The Mobile Genetic Elements and Genome Evolution conference was hosted by Keystone Symposia in Santa Fe, NM USA, 9 March through 14 March 2014. The goal of this conference was to bring together scientists from around the world who study transposable elements in diverse organisms and researchers who study the impact these elements have on genome evolution. The meeting included over 200 scientists who participated through poster presentations, short talks selected from abstracts, and invited speakers. The talks were organized into eight sessions and two workshops. The topics varied from diverse mechanisms of mobilization to the evolution of genomes and their defense strategies against transposable elements. Transposable elements (TEs) are powerful drivers of evolution. TEs constitute the majority of genomic DNA in many eukaryotes, and they dramatically shape genetic content by causing mutations, rearrangements, and sequence duplications. Of increasing significance is the link between these transposon-mediated mutations and disease. Recent advances in the study of TEs motivated Keystone Symposia to host the conference on Mobile DNA and Genome Evolution in Santa Fe, NM, USA, 9 March through 14 March 2014. The topics discussed at the conference often relied on recent innovations in high throughput sequencing and genome analysis. Results presented include discoveries of cellular systems that inhibit transposon activity, mechanisms of TE activity, evolutionary impact of TEs, and connections between TE activity and disease. Also included were intriguing results that documented TE activity during neurogenesis and aging. The symposium fostered ties between scientists in the field of transposon biology with the experts of genome evolution and analysis. The TEs and hosts represented in this meeting included a variety of examples from eubacteria, archaea, protists, plants, fungi, and animals. The meeting featured two keynote addresses by scientists who have made particularly important discoveries in the field of transposable elements.Transposon Regulation and Genome Evolution. He spoke on the role of TEs in regulation and genome evolution. He emphasized that the different insertion patterns of TEs reflect different transposition mechanisms and evolutionary strategies. In his role as director of the Drosophila Gene Disruption Project, Dr. Spradling examined the integration patterns of the TEs used in the project: Minos, piggyBac, and P elements. By dividing the genome into large numbers of equally sized segments, the integration of Minos, a Tc1/mariner-related element from Drosophila hydei, is found to be largely random, while piggyBac, from the cabbage looper moth, has a broad integration profile in Drosophila melanogaster that features integration in the promoter regions of genes. The focus of the talk then shifted to the P element, which displays a highly biased integration pattern that favors promoter sequences. Inserts into just 100 genes account for 40% of all integration events. In searching for a mechanism responsible for P element insertion in specific genes, Dr. Spradling found that in cultured cells the promoters with the highest level of integration events contained binding sites for the origin recognition complex. These data argue that P elements integrate at origins of replication. Dr. Spradling proposed the appealing model that coordination of transposition with DNA replication could increase P element copy number. Excision from recently duplicated DNA coupled with integration into unreplicated DNA would cause an overall increase in copy number because the excised copy could be repaired by homologous recombination using the replicated sequence as template. An additional feature of the model is transposition across replication forks would result in local transposition, a common feature of many TEs that remains unexplained. Regardless of whether integration is local, transposition from replicated into unreplicated DNA would result over time in the accumulation of P elements in late replicating DNA, much of which is heterochromatinized. This trend has been observed for P elements.Allan Spradling  opened the meeting with a keynote address entitled Drosophila larval salivary glands, like many other types of polyploid cell DNA, is highly replicated through a process known as endoreduplication. However, a longstanding mystery is the nature of genome regions that are not fully replicated in polytene cells because they are lower in copy number than flanking sequences. Dr. Spradling examined the replication status of DNA isolated from salivary glands. By applying methods of high throughput sequencing he identified more than 100 regions of DNA from salivary glands that appeared to be under-replicated, compared to DNA from diploid cells. This was many more sites of under-replication than was previously known. Analysis of the sequence reads for deletions revealed a surprising correlation with the DNA found to have low copy number. Dr. Spradling made the provocative proposal that replication forks that stall or fail to complete replication, break and are repaired to generate deletions with diverse breakpoints that are responsible for the copy number changes. He suggested that this process generates genetic diversity in these regions in polytene cells that could be advantageous and that the generation of somatic alterations by incomplete replication might be widespread among polyploid cells in diverse organisms.The DNA of Biology of Human Retrotransposons: A Potential Role for Retrotransposition in Tumorigenesis? Dr. Kazazian highlighted the role of retrotransposition in human disease. He described how in 1985 his studies of hemophilia led to the discovery of two spontaneous insertions of a LINE-1 (L1) element in the Factor VIII gene that resulted in disease. A few years afterwards Dr. Kazazian identified another disease-causing insertion of L1 that in this case was in the dystrophin gene. This insertion was unusual in that it carried non-L1 sequence 3′ to the L1, a so-called 3′ transduction event. These findings represented the first cases of disease that could be directly associated with TE integration. Subsequent studies have identified 101 cases of disease resulting from de novo retrotransposition events: 25 caused by L1 insertion, 61 due to Alu, 10 resulted from SVAs, four due to L1 poly (A) sequence transduction, and 1 processed pseudogene.Haig Kazazian  followed with a keynote presentation entitled The sequences of the disease-causing L1s ultimately enabled Dr. Kazazian to isolate the source copies of L1. His development of genetic assays for transposition in cultured cells demonstrated that the source elements were highly mobile copies of L1. The transposition assay was used to test the activity levels of many L1 elements and these results demonstrated there is just one family of L1s that retains activity. Dr. Kazazian coupled this information with the reference genome sequence in estimating the average human genome has 80 to 100 active copies of L1. While this estimate was based on the reference genome Dr. Kazazian surmised that the sequence and position of active L1s could vary greatly between individuals. This assumption was borne out using PCR methods that specifically detect human families of L1. Dr. Kazazian assayed 16 individuals with this new PCR method and discovered 367 non-reference insertions of L1. In addition, his analysis of sequence from the 1,000 Genomes Project identified another 1,016 L1 insertions not found in the reference genome. These data together with results from other labs clearly indicate