledonous angiosperms like Arabidopsis possess two mitochondrial  and two plastidial  phage-type RNAPs. The dual-targeted enzyme is not found in monocotyledonous plants and the basic angiosperm, Nuphar advena , plastids use a plastid-encoded plastid RNA polymerase (PEP). PEP is not a single-subunit enzyme but related to the bacterial RNAPs. It is composed of four core subunits  and one of several nuclear-encoded sigma factors. In photosynthetically active plastids, the chloroplasts, several additional protein factors are associated with the holoenzyme and essential for transcription  to result in construct pCold-His-RPOTmp. For mutagenesis, RPOTmp point mutations were introduced by site-directed mutagenesis using the PhusionTM Site-Directed Mutagenesis Kit  according to the manufacturer’s instructions. Used primers are listed in Supplementary Table S3. Recombinant hexahistidine-tagged enzymes were overexpressed in E. coli BL21 Codon Plus RIL  from resulting construct pCold-His-RPOTmp (RHR) in parallel with the wild-type enzymes. Protein expression was induced with a final concentration of 1 mM IPTG for 20–24 h at 15 °C. The purification of histidine-tagged proteins over Ni2+-NTA agarose was performed as described previously  into pKL23 upstream of the two bacterial terminator sequences hisa and thra, which efficiently stop transcription by both RNAPs  or −15 to +4 (pKL23-Patp8-C), respectively, and were tested for in vitro transcription by recombinant RPOTm and RPOTp , are transcribed by RPOTm and RPOTp  involves the formation of hydrogen bonds between amino acids of a C-terminal specificity loop with certain nucleotides of the promoter sequence  for specific transcription from PtrnM-98 as well as from the mutagenized PtrnM-98-tata promoter, which enhanced transcription by RPOTm and RPOTp as described above  and has been described to be recognized by RPOTm in vitro  in many mitochondrial promoters and some NEP promoters of Arabidopsis


C. elegans, we show that replication origins are marked with specific histone modifications that define gene enhancers. We demonstrate that the level of enhancer associated modifications scale with the efficiency at which the origin is utilized. By mapping replication origins at different developmental stages, we show that the positions and activity of origins is largely invariant through embryogenesis. Contrary to expectation, we find that replication origins are specified prior to the broad onset of zygotic transcription, yet when transcription initiates it does so in close proximity to the pre-defined replication origins. Transcription and DNA replication origins are correlated, but the association breaks down when embryonic cell division ceases. Collectively, our data indicate that replication origins are fundamental organizers and regulators of gene activity through embryonic development.The primary task of developing embryos is genome replication, yet how DNA replication is integrated with the profound cellular changes that occur through development is largely unknown. Using an approach to map DNA replication at high resolution in DOI:http://dx.doi.org/10.7554/eLife.21728.001 Drosophila and Xenopus embryos, the rapid genome duplication in early cycles is facilitated by the use of many, closely spaced, replication initiation sites  following RNAi depletion of bacteria . Resolutigestion . While tonserved .10.7554/C. elegans apparently initiates within broad zones at most origins, similar to human cells . To investigate this, we harvested pre-gastrula embryos from a hyperactive egg laying strain , where transcription is limited and non essential , would p00 cells . Indeed,00 cells .10.7554/Coupling of DNA replication with gene enhancers within rapidly dividing embryonic cells likely imposes particular constraints upon genome duplication. S phase in pre-gastrula embryonic cells ranges from ~10 to 25 min, depending on the lineage . In an iDrosophila and Xenopus embryos , CG21 egl-30(tg26) I; him-5(e1490) V (RRID:CGC_CG21).The wild-type strain corresponds to Bristol N2 (RRID:lig-1 and control (empty vector) RNAi-expressing bacteria were grown overnight at 37°C in LB medium supplemented with 50 µg/ml Ampicillin and 10 µg/ml tetracycline. Bacterial cultures were transferred to fresh LB medium supplemented with 50 µg/ml Ampicillin and were grown at 37°C until reaching OD 1.0. RNAi expression was induced with 1 mM IPTG for 1 hr at room temperature.We used lig-1 RNAi generated by the Ahringer laboratory and used a modified version of RNAi feeding protocol . lig-1 alig-1 associated genome instability and prevent sterility associated with complete lig-1 depletion, lig-1 RNAi expressing bacteria was diluted 1:10 with control (empty vector) bacteria to a final volume of 500 ml. Diluted bacteria were harvested by centrifugation and added to C. elegans S-Basal liquid media containing 150,000–200,000 well synchronized early L3 staged N2 worms (see below). N2 worms were grown in S-Basal liquid culture containing diluted lig-1 RNAi at 20°C until adulthood. Pre Gastrula embryos were harvested from early L3 staged egl-30 mutants that were grown on 1 mM IPTG plates as described previously . Approximately 150,000 synchronized L1 stage worms were grown in liquid media supplemented with OP50 bacteria until reaching L3 stage. Synchronized L3 staged worms were collected by centrifugation, followed with three times washing with M9 buffer containing 50 µg/ml Ampicillin. For progeny counting L3 staged worms where grown on 1 mM IPTG plates.g for 30 min at 4°C. Genomic DNA was precipitated by adding 500 µl isopropanol and spinning at 16,000 g at 4°C for 10 min. Genomic DNA pellets were washed with 70% ethanol and then resuspended in 300 µl STE . Residual RNA was digested by addition of 5 U RiboShredder RNase Blend (Epicentre) at 37°C overnight. Genomic DNA was precipitated with 30 µl 3 M NaOAc and 1.7 ml ethanol pelleted at 10,000 g for 10 min at 4°C. DNA pellets were washed with 70% ethanol and resuspended in 30 µl TE  and stored over night at 4°C to allow complete resuspension of genomic DNA. Samples were stored at −80°C. Radiolabeling of Okazaki fragments and denaturing gel electrophoresis were followed as previously described . Embryos were fixed with a 1% formaldehyde solution for 30 min and were quenched by adding glycine to a 125 mM final concentration and washed with NP-S buffer  for MNase digestion. Thirty-five thousand fixed embryos were digested with 100 U of MNase  for 3 min at 23°C. After the reaction was terminated, the samples were treated with RNase A and proteinase K before incubation at 65°C for 12 hr to reverse crosslinks. Samples were phenol extracted and the DNA subsequently precipitated  followed by pronase  each for 3 min at 37°C . Embryosipitated . DNA wasSCR_005476). Reads with q < 30 were removed using Samtools (RRID:SCR_002105). Remaining reads were binned in 100 bp intervals using Bedtools (RRID:SCR_006646), maintaining strand identity. Data were partially smoothed by calculating the median with a sliding window of 1.5 kb (http://www.modencode.org/). Regions of ChIP enrichment were calculated using Macs2 (Sequencing was performed using Ion Torrent (Proton) or Illumina (HiSeq) platforms. At least one biological replicate was performed for each DNA replication map; Ion Torrent (Proton) generally had a higher background and lower reproducibility than Illumina so direct comparisons between different embryonic stages were made using only the Illumina data . Sequencf 1.5 kb . Data wef 1.5 kb . Becauseng Macs2  with defng Macs2 ; replicang Macs2  were useAll sequencing data are availed at Gene Expression Omnibus with under accession number GSE90939. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.C. elegans" for consideration by eLife. Your article has been favorably evaluated by Jessica Tyler (Senior editor) and three reviewers, one of whom, Michael R Botchan (Reviewer 1) is a member of our Board of Reviewing Editors.Thank you for submitting your article "Spatiotemporal coupling and decoupling of transcription with DNA replication origins during embryogenesis in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Xenopus and Drosophila development where initiation sites before the mid-blastula transition are dense and poorly defined, perhaps truly random , and then becomes restricted to specific sites only thereafter. They show that origins co-localise with the potential enhancers of genes expressed through the proliferative stages of development. They show that the position of these replication origins correlates with specific histone marks, and that these origins are active before significant levels of transcription are seen. Finally, they show that the cessation of transcription potentially associated with these cis acting enhancers correlates with the cessation of proliferation. The reviewers all felt that certain key points needed to be addressed before publication and these are listed.The Whitehouse lab had previously developed a strategy for mapping eukaryotic DNA replication origins based on the position of Okazaki fragment strand switching, which they used to successfully map replication origins as well as replication termination sites in budding yeast. In this paper, they further develop this technique to map origins in C.elegans. This is an important step forward, and, together with a paper from the Hyrien lab earlier this year using a related approach, represents the first time this technique has been applied to a metazoan. This has great value because this technique has been shown to accurately and sensitively identify origins in budding yeast. For this reason alone the reviewers believe this a significant paper. The authors go on to show that most of the origins used before gastrula are also used later in development. This is in contrast to the well-studied situation in 1) The sites defined as "enhancers" seem to be assigned simply by the histone modification patterns. The reviewers are of course aware of the literature in higher eukaryotes that certain combinations of histone marks are predictive of enhancers but not for the worm. For example: it is not proven that sites of H3K27ac in worms are actually marking enhancers. The authors should either show that some of these sites are indeed transcription enhancers by mutation and effect transcription or nuance their discussion accordingly. If genome wide enhancers have been mapped perhaps they can be identified in a figure. In a related issue is #2.2) The separation between transcription and replication regulation is important but perhaps difficult to prove at this point. Proteins that are binding to the "enhancer sites" may be critical for both functions and mutation of these cis sites could have effects on both processes. If the authors want to establish this as a strong conclusion perhaps they should use inhibitors of transcription  and show that early in development replication patterns do not change. Otherwise the discussion should be modified.3) Have the authors investigated potential phenotypes for ligase depletion at various times in development- such as fecundity or fertility? […]1) The sites defined as "enhancers" seem to be assigned simply by the histone modification patterns. The reviewers are of course aware of the literature in higher eukaryotes that certain combinations of histone marks are predictive of enhancers but not for the worm. For example: it is not proven that sites of H3K27ac in worms are actually marking enhancers. The authors should either show that some of these sites are indeed transcription enhancers by mutation and effect transcription or nuance their discussion accordingly. If genome wide enhancers have been mapped perhaps they can be identified in a figure. In a related issue is #2.C. elegans. To our knowledge there is no published study to systematically and functionally define enhancers in C. elegans. Our definition is based on a large body of data that clearly links enhancers with select histone modifications in other eukaryotes, but we agree with the reviewers comment and have rephrased our discussion accordingly. More descriptive terms such as “enhancer associated chromatin modifications” are now used throughout the manuscript.The reviewers raise an important point and we agree that use of the term enhancers is not directly supported by empirical evidence from studies in 2) The separation between transcription and replication regulation is important but perhaps difficult to prove at this point. Proteins that are binding to the "enhancer sites" may be critical for both functions and mutation of these cis sites could have effects on both processes. If the authors want to establish this as a strong conclusion perhaps they should use inhibitors of transcription  and show that early in development replication patterns do not change. Otherwise the discussion should be modified.C. elegans has been studied intensively and experiments similar to those suggested by the reviewers have been done previously. Importantly, inhibiting transcription with α-amanitin in isolated early embryonic cells was found not to affect timing of cell division, the asymmetric pattern of early divisions, or the pattern of cleavage. Lois G Edgar, Nurit Wolf and William B. Wood, Development 120, 443-451 (1994). To our knowledge, all previous studies indicate that zygotic transcription is inessential in early rounds of division. Due to technical limitations we cannot inhibit transcription by RNAi against Pol II as it will lead to complete sterility. Upon the referee’s suggestion we have emphasized these points in the text.This is a very important point, we agree that it is necessary to functionally separate transcription and replication in early developing embryos. Zygotic activation in 3) Have the authors investigated potential phenotypes for ligase depletion at various times in development- such as fecundity or fertility?LIG-1 using 100% RNAi leads to rapid and complete sterility, therefore, we have optimized partial depletion of lig-1 to achieve maximal viability whilst also enriching for fragments. Knocking down lig-1 with 10% RNAi results in a loss of 30% of viable progeny (i.e. number of hatched embryos). As now shown as Knocking down