L1 elements are highly polymorphic in the human population.Dr. Kazazian has now turned his attention to L1 transposition in somatic cells. With L1-targeted resequencing of colorectal tumor DNAs and matched normal DNAs, he found that certain cancers had large numbers of L1 insertions. Numerous genes associated with tumorigenesis had insertions suggesting somatic retrotransposition may play an active role in colorectal cancer. To test this possibility, Dr. Kazazian is currently analyzing precancerous tissues, including colorectal polyps, and comparing their L1 content to tumors and normal tissue from the same individuals. Many somatic insertions of L1s in colorectal tumors can be identified in the precancerous polyps, indicating the insertions occurred early in the development of the tumors. Dr. Kazazian is also studying precancerous lesions in esophagus resulting from acid reflux. While this study is ongoing, Dr. Kazazian reported that L1 insertion numbers were higher in the precancerous tissue than in the normal samples. Importantly, the esophageal cancers had greater numbers of L1 insertions than the precancerous lesions. These data argue L1 is active in somatic tissue and that the numbers of L1 insertions parallel the conversion of normal tissues into precancerous cells and finally into cancerous tumors. Dr. Kazazian is currently examining the positions of these insertions to assess whether L1 may contribute to the progression of cancer. In other studies Dr. Kazazian is studying the L1 insertions in metastases. Here, he reports that specific insertions in source tumors can be identified in metastases. These data suggest that L1 insertion sites could be used as biomarkers that could allow clinicians to determine the progression of primary tumors and metastases.de novo retrotransposition events that were absent from the respective parental cells. Both up-regulation of TE activity and concomitant demethylation of endogenous L1 elements were observed upon induction. Retrotransposon capture-sequencing (RC-Seq) was used to discover insertion sites within the host genome. Their experiments showed that endogenous L1-mediated retrotransposition is highly dynamic during reprogramming and hiPSC cultivation. Future in-depth experiments are required to assess whether this phenomenon alters the phenotype of hiPSC derivatives sufficiently to impact their use in medical or research applications.Gerald Schumann , explored how reprogramming to create human induced pluripotent stem cells (hiPSCs) and their subsequent cell culture induced genomic changes mediated by the mobilization of endogenous TEs. In particular, his lab analyzed the genomes of eight different hiPSC lines generated by Sleeping Beauty or lentiviral-based introduction of reprogramming genes, for the presence of endogenous Frederic Bushman  explored the role of the human virome in human health. His lab explored the diversity of the virome within the gut across a dozen different individuals. They assessed longitudinal evolution of the gut virome by collecting a series of samples over 2.5 years from the same individuals. After sequencing and assembling more than 56 billion bases from the study, they identified 478 viral contigs of mostly bacteriophage origin. About 80% of the contigs were present throughout the longitudinal study, but a subset of contigs, as exemplified by elements of the circular Microviridae genome, showed rapid evolution with a near 4% speciation divergence over the 2.5-year study. This rapid divergence occurred through a number of different mechanisms including steady substitution, use of Diversity Generating Retroelements , and use of bacterial CRISPRs.Shawn Burgess  described the use of high throughput sequencing to identify the sites of murine leukemia virus integration in the human genome. His lab constructed libraries using restriction enzyme digestion of gDNA in combination with ligation-mediated PCR to enrich and identify viral insertion sites. In particular, they studied insertion sites in two ENCODE cell lines, K562 and HepG2. They found an insertion site preference in promoter and enhancer regions with a particular preference for strong promoters/enhancers characterized by profiles of active chromatin marks.Kevin Gunderson  described how transposase biochemistry is being used to streamline library preparation for high throughput sequencing and to create novel sequencing libraries supporting synthetic long read (SLR) technology. In particular, he presented recent results on an imputation-free SLR method that used Tn5 transposase to tagment (fragment and add adapters) the genome while maintaining contiguity information. Contiguity is preserved since transposases physically hold the fragments together until the protein is removed or denatured after compartmentalized dilution. These libraries were sequenced and haplotype phases reconstructed (imputation-free), with over 95% variants phased at a low switch error rate (1 in 10 Mb) and an average N50 contig length of over two Mb. This approach was also combined with 3 kb Mate Pair libraries to create assembly scaffolds of over 27 Mb. Such SLR technology approaches should greatly expand the utility of short-read sequencing by synthesis platforms for obtaining long-range information.Retrotransposon insertions generate both intra- and inter-individual genetic diversity in mammalian genomes. The talks in this session focused on exciting, unpublished discoveries in four major areas: 1) the identification of polymorphic retrotransposon insertions in primate genomes; 2) the discovery of somatic retrotransposon insertions in the human brain; 3) the identification of host mechanisms to restrict retrotransposition; and 4) the elucidation of mechanistic steps of L1 retrotransposition.Mark Batzer  identified polymorphic retrotransposons in primate genomes and demonstrated that their insertion rates among primates are relatively constant. However, he noted the striking exception of how Alu insertions have dramatically declined in the orangutan genome. Dr. Batzer then described how the polarity and homoplasy-free (the unlikely event where independent insertions occur at the same genomic site) characteristics of many polymorphic retrotransposon insertions allow their use as genetic markers to resolve phylogenetic relationships among human and non-human primates. Importantly, Dr. Batzer reported that approximately 4% of retrotransposon insertions among primates are not identical by descent, but instead represent closely spaced, parallel, independent insertions into a genomic locus. The identification of such events is of paramount importance to allow accurate use of polymorphic retrotransposons as genetic markers in phylogenetic studies.SAMHD1 gene. Once again, he identified somatic L1 insertions in the AGS brain tissue, though the relative numbers of insertions were not increased when compared to wild type controls. Together, these data provided compelling evidence that L1 retrotransposition continues to create somatic mosaicism in the human brain.Geoffrey Faulkner  described enhancements to a retrotransposon capture sequencing (RC-seq) protocol, and used them to uncover somatic L1 retrotransposition events in ‘bulk’ human brain samples and single hippocampal neurons. Dr. Faulkner uncovered greater than 300,000 somatic L1 insertions in hippocampal tissues, confirming earlier studies from his laboratory. He then used RC-seq to identify somatic L1 retrotransposition events in single hippocampal nuclei, and estimated that hippocampal neurons may contain approximately 15 somatic L1 retrotransposition events per cell. Dr. Faulkner also discussed preliminary RC-seq experiments conducted on genomic DNAs derived from frozen hippocampus and matched fibroblast samples from an Aicardi-Goutieres Syndrome (AGS) patient that harbor mutations in each allele of the David Greenberg  described how the two human Krüppel-associated box zinc-finger proteins (KRAB-ZNF), ZNF91 and ZNF93, restrict the transcription of certain SVA and L1 retrotransposons and may protect primate genomes from unabated retrotransposition. The team found KRAB-associated protein 1 (KAP1/TRIM28) and human ZNF91 were enriched at SVA elements in mouse embryonic stem cell lines that contain a single human chromosome. Reporter assays demonstrated that ZNF91 inhibited SVA expression by binding to the SVA variable nucleotide tandem repeat sequence. Similar analyses suggested ZNF93 could bind within the 5′UTRs of evolutionarily older (PA3-PA6) L1 subfamilies. Notably, the ZNF93 binding sequence is absent from the evolutionarily younger (PA1 and PA2) L1 subfamilies, suggesting a mechanism for how younger L1s have evaded the repressive effects of ZNF93.in vivo.Pam Cook  showed that purified L1 ORF1p is phosphorylated on multiple residues. A subset of these residues was found to be critical for retrotransposition in HeLa cells. Remarkably, each residue in that subset lies within canonical target and docking motifs of proline-directed kinases (PDKs) and is highly conserved in both primates and mouse. Mutations of these residues inhibited L1 activity, but substitution with the phosphomimetic aspartic acid could restore L1 function. Thus, phosphorylation may represent an important mechanism to regulate L1 retrotransposition in cis. However, proteins translated from L1/MALAT mRNA could promote the retrotransposition of mutant L1 and Alu RNAs that contain poly (A) tails in trans. These data suggest that the presence of a poly (A) tail is required for efficient L1-mediated retrotransposition.John Moran  discussed preliminary data on the identification of sequences within L1 mRNA required for retrotransposition. The team replaced the 3′ poly (A) signal of an engineered human L1 with a sequence cassette derived from the 3′ end of a long non-coding RNA (MALAT1), which previously was shown by Wilusz and colleagues to form a stabilizing triple helical structure at the 3′ end of an mRNA. The resultant L1/MALAT RNAs accumulate in cells, serve as translation templates for the L1 encoded proteins, but lack a poly (A) tail and cannot undergo retrotransposition This session focused on the different roles that mobile elements have played and will continue to play in genome evolution. The talks highlighted the diversity of contributions mobile elements make in genome evolution.Marlene Belfort  talked about group II introns that are the putative ancestors of both the spliceosomal introns and retrotransposons. She highlighted the absence of group II introns from eukaryotic nuclei and discussed the conundrum this implies with respect to their putative ancestral relationship to spliceosomal introns. She also talked about how retrotransposition is promoted by the synergistic relationship between the intron and the enzyme relaxase, whose gene is split by the intron.Lynne Maquat  spoke about mRNAs that are regulated post-transcriptionally via Staufen-mediated mRNA decay due to a STAU-binding site in their 3′UTR. Interestingly, she showed that in many cases these binding sites consisted of a 3′UTR Alu element or another type of SINE that base-paired with a partially complementary SINE within an lncRNA or another mRNA. She presented results that suggest a role for mobile elements in the intricate network of post-transcriptional interactions that regulate gene expression.Drosophila brain. He also showed that loss of piRNA proteins correlated with even higher levels of transposon expression in brain. He reported over 200 de novo transposon insertions in neurons through paired-end sequencing. His observations indicate that genomic heterogeneity is a conserved feature of the brain.Scott Waddell,  reported on transposition events in the memory-relevant neurons in the Tetrahymena, which is a model for programmed transposon-containing DNA elimination. The main analysis by high throughput sequencing involved measuring or analyzing the production and turnover of scan RNAs (scnRNAs) during the conjugation process. Notably, they found that there were two types of Internal Eliminated Sequences (IESs): those producing sncRNAs and those eliminated at a later stage potentially because of sequence similarity to the ones of the first type.Kazufumi Mochizuki  presented results on the ciliated protozoan Syncytins that function in placenta formation. He described three hitherto independent captures of endogenous retrovirus-derived envelope genes in Carnivora, Ruminantia, and Marsupial genomes. Together with previously reported cases, the data suggest that this exaptation process has been a major driving force in placenta evolution and diversity.Guillaume Cornelis,  talked about the exaptation during mammalian evolution of multiple retroviral envelope genes called Finally, Guillaume Bourque  discussed the impact that transposable elements have had on the evolution of human gene regulation. He showed that mobile elements have been a major contributor to host regulatory and transcript innovation, but also highlighted that these elements have been a significant source of ‘biochemical noise’ with limited impact on phenotype.Workshop 1 represented a mixture of TE structural studies, TE insertion targeting studies, and TE regulation by small RNAs. The first two talks shed light on key aspects of the protein dynamics involved in TE duplication.Stuart Le Grice  presented the structure of the budding yeast Ty3 reverse transcriptase (RT). He showed that this protein is actually a dimer, but only in the presence of its polypurine tract DNA/RNA hybrid substrate. He demonstrated that the Ty3 RT structure is topologically highly similar to HIV-RT and XMRV-RT. However, a critical difference is that unlike other RT proteins, Ty3 RT adopts an asymmetric homodimeric structure when bound to its substrate and unlike dimeric retroviral enzymes, the DNA polymerase and RNase H catalytic centers of Ty3 RT reside in different subunits.Tn1549 and demonstrated that this protein is active as a dimer and triggers formation of heteroduplex DNA intermediates during recombination.Orsolya Barabas  spoke about the critical need to understand bacterial conjugative transposons, as they are responsible for the transmission of antibiotic resistance. Dr. Barabas’s lab has determined the crystal structure of the conjugative transposon integrase enzyme from the vancomycin-resistance carrying Schizosaccharomyces pombe is targeted away from genes and towards stalled replication forks. The Zaratiegui lab identified a strong correlation of Tf1 insertion at sites of Sap1 protein binding, which suggests Tf1 is directed to integration sites via the Sap1 protein. Jacobs showed that Sap1 binds DNA and creates a replication fork barrier. He used an elegant two-plasmid transposition assay to determine that Sap1 is important, but not necessary, for targeting Tf1 insertion, and that the fork barrier activity of Sap1 binding sites is an important secondary requirement for Tf1 transposition at stalled replication forks.The focus of Workshop 1 then changed to understanding the mechanism of the targeting of TE insertions. Jake Jacobs  described a model suggesting the integration of Tf1 retrotransposons in Next, Bao Ton-Hoang  investigated the mechanism of targeting the