Python bivittatus. Our results provide insight into pigment phenotypes in pythons.Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors  that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarily conserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Color and pattern variation within, and across, species provide a unique opportunity to investigate the genetic basis of evolution and selection. Pigmentation variation in snakes and lizards serves a variety of ecological roles. Pigmentation patterns on dorsal surfaces can provide camouflage from visual predators, such as birds. Pigmentation can also provide warnings to potential predators regarding specific toxins or venoms. Additionally, sexually dimorphic pigmentation may facilitate communication among conspecifics relating to reproduction and territorial control .To date, the mouse has been the most studied organism in the dissection of genes modulating coat color and pattern. Although more than 100 genes have been identified which contribute to pigmentation and color phenotypes, the emerging view is that although some genes specifically control color while other genes affect patterns, the tremendous diversity in color and patterns arises through the epistatic interaction between color and pattern genes .Unlike many phenotypes, in which mammals exhibit greater complexity than amphibians, reptiles, and fishes, mammalian color determination is simpler because mammals exhibit much less color variation than other classes of vertebrates. In contrast to mammals, in which color is determined by a single cell type (melanocyte), color in reptiles, amphibians, and fishes arises from three cell layers: the melanophore, the xanthophore, and the iridophore .Python regius), have rods with peak absorption at 495 nm representing 90% of their photoreceptors while the remaining 10% of photoreceptors are comprised of two types of single cones absorbing at 360 nm and 550 nm [Color and pattern phenotypes are valuable provided they can be perceived. Eye development in squamates involves formation of the retinal pigmented epithelium (RPE), which is required for normal physiological function of the eye . In mammhttp://www.worldofballpythons.com/morphs/). Many of these color and pattern phenotypes exhibit Mendelian modes of inheritance such as simple dominant, incomplete dominant, codominant, and recessive modes. Moreover, the breeding data from some color and pattern morphs suggests sex linked modes of inheritance are also represented [As of 2016, ball python breeders have produced over 4600 distinct genetic strains of color and pattern morph variants (resented . AltogetMc1R) and Agouti, the receptor's antagonist. Mc1R normally produces dark brown to black pigment called eumelanin, while binding of Agouti results in production of the yellow-red pigment pheomelanin [Color phenotypes in vertebrates arise through specific type of pigments deposited within the ectodermal appendage  and the spatial distribution of those pigment types throughout the organism's body. Genetic factors influencing the types of pigments produced include two genes of main effect, the melanocortin receptor , as well as biopterin (found in both the yellow and red chromatophores), contribute to the red and green colors of the skin [A recent study investigating the mechanistic basis for the extensive color variation observed inthe skin . AdditioIndeed, the cellular, molecular, and biochemical mechanisms contributing to color and pattern variation in reptiles are complex and may utilize processes that are distinct from those found in mammals. Nevertheless, computational approaches aimed at analyzing the python genes known to modulate pigmentation in other species, such as horses, dogs, and mice, and people can provide additional insight into those mechanisms that might be present in mammals and conserved across vertebrates.Agouti, Tryp-1, Myo5a, Mc1R, Oca2, DCT, and Hps5), the keratinocyte derived factors endothelins 1, 2, and 3 , leukemia inhibitory factor (LIF), and kit ligand (kitlg), as well as ultraviolet light [ Mc1R, EDNRB, Rab27A, OA1, PMEL, MLANA, and AIM1 [In mammals, melanocytes synthesize the pigment melanin (eumelanin and pheomelanin) via the action of three main enzymes: tyrosinase (TYR), TYR-related protein (TRP-1), and dopachrome tautomerase (DCT). Once produced, melanin accumulates in melanosomes which ultimately are transferred to keratinocytes. Numerous factors control melanocyte differentiation and proliferation, including coat color genes  has genomic sequence publicly available along with 20,392 genes encoding 25,943 protein sequences [Although relatively few genomic resources are available for investigating genetics in the ball python, the Burmese python .A total of 24 genes were listed in the table. A set of 23 genes were identified for which python protein sequences were available in NCBI protein repository see . Each gehttp://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) [ Homo sapiens, Canis lupus familiaris, Mus musculus, Equus caballus, Bos taurus, Gallus gallus, Anolis carolinensis, and Thamnophis sirtalis). In some cases when the an ortholog was not identified for horse or cow, a subsequent search using a related species in the same genus occurred, for example, Equus przewalskii in place of Equus caballus.Protein orthologs for each python coat color gene were identified using the NCBI protein BLASTP server (roteins) . For eacThe BLAST results reported by NCBI were manually reviewed to identify the true ortholog and exclude any paralogs. When a specific species lacked the true ortholog, a false positive result corresponding to a paralog was typically provided by BLAST. Visual inspection of results helped exclude paralogs and other undesired proteins during the ortholog detection process. The percent identity for each species specific ortholog pair was obtained from the results. The reported percent identity for each query-subject species pair corresponds to the best blast hit produced during the BLAST search. In some cases, the percent identity was calculated for an alignment length less than the actual length of the protein sequence.http://www.broadinstitute.org/cancer/software/morpheus/) available from the Broad Institute. The file was formatted to conform with the expected column headings as described in the input file documentation. Custom colors were selected to represent high sequence identity (red), moderate sequence identity (magenta), and low sequence identity (blue). The heat map is displayed in The multispecies, pairwise, ortholog, percent identity data was formatted as displayed in The pigmentation gene expression network components were identified by reviewing the relevant literature covering transcriptional regulation of melanocyte differentiation and melanin production. In total, thirteen papers spanning 20 years (from 1994 to 2014) provided detailed information about the specific transcription factors, promoters, regulatory elements, transcription factor binding sites, and target genes .Transcription factors: CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1.Target gene promoters: AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR.Upstream regions: 1500 bases upstream of the transcription start site.After reviewing the relevant information contained in the references, the following pigmentation gene expression network components were selected for inclusion in subsequent analysis:http://www.clcbio.com/products/clc-sequence-viewer/). Phylogenetic trees were produced using the tree constriction algorithm in CLC Viewer 6. Alignments were colored to reflect regions of conservation (blue) and regions of divergence (red); some alignments depict moderate sequence similarity (magenta).Alignments for each python pigmentation associated transcription factor were generated in order to assess the extent of amino acid similarity across a variety of vertebrates. Multiple sequence alignments were generated using CLC Sequence Viewer 6 (http://www.ebi.ac.uk/interpro/) [The python transcription factor proteins were analyzed to identify the start and end positions of functional domains within each protein sequence. The online InterPro 54.0 server was used to identify protein domains. FASTA formatted protein sequences were submitted to the InterPro domain server (terpro/) .  