replication fork by bacterial ‘HuH’ single-strand transposases encoded by the IS200/IS605 family. Ton-Hoang’s results suggested that the HuH transposase might be targeted to replication forks by direct interaction with structured DNA and that the interaction may be mediated by the single-strand DNA-binding proteins Ssb and RecA.piwi-interacting small interfering RNA (piRNA)-dependent and -independent mechanisms responsible for the TE epigenetic programming of mouse primordial germ cells. Dr. Molaro demonstrated that TE de novo methylation could be separated into two independent waves during germ cell development. The first wave appears unspecific and targets methylation genome-wide. However, some TE promoters escape this initial silencing and instead become the target of a piRNA-dependent secondary wave.The final section of Workshop 1 focused on the regulation of TEs by small RNAs. Antoine Molaro  showed that there are both Drosophila germline. Dr. Shpiz demonstrated that new TE insertions into euchromatic regions of the genome become piRNA producing loci, and produce piRNAs from the surrounding genic sequences. Most of the insertions produced piRNAs from both sides of the insertion and from both strands. Importantly, Dr. Shpiz demonstrated these new genic piRNAs can regulate the mRNA levels of the genes from which they originated.Sergey Shpiz  discussed piRNA formation in the R. Keith Slotkin  showed that genic mRNAs can be regulated by the small RNAs that TEs produce in Arabidopsis. Dr. Slotkin demonstrated that upon TE transcriptional activation the TE mRNAs are degraded into abundant small interfering RNAs. These siRNAs are incorporated into gene-regulating Argonaute proteins and function to regulate partially complementary genic mRNAs akin to a microRNA. In addition, Dr. Slotkin showed that TEs carry non-protein coding fragments that give rise to small RNAs that can target specific host genes and potentially favor TE propagation.David Kingsley  summarized over a decade of work conducted in his laboratory to elucidate the genetic basis of ecological adaptation in stickleback fish. One of the lessons learned is that the molecular changes underlying the evolution of new morphological traits involve noncoding cis-regulatory sequences such as enhancers, rather than mutations in protein-coding sequences. In some recurrent instances, changes in regulatory elements include the deletion of a pre-existing enhancer or the co-option of transposable element sequences to form a novel enhancer. Kingsley argued that such cis-regulatory modifications also play a prominent role in the adaptation of other vertebrates, including humans, to rapid changes in their environment.Drosophila species. Genetic, genomic, and biochemical data suggest a model whereby the two encoded proteins from these speciation genes act together to suppress a broad range of repetitive elements in the Drosophila germline. Interestingly, these proteins also exhibit a gain-of-function phenotype in species hybrids that may contribute to transcriptional reactivation of previously silent transposons and chromosomal abnormalities.Daniel Barbash  reported new results on the molecular mechanisms by which two ‘speciation’ genes normally function within Anne-Marie Dion-Côté  presented data from another speciation model, the lake whitefish, supporting the notion that many transposable elements are massively activated transcriptionally in interspecific backcross hybrids of closely related species. There appears to be a positive correlation between the level of transposon transcript upregulation and the appearance of malformations or deformities in the backcross embryos, suggesting that hybrid breakdown in part may be caused by a failure to repress the transcriptional activity of transposable elements.A presentation by David Kelley  illustrated one of the potential mechanisms that could contribute to phenotypic consequences when the transcription of mobile genetic elements goes awry. He showed that transposable elements are frequently transcribed as part of long noncoding RNAs (lncRNAs) in various human cells. Transposon sequences embedded in lncRNAs are not biochemically inert but interact directly with a variety of RNA-binding proteins, as determined by cross-linking immunoprecipitation experiments followed by high-throughput RNA sequencing (CLIP-seq). Though it remains to be seen whether these interactions are relevant to cellular function, the data indicate that transposons are integral components of the intricate network of ribonucleoproteins found in human cells.Cédric Feschotte  explored the evolution and genomic impact of mobile DNA in yet another group of organisms, the bats. While bat genomes are relatively small and show little size variation among extant species, analyses of ten bat species representing five major chiropteran families suggest dramatic variation in transposable element activity among different bat lineages. Transposon expansions are counteracted by a high rate of DNA loss compared to other mammals and predominantly via large-scale deletion events, uncovering a previously underappreciated mechanism for genome size homeostasis.Suzanne Sandmeyer  discussed research showing that the budding yeast, gypsy-like long terminal repeat retrotransposon Ty3 and its long terminal repeats act as pheromone-inducible promoters throughout the yeast genome. Work presented also showed that the RNP granules where Ty3 assembles have components in common with the animal germ cell granules that are known to be sites where RNA interference suppresses retrotransposons.Jef Boeke  described studies of L1 host factors discovered through proteomic screens, including the UPF1 RNA decay protein and the PCNA sliding clamp. Studies suggest the PCNA is required after endonucleolytic cleavage of the target DNA. Preliminary studies suggesting cell cycle regulation of L1 through imaging and synchronization were also described.Axel Horn  presented data from yeast (Zorro3) and human (L1) non-LTR retrotransposons showing that an interaction with the ESCRT membrane budding complex plays an important role during the life cycle of LINE-like retrotransposons.Alan Engelman  discussed allosteric inhibitors of the HIV-1 integrase that engage the catalytic core domain dimer interface at the binding site for the cellular LEDGF/p75 protein. The compounds induce integrase multimerization and compete for LEDGF/p75 binding. Interestingly, they were most potent during virus assembly, a point in the virus life cycle where LEDGF/p75 is apparently unable to shield the integrase from multimer induction.Pascale Lesage  showed a functional interaction between Ty1 integrase and a subunit of Pol III, which is essential for Ty1 integration site determination, thus answering the long-standing question of why Ty1 integrates preferentially at genes transcribed by Pol III.Paramecium. She presented work showing that a prerequisite to IES end cleavage by PiggyMac, a domesticated piggyBac transposase, is the availability of a specific Ku heterodimer, which forms a complex with PiggyMac in cell extracts.Mireille Bétermier  studied the molecular mechanism involved in the precise excision of Internal Eliminated Sequences (IES) during assembly of the somatic genome of the ciliate piggyBat transposon but is not required for repair after transposon integration at the insertion site.Nancy Craig  presented work in yeast showing that the non-homologous end-joining (NHEJ) DNA repair pathway is required for rejoining of the donor site after excision of the S. pombe heterochromatin formation can be induced at specific sites by environmental conditions. Regions of heterochromatin termed ‘Islands’ include meiotic genes and this heterochromatin forms independently of RNAi factors. Other clusters of heterochromatin termed ‘HOODS’ include meiotic genes and retrotransposons but formation of this heterochromatin relies on RNAi factors. Grewal reported that the heterochromatin in Islands and HOODS both rely on an RNA processing network that includes a core module (MTREC) composed of Mtl1 and Red1. MTREC degrades meiotic and retrotransposon mRNA, and through interactions with Nrl1 and splicing factors, recognizes cryptic introns and creates heterochromatin, whereas Mtl1 and Ctr1 promote proper processing of intron-containing telomerase RNA to maintain telomeres.Shiv Grewal  presented his finding that in Drosophila that requires the condensin II subunit dCAP-D3. This protein plays a key role in genome integrity by inhibiting double strand breaks in transposable elements. 3D chromatin data indicated that dCAP-D3 causes the formation of rigid chromatin loops that exclude transposable elements. This organization of chromatin inhibits the expression of retrotransposons and reduces homolog pairing of chromosomes that leads to double strand breaks.Michelle Longworth  presented a new mechanism of silencing retrotransposable elements in MuDr. This mechanism involves the DNA methylation and histone modification of promoter sequences. The silencing of MuDr is relieved in young leaves but returns during specific stages of development. Lisch proposed an intriguing model arguing that maize turns off its defense against transposable elements temporarily in leaves to allow the transmission of newly produced MuDr small RNAs into the germline where they can repress transposition.Damon Lisch  described his studies of the diverse family of Mutator elements in maize that rely on the autonomous element for transposition activity. He reported the discovery of Muk, a Mutator element in maize that, as the result of a rearrangement, produces a hairpin transcript that silences Drosophila that is proposed to cause speciation. Lhr and Hmr form a centromeric complex that mediates chromosome segregation and inhibits expression of transposable elements. D. melanogaster and D. simulans express different levels of Lhr and Hmr. In species hybrids, changes in the Lhr to Hmr ratio cause extensive mis-localization of the complex, increased expression of transposable elements, and impaired cell proliferation. These results argue that centromere binding proteins can play a surprising role in generating biodiversity.Axel Imhof  presented evidence for a unique system of reproductive isolation in S. pombe integrates into the promoters of stress related genes. Importantly, Tf1 increases the expression of adjacent genes in 40% of the insertions tested. The impact of Tf1 integration on growth in conditions of stress was examined with cobalt chloride. Large cultures of cells with diverse profiles of Tf1 insertions show that integration in specific intergenic sequences allows these cells to out compete others when cobalt chloride is present. These results argue that Tf1 integration possesses specific features that result in resistance to cobalt chloride and provide evidence supporting McClintock’s hypothesis.Henry Levin  presented results that test Barbara McClintock’s hypothesis proposing that transposable elements benefit the host by rearranging the genome in response to stress. The LTR retrotransposon Tf1 of Drosophila by piRNAs that silences transposons during germline development. He showed evidence that the HP1 homolog Rhino directly binds to the heterochromatic piRNA clusters and triggers piRNA production, leading to silencing of other sense and antisense transcripts. He also presented data indicating Rhino might anchor a nuclear complex that suppresses piRNA cluster transcript splicing, which may be required to differentiate piRNA precursors from mRNAs. The Drosophila findings were quite reminiscent of a theme introduced earlier in the meeting by Shiv Grewal  from his work in S. pombe, in which he also concluded that stalling splicing might be sufficient to induce the production of siRNAs.William Theurkauf  discussed the adaptive genome defense mounted in Continuing the theme of adaptive RNA-guided defenses, Luciano Marraffini  discussed how prokaryotic CRISPR-Cas immune systems might distinguish pathogenic from self and commensal elements. He discussed temperate phages that can integrate into the bacterial chromosome and could be considered ‘commensals’ of bacteria because they can carry genes that provide a fitness advantage to their host. Luciano elucidated an elegant means by which CRISPR-Cas might tolerate beneficial temperate phages through transcription-dependent DNA targeting, leading to no action against lysogenic prophages but targeting during the prophage lytic cycle. His results highlighted the myriad of means used by defense systems not just to distinguish ‘self from non-self’ but also to encode ‘tolerance to non-self’ in this exciting prokaryotic branch of adaptive immunity.Grace Wyngaard  reported on genome elimination in the somatic cells of copepods. Germline genomes in copepods can be 5-75X larger than somatic cells, due to a massive elimination of mobile elements from the presomatic cell lineage. She raised the speculation that the billions of basepairs of excised DNA might serve an adaptive role that provides the embryo with much of the DNA material needed to replicate the genome during subsequent cleavage divisions until the embryo hatches into a feeding larvae. Both the phenomenal genome diminution and the speculation that this could be adaptive resulted in much discussion among the meeting participants.Todd MacFarlan  discussed his work on the intriguing hypothesis that some members of the large and rapidly evolving KRAB-ZFP transcription factor family might function to silence endogenous retroviruses (ERVs). He showed that the mouse KRAB-ZFP protein Zfp809 binds and silences a sub-group of class I ERVs that utilize a proline primer binding site (Pro-PBS) via recruitment of heterochromatin establishment machinery. This work extends previous findings from Steve Goff’s lab, which originally identified Zfp809 in a biochemical screen for repressors of murine leukemia virus.Vasavi Sundaram  presented data on comprehensive mapping of binding sites for 26 pairs of orthologous transcription factors (TFs) in human and mouse cell lines. She presented data that on average 20% of TF binding sites were within TEs. TF binding peaks derived from TEs had epigenetic signatures consistent with regulatory activity. A substantial fraction of TE-derived TF binding events showed cell type-specificity. The results significantly extend previous findings that TEs have continuously shaped gene regulatory networks during mammalian evolution by their widespread contribution of TF binding sites.Harmit Malik  presented work from his lab on using evolutionary signatures of genetic conflicts in host-virus ‘arms-races’ to dissect the basis of differential viral susceptibility between different closely related host species. He presented findings about the antiviral protein MxA, which is remarkably broad in its antiviral range despite having to specifically recognize diverse, unrelated viral proteins in order to mount its antiviral action. In an analysis of primate MxA proteins, he showed that there were significant clusters of positive selection, which may each have specialized to be recognition interfaces for proteins from unrelated viruses. He concluded by suggesting that