Table http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) [http://www.biobase-international.com/product/transcription-factor-binding-sites) [Transcription factor binding sites (TFBS) were identified in the 1500 nucleotide upstream sequences from the target genes  using the PROMO virtual laboratory (=TF_8.3) , 23. Theg-sites) .Transcription factor binding sites were identified in each 1500-nucleotide upstream sequence separately and also within the group of all nine upstream regions. When the upstream regions were analyzed as a group, the following parameter settings were used: (1) sites found in one or more sequences and (2) maximum matrix dissimilarity rate = 15%. The decision to use 15% dissimilarity rate was based on the fact that the python regulatory regions and transcription factor DNA binding regions may diverge from the mammalian counterparts. Subsequently, stringent matrix (dissimilarity ≪ 15%) may result in false negatives. It is also true that the higher dissimilarity rate may result in false positives within the data set. Since these are computational predictions of protein-nucleic acid binding sites and potential macromolecular complexes, the predicted molecular binding results should be treated as candidate binding sites until being experimentally verified.Conserved clusters of transcription factor binding sites located within the pigmentation associated target genes were identified using sliding windows of variable size to identify intervals within the upstream regions that were enriched for two or more distinct transcription factor binding sites. Although TRANSFAC represents each instance of a particular transcription factor's binding site as a unique entry in the binding site database, we collapsed the numerous versions of each binding site into a single class of binding site that was associated with each unique transcription factor. The criteria requiring at least two distinct transcription factors provide a mechanism for identifying regulatory regions that potentially function combinatorically to modulate transcription synergistically. In order to maximize the significance of the binding site clusters, we chose to identify relatively small intervals, of approximately 200 nucleotides for which the greatest number of pigmentation transcription factor binding sites occurred. We employed an algorithm in which the interval containing the greatest number of binding sites was identified first, and then the process was repeated two more times, for each upstream region. Correspondingly, we identified three ~200-nucleotide intervals within each upstream region. CREB binding sites are green, FOXD3 binding sites are magenta, LEF-1 binding sites are orange, MITF binding sites are blue, POU3F2 binding sites are red, and USF-1 binding sites are purple.To better visualize the candidate pigmentation enhancer modules within each upstream region, we developed a simple schema for representing the evolutionarily conserved transcription factor binding sites within each identified promoter interval. First, each transcription factor binding site was color coded with a single color mapping to each distinct transcription factor such that total number of binding sites within the interval; (3) the linear arrangement of binding sites within the interval; (4) the specific transcription factor that binds to each site; (5) binding sites that overlap with binding sites for a different transcription factor; (6) the relative location of the candidate enhancer modules within each upstream region; (7) the relative density of binding sites within each candidate enhancer module; and (8) the relative nucleotide length for each candidate enhancer module.Next, we chose to visualize the candidate enhancer modules such that the following information would be quickly and easily extracted from each interval's visual representation: (1) the type and frequency of binding sites within the interval; (2) theIn order to develop visualization schema that accomplished those eight goals, we recognized that other types of information would not be easily extracted from the visualization. We willingly accepted these trade-offs in information content related to the visualization. Specifically, the following types of information are not easily inferred from our enhancer module visualization method: (a) the orientation of the transcription, (b) the strand that the transcription factor binds to, (c) the relative spacing between the binding sites within each interval, (d) the exact distribution of binding sites within the candidate enhancer module, (e) the length in nucleotides of each binding site, (f) the linear nucleotide distance between specific binding sites within the candidate module, and (g) the nucleotide composition of the binding sites and/or module.http://dx.doi.org/10.1155/2016/1286510). Additionally, all of the raw transcription factor binding site data is also freely available to the research community. The table describing the gene expression network components  is included in this paper in order to facilitate public access to resources needed to further investigate the role of these candidate regulatory enhancers and their associated transcription factor binding sites.The nucleotide and amino acid sequences described in this study are publicly available in the form of supplemental files associated with this paper  for the transcription factors described in this study.Ball pythons exhibit a large number of color and pattern phenotypes Figures  and 2. TThe piebald phenotype modifies both the pattern and the color Figures . Note thConsidering other morphs besides piebald Figures , one canThe spider morph  causes aThe animal in The phenotypic plasticity of color and pattern morphs has contributed substantially to the rapid popularity of the ball python as a pet. Interestingly, some morphs are associated with an increased risk for neurological signs, such as the spider morph. Other morphs have produced offspring with ocular abnormalities and craniofacial phenotypes. Additionally, reports of animals with abnormal spinal morphology underscore the importance in gaining a better understanding of the genetics underlying color and pattern variability in this species. Currently, it is not clear which specific undesirable traits are caused by the alleles responsible for the color and pattern variation and which undesirable traits are the result of linked genes,Another consideration in ball python breeding programs is the decreased time needed to produce double or triple