signatures of genetic conflicts may also allow the specific dissection of host defenses against their resident mobile elements.Aedes aegypti called Muta1. The authors cloned this element from the genome and showed it is transpositionally active in a range of insect species, including in its own host, in which it can be used to achieve genetic transformation.Peter Atkinson  reported the identification, of a new active Mutator superfamily transposon from the mosquito mariner transposon. Miskey and colleagues used chromatin immunoprecipitation experiments to map more than two hundred SETMAR-bound loci in the genome of human cells. The presence of SETMAR at its binding sites was associated with increased levels of local H3K36 di-methylation due to the methyltransferase activity of the SET-domain. These results support the hypothesis that the evolutionary recruitment of SETMAR’s transposase domain resulted in the emergence of an anthropoid-specific gene-regulatory network.Csaba Miskey  described a primate gene called SETMAR that emerged from the fusion of a histone methyltransferase SET-gene and the transposase of a Keizo Tomonaga  focused on endogenous Bornaviruses, which are non-segmented negative strand RNA viruses that have left their own endogenous elements in the genomes of many animal species. Tomanaga demonstrated that mouse endogenous bornavirus-like elements (EBL) produce the pachytene piRNAs in the testis, and also demonstrated that an EBL element in ground squirrel could efficiently inhibit the replication of exogenous bornavirus in cultured cells. This talk provided insights into the interaction between RNA viruses and hosts but also the biological significance of endogenization of non-retroviral viruses.Drosophila melanogaster. A Bari1 insertion was reported to mediate resistance to oxidative stress by adding CncC transcription factor binding. And a pogo insertion was found to increase expression of CG11699, which leads to increased ALDH-III enzymatic activity and resistance to xenobiotic stress. These results contribute to our understanding of eukaryotic stress response and highlight the role of TEs in environmental adaptation.Josefa Gonzalez  reported the molecular mechanism underling the phenotypic effects of two putatively adaptive transposable elements discovered in Inigo Narvaiza  and colleagues investigated the function of L1s in induced pluripotent stem cells (iPSCs) from human and non-human primates (NHPs). They used iPSCs from humans, chimpanzees and bonobos to explore factors that could have contributed to primate evolution and genomic diversity. Comparative gene expression analysis of human and non-human primates (NHP) iPSCs revealed differences in the levels of APOBEC3B (A3B) and PIWIL2, two regulators of L1 transposition. Higher levels of A3B and PIWIL2 in human iPSCs correlated with decreased L1 mobility compared to NHP iPSCs. These results were consistent with an analysis finding increased copy number of species-specific L1 elements in the genome of chimpanzees compared to humans.Liana Fasching  presented data demonstrating that transcription of ERVs in neural progenitor cells is dynamically regulated and activation of ERVs resulted in increased expression of nearby genes as well as the production of novel long non-coding RNAs. These data suggest that ERVs play a role in controlling genetic networks in neural progenitor cells. Since elevated levels of ERVs have been found in several neurological disorders, further studies on the role of ERVs in the control of gene expression are warranted in both the healthy and diseased brain.de novo insertions in neurons. Dubnau and colleagues also demonstrated that genetically activating LINE-like and gypsy retrotransposons by disruption of the Drosophila argonaute-2 gene, leads to rapid age-dependent memory impairment, defects in locomotion, and ultimately to shortened lifespan. Finally, Dubnau showed evidence that links this transposon activity to the neurodegenerative effects seen with TDP-43 pathology both in flies and in mammals, including humans. These findings have implications for the mechanisms of neurodegeneration seen in amyotrophic lateral sclerosis and frontotemporal dementia, where the TDP-43 protein’s pathology is central.Finally, Josh Dubnau  discussed the potential role of retrotransposons in neurodegeneration and age-related cognitive decline. He showed evidence that a diverse set of retrotransposons is transpositionally active in specific sets of post-mitotic neurons and glia in the fly brain. Some of these retrotransposons become aggressively active with age, leading to accumulation of In the final session of the meeting, three talks showcased the versatile nature of the transposase family used by DNA transposons. A fourth talk focused on an interesting enzymatic collaboration between serine recombinases in site-specific recombination.Rasika Harshey  presented work on an under studied area of transposition involving the repair of DNA following transposition with bacteriophage Mu. Mu uses transposition as a mechanism for replication. During the lytic cycle, integration is via a ‘cointegrate’ mechanism where replication of the element is primed from the target DNA. However, when forming a lysogen in a new host, repair at the ends of the element and removal of the ‘flaps’ of DNA from the previous host occurs by an unresolved mechanism. Previous work indicated that, unexpectedly, host double-strand break repair was involved in flap removal. In surprising new work, evidence was presented indicating that the replisome involved in normal chromosomal DNA replication is actually responsible for repair. A model was presented attempting to reconcile this finding with what we know about bacteriophage Mu and other transposable elements.hAT superfamily of DNA transposases. A crystal structure of the Hermes transposase complex with transposon end DNA sequences was presented. The transposase in the crystal structure is an octamer, a tetramer of four dimers, an oligomerization state that was confirmed both by electron microscopy and small-angle X-ray scattering technique. Biochemical data suggested that the octamer is needed to provide an array of DNA binding domains to find Hermes’ ends and to give sufficient binding affinity. Analysis indicated that the transposon ends are situated in a configuration such that the 3′-OH ends of the transferred strands orient to the suitably charged cavity in the complex where the target DNA likely resides. Further DNA-transposase crystal structures indicated that the configuration is consistent with double-strand break formation during excision by flanking-hairpin formation via a strand-switching mechanism. It is also consistent with the 8-bp duplication found in vivo at the sites of integration.Fred Dyda  presented work on the architecture of the Hermes transposase, a member of the mec element encoding methicillin resistance in Staphylococcus aureus. Unlike more typical site-specific recombinases where a single recombinase recognizes a symmetric target site, SCCmec uses two serine recombinases that collaborate to recognize an asymmetric site. This asymmetry may have broadened the options for evolving an integration site given that site-specific recombinases that use a single enzyme are constrained to inverted repeats found naturally in the host. Preliminary work also gave provocative possibilities for other proteins encoded on the element.Phoebe Rice  presented work on a pair of large serine recombinases that are responsible for the integration of the SCCJoseph Peters  presented work on Tn7, an element that is notable because of the level of control it has in target site selection and the broad diversity of bacteria that it uses as a host. The element uses five transposon-encoded proteins, including a heteromeric transposase (TnsA and TnsB), a regulator protein (TnsC), and target site selecting proteins that can choose a single site found in bacterial chromosomes (via TnsD) or target a complex found during lagging-strand DNA replication (via TnsE). TnsE was previously shown to recognize DNA replication by associating with 3′ recessed ends and the sliding clamp processivity factor. Current work suggests that TnsE adapts to the host sliding clamp protein and that this adaptation provides species specificity to Tn7-like elements. Bioinformatics analysis was presented indicating that elements with heteromeric transposases are more widespread than previously appreciated and have adapted new target site selection programs distinct from Tn7-like elements.Understanding transposition and its interaction with host genomes will not only explain the host response to the environment but also help to reveal its concealed role in evolution. The results presented at this conference show that much progress has been made in achieving these goals.The tradition of a large international meeting on ‘mobile DNA’ will continue. In the spring of 2016 (16 April to 19 April), the International Congress on Transposable Elements will be held in St Malo, France. Stay tuned for more details!


CDYL, MEIS1, STAT3 and PAX6. A significantly lower expression level for PAX6 in HSCR patients, would suit with the finding of an over-representation of the larger tandem (AC)m(AG)n repeats within the PAX6 promoter in HSCR patients, with the subsequent loss of protein P300 binding. Alternatively, PAX6 is a target for DNMT3B-dependant methylation, a process already proposed as a mechanism with a role in HSCR. Such decrease in PAX6 expression may influence in the proper function of signaling pathways involved in ENS with the confluence of additional genetic factors to the manifestation of HSCR phenotype.Hirschsprung disease (HSCR) is attributed to a failure of neural crest derived cells to migrate, proliferate, differentiate or survive in the bowel wall during embryonic Enteric Nervous System (ENS) development. This process requires a wide and complex variety of molecules and signaling pathways which are activated by transcription factors. In an effort to better understand the etiology of HSCR, we have designed a study to identify new transcription factors participating in different stages of the colonization process. A differential expression study has been performed on a set of transcription factors using Neurosphere-like bodies from both HSCR and control patients. Differential expression levels were found for  Hirschsprung disease , the most common neurocristopathy in humans (1:5000 newborns), is characterized by the absence of enteric ganglia along variable lengths of the distal gastrointestinal tract, resulting in severe intestinal dysfunction42623, th34RET proto-oncogene (OMIM 164761) is the main gene associated to HSCR with differential contributions of its coding and non-coding mutations7The ne OMIM 14761 is tSOX10, PHOX2B and NKX2-1 (TTIF1), previously associated to some isolated or syndromic forms of HSCR, act as potential regulators of RET expression and SOX10 also modulate EDNRB expression91011Pax3 is required for the activation of Ret transcription co-operatively acting with Sox1013Zfhx1b in mice neural crest prevents ENCCs migration beyond the proximal duodenum, and mutations in this gene are a cause of the Mowat-Wilson syndromic form of HSCRSeveral transcription factors are critical for the correct time course of ENS development. In humans Nevertheless, mechanisms underlying enteric precursor cell fate decisions during colonization of the bowel are relatively poorly understood. In an effort to better understand the etiology of HSCR and the signals required for a proper ENS development, we have designed a study to identify new transcription factors participating in different stages of the colonization process. With such purpose, we have performed a large-scale real-time PCR expression study of transcription factors participating in Embryonic Stem Cell networks that might be involved in ENS development, specially focusing on cell proliferation and/or differentiation process. For this study we have used human enteric precursor cells isolated from colon tissue of HSCR patients and control individuals as Neurospheres-like bodies (NLBs).CDYL, MEIS1, STAT3 (up-regulated) and PAX6 (down-regulated). Among these results, the most significant finding was the reduction by 4.67 fold of (cDNA)PAX6 expression in HSCR-NLBs compared with Control-NLBs . PAX6 is a regulator required for a correct differentiation and proliferation in the signaling and formation of the Central Nervous System (CNS). Therefore, in light of these results we further focused on PAX6 as a candidate gene in the pathogenesis of HSCR.We have evaluated the gene expression profile of 48 transcription factors, all of them previously described as cell proliferation or differentiation modulators, which may be potential participants in the interactome network of enteric precursor cells during ENS development. Analysis and display of quantitative gene expression assay was positive for 14 transcription factors  online, PAX6 in HSCR-NLBs compared to Control-NLBs were also verified by immunocytochemistry. The percentage of cells expressing PAX6 was 36% in HSCR compared to controls, which represents a 64% of reduction in the basal values of expression  supporting that these cells are neural precursors. We have also analyzed the conditional expression of the two major isoforms of PAX6 in HSCR-NLBs, given that changes in the relative concentrations of these isoforms of PAX6 [PAX6/PAX6(5a)] during development and neurogenesis, result in changes in the functions of its downstream regulated genes. However, we did not detect different expression levels between cDNA of PAX6/PAX6 (5a) (HSCR-NLBs Ct-PAX6:32.7 and Ct-PAX6(5a):33.16; Control-NLBs Ct-PAX6:23 and Ct-PAX6(5a):22). The (cDNA) PAX6/PAX6(5a) values after normalization were 1,0175 and 1,09 in HSCR and controls respectively.The reduced expression levels of (cDNA) pression . It is rPAX6 sequence in 196 HSCR patients, revealed no pathogenic variants, ruling out therefore the presence of deleterious mutations in this gene as a mechanism leading to HSCR . The most common allele for both groups was the one with 26 repeats, although its frequency was 11.4% lower in HSCR in comparison to controls (44.89% vs 56.3%). Moreover, when analyzing the distribution of each specific allele, we found significant under-representation of the 26 repeats allele  and over-representation of the 29 repeats allele  in the group of patients. These findings led us to set up a cut-off point at 26 repeats, and a detailed inspection showed that alleles with 26 or less repeats accounted for 52.29% of patients and 63.76% of controls, whereas the >26 repeats alleles accounted for 47.71% of patients and 36.24% of controls  . Statistical significance was maintained when analyzing the genotypic distribution after assembling heterozygous individuals and homozygous individuals with >26 repeats alleles  m(AG)n repeat at the  0.0033) . In addi 0.0020) . These fPAX6 P1 promoter, we used in silico approaches aiming to analyze any differential binding of regulator molecules. We submitted different alleles containing variable repeats to TFSEARCH and, noteworthy, the analysis revealed a loss of the