homozygous recessive morphs when breeders choose to back cross offspring and breed siblings together. Such practices can simultaneously decrease the time to produce complex morphs and also increase the frequency of desired genotypes in the offspring. However, continued inbreeding results in a loss of heterozygosity and over time, without intending to, breeders may be faced with triple or quadruple recessive morphs which suffer from a number of undesirable traits resulting from autosomal recessive disease alleles acquired through multiple generations of inbreeding.Unfortunately, attempting to breed out such disease alleles in ball python lines bred specifically for homozygosity at three, four, or even five loci will prove quite challenging, unless the animals are outbred, and the resulting multilocus homozygous recessive phenotypes will be lost.Increasing knowledge of the specific genetic mechanisms underlying ball python development and coloration will inevitably lead to enhanced genetic tools and resources for improving breeding programs and actively reducing the frequency of undesirable phenotypes in subsequent generations. Although the identification of alleles responsible for encoding specific color and pattern phenotypes in ball pythons is of interest to breeders and the research community, the goal of this paper is to utilize a bioinformatics based comparative genomics approach to identify genomic sequences likely to underlie aspects of wild type python pigmentation phenotypes. As a first step in investigating the genetic regulation of pigmentation in pythons, it is worthwhile to try and determine whether some of the same genes and transcriptional regulatory elements are conserved between pythons and mammals.A set of mammalian coat color associated genes with known pleiotropic effects were identified from the literature  and usedEye-related phenotypes include dark eyes, ocular hypopigmentation, and iridis, as well as black, blue, hazel, red, and dilute eye colors. Numerous genes within this set shift the balance between light and dark pigmentation within the eye. It is not surprising that phenotypic variation in ocular pigmentation can give rise to the undesirable phenotypes of pigment disturbances, heterochromia, reduced pigmentation patches, and both transparent and dark iris phenotypes.Because we ultimately want to understand the relationship between color variation and health versus disease, we next explored the pleiotropic effects these genes exert on the rest of the body. Because these genes are widely expressed in multiple cell types, tissues, organs, and body systems, the phenotypic impact of mutations within this gene set is not limited to color variation. In fact, the phenotypes affect sensory organs and nerves, behavior, immune function, tumorigenesis, fitness, reproduction, and metabolism.Clinically relevant phenotypes have been described in mice exhibiting diluted pigmentation phenotypes. Ocular hypopigmentation is associated with altered ocular development and visual physiology. Diluted eye color contributes to altered iris patterns which correlate with eye diseases. Oculocutaneous albinism (OCA) occurs when mutations in the tyrosinase gene (TYR) lead to oculocutaneous albinism type 1 (OCA1) giving rise to nystagmus and hypoplasia as well as decreased visual acuity.Phenotypes caused by the genes endothelin 3 (END3), endothelin receptor B (EDNRB), microphthalmia-associated transcription factor (MITF), paired box 3(PAX3), SRY-box 10 (SOX10), and snail homolog 2 (SNAI2) can result in decreased melanocytes, white coat color, and ultimately deafness. Because these genes play important roles in cell differentiation and development, homozygous white animals are frequently nonviable. Horses that are homozygous for the overo mutation in EDNRB exhibit a white coat phenotype and an absence of ganglia of the myenteric plexus. Foals typically develop colic at birth and die within 48 hours.Reproductive phenotypes are associated with genetic variation in both the KIT receptor and the KIT ligand. Decreased function of these genes results in deficiencies of melanogenesis, hematopoiesis, and gametogenesis. The KIT genes are associated with the piebald phenotype in mammals. Male mice exhibiting a dominant white spotting coat color phenotype are sterile. Similarly, homozygous roan cattle have a white coat color and females lack Mullerian ducts.An autosomal recessive disease, Chediak-Higashi syndrome (CHS), occurs in humans, American mink, fox, mouse, rat, cattle, cats, orca, tiger, and American bison. The disease is associated with oculocutaneous hypopigmentation, photophobia, increased susceptibility to infections, and bleeding disorders. Immune related phenotypes can arise through mutations in lysosomal trafficking regulator (LYST) and RAS oncogene family member 27A (RAB27A).Metabolic disorders are also associated with pigmentation phenotypes. The dalmatian urinary metabolism phenotype may have been maintained in the breed through selection for the dalmatian's spots. Another example of metabolic disease arising from these genes was discovered in yellow mice produced from a dominant mutation in agouti signaling protein (ASIP) which is responsible for controlling the relative amounts of eumelanin (dark pigment) and pheomelanin (yellow pigment) produced via signaling through the melanocortin 1 receptor (MC1R). Mice with the ASIP mutation had a yellow coat color but also suffered from obesity, hyperglycemia, hyperinsulinemia, an increased susceptibility to hyperplasia and carcinogenesis, and ultimately lethality.It is important to note that this relatively small set of important genetic pigmentation mediators are also implicated in a large number of clinically important phenotypes ranging from sensory defects to immune dysfunction and encompassing reproduction, cancer susceptibility, and metabolism. Unlike mammals, which are relatively well studied and well cared for as pets, reptiles in general, and snakes in particular, are likely to be significantly underdiagnosed for the types of undesirable phenotypes associated with these genes. Very few snake owners routinely run clinical pathology tests to assess their snake's health during routinely scheduled wellness exams. Furthermore, ball pythons evolved to be solitary subterranean creatures that, accordingly, spend the majority of their lives in isolation from their owners/breeders. Subsequently, even if one were aware of the presenting signs associated with python metabolic, sensory, reproductive, or immunological disorders, it would be a truly rare occasion when an owner or breeder brought his snake to the veterinarian to address such a disorder.Since our goal in this project is to identify genomic signals associated with conserved transcriptional regulatory networks between mammals and reptiles, we next wanted to assess the extent of conservation between pythons and mammals within these pigmentation associated genes. We were curious to know if these represent highly conserved proteins or fairly divergent ones.On the one hand, it seems likely that pigmentation genes might represent a highly divergent set of genes. Many animals, including ball pythons, exhibit unique and diverse coloration patterns. However, with the association these genes have with key biological processes, such as nerve formation, reproductive viability, and sensory perception, it is also likely that these genes may be highly conserved across vertebrates.In order to assess the extent of conservation between mammals and pythons, we identified the orthologous proteins for the 23 python pigmentation genes for human, mouse, dog, horse, cow, chicken, anole lizard, and the garter snake. We were able to identify all 23 orthologs for each species except for the garter snake, for which the orthologs for RAB38 and SLC45A2 were present in the NCBI protein database. Python bivittatus and each of the other species. All percent identity values were recorded , RAB27A (90%–95%), MYO5A (89%–92%), and OCA2 (83%–93%). In contrast, the most divergent proteins were extracellular secreted ligands for receptors, including ASIP (46%–86%), KITLG (40%–85%), and EDN3 (42%–78%). The extracellular transmembrane glycoprotein, PMEL, exhibited sequence similarity comparable to the extracellular ligands (39% to 77%).Compared to the secreted ligands, the corresponding membrane bound receptors display considerable more identity. For example, KIT (64%–89%) and EDNRB (64%–79%) exhibit approximately 64% identity between python and mammals but 71% between python and chicken and 83% to 89% among reptiles. EDNRB exhibits comparable patterns of identity with approximately 61% identity between python and mammals and 80% between python and chicken.The tyrosinase enzyme exhibits slightly lower identity (71%–87%) than the most conserved proteins  and is more similar to the transporter SLC2A9 (71% to 87%) than to either ligands or receptors. Based on the pattern we observed across different classes of protein, such as ligands, versus receptors, we were curious to determine if a similar relationship was associated with the undesirable traits associated with these pleiotropic pigmentation genes.Unlike the striking pattern identified in the types of proteins, the relationship between protein identity and phenotype is not as tightly linked. For example, both KIT receptor and KIT ligand are associated with sterility and lethality; however, the receptor exhibits about 62%–64% identity among the mammals while KIT ligand exhibits approximately 45% identity between the python and mammals. Also, the very highly conserved sequence (MYO5) has an association with lethality, as SLC2A9, although SLC2A9 exhibits almost 10 percentage points less identity than MYO5A.Nevertheless, the identification of these genes in pythons provides a unique perspective to view undesirable abnormalities produced by breeders. Furthermore, this set of genes represents high confidence candidates to genomic locations of polymorphisms associated with phenotypic variation in coloration.Transcription factors: CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1.Target gene promoters: AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR.Upstream regions: 1500 bases upstream of the transcription start site.The following gene expression network components were identified:Because the foundation of our hypothesis is that shared pigmentation physiology and cell biology between pythons and mammals may be detectable as conserved transcriptional regulatory fingerprints in the respective genomes, it is worthwhile to assess the identity of these pigmentation transcription factors across species. Although the particular DNA sequence recognized by a sequence specific transcription factor may diverge in different evolutionary lineages, we reasoned that the extent of divergence between the transcriptional regulatory elements (between the species) may be inversely related to the conservation among the transcription factors (between the species) that recognize those regulatory binding sites. Therefore, we expanded the set of species considered for the alignments of orthologous pigmentation transcription factors to broaden the representation of reptile transcription factor sequences.In order to most effectively interpret the sequence alignment data, we first identified functionally important domains within each protein . Domain Analysis of CREB protein sequence revealed the presence of a coactivator domain from position 87 to position 146 and a basic-leucine zipper domain spanning amino acids between residue 284 and residue 342. Analysis of FOXD3 identified a winged helix-turn-helix DNA binding domain beginning at position 71 and ending at position 173. Within the LEF-1 sequence, three domains were identified; the first spans residues 1 through 65 and encodes a catenin binding domain. The second LEF-1 domain detected was CTNNB1 binding, N-terminal region beginning at the first residue and ending at position 209. The final functional region detected in LEF-1 was the high mobility group box domain spanning the region from 293 to 375.MITF contains an N-terminal MiT/TFE transcription factor region from position 4 to position 142 and a MiT/TFE transcription factor C-terminal motif beginning at position 393 and ending at position 519. Additionally, MITF encodes a Myc-type basic helix-loop-helix (bHLH) domain beginning at position 303 and ending at position 370. Domain detection with POU3F2 identified a POU-specific domain from residue 51 through residue 125 and a homeodomain-like region from position 125 to position 205. The USF-1 protein has an Myc-type helix-turn-helix-loop-helix (bHLH) domain beginning at amino acid 193 and continuing until amino acid 286.The MITF alignment  includesThe FOX3D alignment  includesWithin the LEF-1 alignment , there aCREB sequences exhibit considerable conservation throughout much of the alignment  with a fThe POU3F2 alignment  shows maTHE USF-1 alignment  shows paThe 1500-nucleotide upstream regions immediately adjacent to the transcription start site of python genes were analyzed to identify evolutionarily conserved transcription factor binding sites associated with gene expression regulation of pigment related transcripts.The upstream region from the python tyrosinase gene (TYR)  containsThe upstream region of python dopachrome tautomerase (DCT)  containsThe upstream region from Absent In Melanoma 1 (AIM1)  containsThe upstream region from melan-A (MLANA)  containsThe upstream region for RAS oncogene family member 27A (RAB27A)  has a clThe upstream region for melanocortin 1 receptor (MC1R)  containsThe upstream region for OA1-melanosome membrane protein (OA1)  containsThe upstream region for microphthalmia-associated transcription factor (MITF)  has a clThe upstream region for premelanosome protein (PMEL)  containsPigmentation phenotypes have been produced through both natural selection and artificial selection. Naturally occurring color and pattern variation provide advantages for organisms that include thermoregulation, camouflage, mimicry, sexual fitness, and warnings. Naturally selected pigmentation variation in domestic animals offers a breadth of morphological phenotypic variation that can be used to produce visually appealing lineages and breeds. Undesirable health traits associated with perturbations in some pigmentation genes have been observed in certain domesticated species bred for particular color and pattern phenotypes.Moreover, malignant melanoma arises from the neoplastic transformation of melanocytes and results in the most aggressive form of skin cancer. As of 2004, the incidence was 27.5/100,000 for Caucasian Americans and less than half that for African Americans. Although early surgical intervention cures 90% of patients, metastatic melanoma is resistant to treatment. The five-year survival rate for patients in stage III ranges from 78% to 48%, and for M1 patients, the prognosis is even worse, with a 1-year survival rate between 62% and 33% .As of 2013, the incidence of melanoma had grown to 5% of the population. A number of pharmacological treatments target inhibition of BRAF, MEK, c-KIT, and PI3K pathways. BRAF is a serine/threonine kinase involved in the RAS-RAF-MEK-ERK signaling cascade which promotes proliferation and antiapoptotic cellular programs. Mutations in BRAF and N-RAS account for roughly 50% and 20% of melanoma patients, respectively. MEK is a component of the MAPK signaling cascade which also promotes proliferation and migration. c-KIT mutations and amplifications are potent drivers of melanocyte tumorigenesis. Phosphatidylinositol-3-kinase (PIK3) is ectopically activated in most metastatic melanomas . AltogetChelonia mydas), are threatened by herpes-associated fibropapillomatosis, as the endangered leather back turtle (Dermochelys coriacea), Kemp's ridley turtle (Lepidochelys kempii), and the hawksbill turtle (Eretmochelys imbricata) [Cancer is responsible for approximately 15% of human deaths worldwide each year. In wildlife, trauma and starvation have been considered the leading causes of death. However, more recently, the impact of cancer on wildlife has become more recognized. For example, endangered reptiles, such as the green sea turtle (bricata) .Neoplasia in reptiles was once thought to be uncommon, but in the last decade this view has evolved. Prevalence is the highest in snakes (15%), followed closely by lizards (9%) and then chelonians (3%), and is the lowest in crocodiles (2%). Out of 43 different types, the top 5 are lymphoma (11%), soft tissue sarcoma (11%), adenocarcinoma (8%), fibrosarcoma (6%), and melanoma (6%). Among snakes, melanoma prevalence has been characterized across colubrids (2%), crotalids (1%), boids (0.5%), and vipers (0.5%) .Tumors associated with pigment cells of skin tend to be rarely reported in reptiles. One recent study from 2012 characterized the frequency of chromatophoromas in an archive of 179 reptilian tumors. The results identified a frequency of 15% across all reptiles, with melanophoromas representing 11% while iridophoromas had slightly over 3%. When compared across species, lizards exhibited the highest frequency with 20%, corresponding to 14% melanophoromas and 6% iridophoromas. Snakes had the second highest frequency (10%) with all being melanophoromas, followed by turtles/tortoises (8%), also having all melanophoromas . shared pigmentation biology between species can be identified through the detection of genomic fingerprints associated with conserved transcriptional regulatory networks underlying melanocyte biology and melanin formation. The idea that conserved physiology will be encoded in conserved genomic signals is the foundation of comparative genomics.Reptiles are less well studied than mammals. Increasing knowledge of the shared physiological and molecular mechanisms underlying important phenotypes is valuable. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. Our working hypothesis for this study is thatSince melanocytes are conserved cell types in both mammals and reptiles, we hypothesized that the transcriptional regulatory mechanisms would be conserved for numerous cellular processes common to melanocytes such as melanocyte differentiation, melanosome formation, regulation of enzymes involved in pigment biosynthesis, and melanocyte signaling pathways. Because many of the mammalian genes involved in these processes have corresponding orthologs in the Burmese python, it seemed plausible that some of the transcriptional regulation of these genes would also be shared between mammals and pythons.The underlying principle for eukaryotic gene activation is cooperativity whereby nucleosomes inactivate chromatin and transcriptional activators activate chromatin. This “chromatin switch” provides a molecular regulatory mechanism for gene level transcriptional control. The switch between active and inactive state is determined by the competition between the factors contributing to the active and inactive states. In this model, enhancers are contiguous DNA sequences containing binding sites for transcriptional activators and thereby enable specific combinations of transcription factors to flip the “chromatin switch” towards gene activation .Because transcription factor binding sites are much shorter than full length amino acid and nucleotide sequences, identifying evolutionarily conserved binding sites is not effectively accomplished using sequence alignment methods, as is typically done with proteins. Moreover, the fact that transcription factors can recognize a variety of consensus binding site perturbations contributes to the difficulty in successfully identifying true positive binding sites. Additionally, selective pressures acting on the two species since their divergence from a common ancestor may have resulted in corresponding changes in the transcription factor DNA binding domains resulting in correlated divergence in the transcription factor binding site sequences in the upstream regions of genes regulated by these transcription factors.Identifying enhancers in the python genome represents a cryptographic approach to decoding biological information. Specifically, the function of mammalian melanocyte associated transcription factors was employed to identify multiple binding sites clustered within a relatively small interval in the upstream region of a particular gene within the python genome. These mammalian transcription factors and associated target genes provide the “language of the cipher” needed to decode the evolutionarily conserved melanocyte biology encoded in the reptile genome .Our strategy for identifying genomic signals associated with python pigmentation biology parallels the method described by Bodnar and Bradley to decode the genetic program underlying developmental phenotypes during drosophila embryogenesis. Although our end product differs substantially from the integrated developmental maps characterizing gene regulation of development, our result integrates a variety of comparative genomic and published knowledge to produce an initial model of portions of the gene expression network underlying pigmentation phenotypes in the python .pigmentation OR melanocyte” as of 2016 is 54,319, with 9,254 publications specifically containing “gene OR genetic OR genome OR genomics” in the title or abstract. The information content of this dataset is overwhelming and so complex that it is not possible to use in its entirety. However, a workable approach was adopted as described by Bodnar which provides experimentalists with testable hypotheses about pigmentation and melanocyte genes, upstream regions, transcription factors, cis elements, and putative enhancers [The total number of indexed publications in PubMed associated with “nhancers .One method for increasing the signal to noise ratio in detection of regulatory binding sites within noncoding regions of the genome is to limit the search to those regions in close proximity to the transcription site of a gene. Although this can facilitate the identification of true positive transcription factor binding sites, it also causes an increase in false negatives due to the true binding sites that are not detected which lie outside of the search region. Another method for increasing true regulatory motifs is to look for combinations of transcription factor binding sites. Since transcription factor binding sites are relatively small, on the order of 4 to 20 nucleotides each, the search for combinations of binding sites adds the benefit of increased specificity of detection through the reduced frequency associated with the reduced joint probability of finding a combination of sites with close proximity of one another.Further success in identifying true regulatory motifs in genomic sequence can be accomplished by limiting the search to transcription factors that are known to regulate or contribute to the regulation of genes. This also suffers from the production of false negatives because transcription factors not considered in the search will not end up in the results.Ultimately, the specific methodological approach used to identify cis-regulatory elements in genomes will be informed by the specific overarching goal of the project and the subsequent anticipated downstream applications of the data. For example, a goal of identifying any possible transcription factor binding site within a genomic sequence is quite different from the goal of identifying at least one highly likely true transcription factor binding site in a genomic sequence. When considering downstream applications of the predicted data, it is important to consider the associated time and cost of validating predictions which is directly proportional to the number of prediction and affected by the prediction success rate.For the purpose of investigating the genetic basis of common pigmentation and morphological pattern phenotypes that may be shared between mammals and pythons, we were much more interested in identifying fewer candidates with very high true positive rates rather than massive numbers of predictions that are dominated by false positive rates. Subsequently, we used a variety of constraints that helped shift the outcome towards true positives with the trade-off of identifying fewer numbers of candidates and also having a relatively high rate of false negatives .We believe that the constraints we imposed shifted our predictions towards our desired goal of producing a higher true positive rate in a smaller set of total predictions. First, we selected as starting orthologous python genes, only genes for which known coat color and pattern phenotypes were known to occur in other species . AdditioTogether, these constraints have produced a set of 27 candidate enhancer modules likely to underlie pigmentation and pattern phenotypes in pythons. While bioinformatics approaches ultimately produce predictions, for which subsequent experimental validation must be performed, we noted a number of features in our predictions suggesting some of them are true positive regulatory control elements driving expression of python genes contributing to the types of color patterns observed in ball pythons. Specifically, many of our clustered transcription factor binding sites contain multiple instances of the same transcription factor in tandem , 35, whiThe results of our work provide a starting frame work for investigating the genetic regulation of python color and pattern variation. In combination with the hundreds of color/pattern variant lines of ball pythons, our set of color/pattern enhancer modules creates an ideal starting point to investigate the genetic factors contributing to observed color and pattern variation in pythons. Although genes and alleles contributing to the specific phenotypes observed in the ball python may lay outside the set we have described, our work implicates a number of genes as likely to be involved in python melanocyte biology.Another important application of our work is a better understanding of the anticipated undesirable phenotypes that may be associated with specific ball python color variants based upon the pleiotropic effect these genes play in biology .The results of this study should be interpreted cautiously based on the fact that there are hundreds of genes involved in pigmentation, for which subtle and complex regulatory mechanisms have yet to be elucidated. Moreover, the regulatory elements underlying eukaryotic gene expression are distributed across very large landscapes that may cover thousands and even millions of bases along a chromosome. Therefore, further experimental investigations into our predictions are necessary to not only validate each prediction but also relate each cluster of evolutionarily conserved transcription factor binding sites to its regulatory purpose and association (if any) with specific wild type python color/pattern phenotypes. Ultimately, through future studies, the genetic correlates of ball python color and pattern morphs may be elucidated.We describe a novel application of bioinformatics to infer conserved cellular genetic programs in melanocyte biology based on comparative genomics, supervised literature mining, and regulatory motif analysis resulting in the identification of 27 candidate enhancer modules, within 9 orthologs of mammalian pigmentation genes likely to contribute to pigmentation and pattern phenotypes in pythons. Our results focused on a set of 6 transcription factors that have been previously demonstrated to modulate melanocyte and pigmentation in mammals. The integration of supervised literature mining, phenotypic characterization of pigmentation in pythons, comparative genomics, orthologous protein sequence analysis, phylogenetic analysis, sequence similarity, detection of TF binding sites, clustering of TF binding sites, and regulatory module identification provides an example of an integrated bioinformatics framework for extending biological knowledge from one species to another. Specifically, we searched for CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1 binding sites within the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR. Our results provide initial evidence for aspects of conserved melanocyte and pigmentation biology between mammals and reptiles. Among our findings, we identified binding sites for the MITF transcription factor in upstream regions for DCT, TYR, AIM1, MC1R, MITF, PMEL, and OA1. Interestingly, we detect evidence of a MITF positive feedback circuit in reptiles, as has been characterized in mammals. We briefly review the prevalence of melanoma in both humans and reptiles, with a particular focus on snakes. Overall, our findings provide a set of computationally inferred transcriptional network models for melanocyte mediated pigmentation biology in pythons. These models are valuable for future experimental approaches aimed at dissecting the genetic regulation of this interesting and important biology in pythons and other reptiles. FASTA nucleotide and protein sequences described in this paper are provided in the supplemnetal file.