binding of E1A Binding Protein P300 (P300) for PAX6 alleles containing ≥29 repeats. Furthermore, such loss was observed with different combinations of (AC) and (AG) units leading to alleles of 29 or more repeats.To further investigate the possible impact of the (AC)m(AG)n differential representation at Gene-Mania and Var-Elect tools were then used to analyze interactions between PAX6 and P300, and predictions revealed a direct physical interaction. Moreover, both proteins were found to be indirectly related to RET and other proteins encoded by genes previously associated to HSCR .PAX6, we checked first for the expression of P300 in HSCR and control NLBs obtaining positive expression m(AG)n tandem in control-NLBs. However, the band was dramatically reduced in the HSCR patient  DNMT3B and (cDNA) PAX6 compared with Control-NLBsPAX6 is a direct target of DNMT3B and therefore its expression might be regulated by methylation. For this purpose, ChiP-PCR assay was performed in NLBs from mice. Samples after ChIP assay with anti-Dnmt3b antibody were amplified by PCR. Two out of four Pax6 studied regions were detected in ChiP samples    online.CDYL, MEIS1, STAT3 and PAX6) that might be involved in human enteric precursor cells interactome networks have been identified. It comes as no surprise that all transcription factors found to be deregulated in ENS precursors from HSCR, are implicated in the transition of proliferation/differentiation state, a critical process for the correct ENS formation. CDYL is a chromodomain-containing transcriptional co-repressor ubiquitously expressed. A study conducted in mice suggests that during neural development, Cdyl might be inhibiting the neuronal differentiation of induced pluripotent stem cells (iPS)17MEIS1 is a conserved transcription factor that specifically acts as a direct molecular regulator of PAX6 expression during vertebrate’s development19STAT3 is part of the JAK-STAT signaling pathway and plays an important role in the coordination of the succession of steps along the cell cycle and the differentiation process during neural crest cell specification21PAX6. This gene is a highly conserved transcription factor belonging to the family of the so-called “paired-box” with functions in the eye, central nervous system and pancreas development. PAX6 is a regulator of the neural precursor proliferation and differentiation which acts by modulating the expression of different downstream effectors. Its main role is the generation of new neurons from stem cells and neural progenitors during the initial stages of the CNS development2324PAX6 initially modify the proliferation of progenitor cells and later neural differentiation in a highly context-dependent manner. This changing role might be due to the relative balance of the two major isoforms PAX6 and PAX6(5a) expression levels during developmentPAX6 isoforms, leading to the conclusion that the ratio PAX6/PAX6(5a) does not seem to be essential for the enteric precursor decision (proliferation/differentiation).The ENS arises from neural crest-derived cells with multipotency and migratory capabilities leading to the formation of a complex network of enteric neurons and glial cells that colonize the gastrointestinal tract. This complex process is carefully regulated by interacting signals and transcription factors that confer cell characteristics in each moment of the ENS development, and it is accepted that failures in this process are responsible for HSCR pathogenesis. Understanding underlying transcriptional programs required for enteric precursors normal development might help us to identify new genes and pathways involved in HSCR pathogenesis. With this aim, through the expression study performed among HSCR and controls NLBs, four new transcription factors , for instance, GAA-repeat expansion in the first intron of PAX6 expression is regulated by alternate usage of two promoters (P0 and P1) which are differentially regulated in a tissue-specific mannerPAX6 transcripts expression. P1 promoter contains distinct cis elements and several potential binding sites for transcription factors involved in tissue-specific expression in the eye, central nervous system and pancreas282931versus control individuals, showed an over-representation of >26 repeats alleles in HSCR patients in comparison with control subjects. In silico analyses revealed the existence of two putative binding sites, just flanking the repetitive sequence, recognized by P300. This protein is a histone acetyltransferase essential in the processes of cell proliferation and differentiation regulating transcription via chromatin remodeling. Subsequently, ChIP analyses revealed the existence of that physical interaction between PAX6 and the protein P300 in enteric precursors. However, the ability of P300 to bind its target sequence was dramatically reduced when the number of repeats was increased. Changes in the repeat number might alter the distance between P300 binding sites, thus modifying this protein binding affinity. This specific DNA region within PAX6 promoter sequence must undergo structural changes to either facilitate or hinder the accessibility to P300. The increase of repeats may also have influence on the conformation of the sequence and generate different structures for other proteins recognition. This mechanism might contribute to a lower expression of PAX6 and, probably leads to a decrease in regulation of the signaling pathways related to the ENS development in which this protein is involved. Regarding to the complex genetic basis of HSCR disease, we propose this mechanism as a necessary event contributing to PAX6 down-regulation observed in patients, even though other conditions are required to completely elucidate this finding.et al., an aberrant methylation status in HSCR-NLBs, was proposed as a mechanism responsible for incorrect gene expression patterns in these patientsEpigenetic programs are essentials to drive the transcriptional profiles required for a normal neural development. As described by Torroglosa PAX6 expression levels may be disturbed by methylation35PAX6 is a target of DNMT3B and therefore susceptible of being regulated by methylation, we performed a new ChIP-PCR analysis. Pax6 was shown to be a target for Dnmt3b, as well, as we identified two specific regions of this gene to where Dnmt3b is attached. Such binding sites are within CpG-rich regions in the human PAX6 sequence.Furthermore, it has been shown that PAX6 isoforms as it has been discussed previously.We hypothesize that enteric precursors from HSCR-NLBs seem to be non-responsive to external signals that lead to the correct neuronal differentiation, so that this event cannot occur at the right time, regardless of the balance between PAX6 expression levels in HSCR-NLBs supports that PAX6 operates and regulates transcriptional network during the ENS development. We report the first association study of the transcription factor PAX6 with HSCR and that its low expression levels may result in an aberrant neurogenesis, which is directly related with manifestation of HSCR phenotype.We have evaluated the gene expression profile of transcription factors which may be potential participants in the gene regulatory network of enteric precursor cells in the context of ENS development and HSCR. Findings of reduced ENS progenitor cells were obtained from human postnatal tissues of ganglionic gut of six sporadic non-related patients diagnosed with isolated HSCR  and six patients of other gastrointestinal disorders undergoing gut resection surgery at Hospital were used as controls . For both HSCR patients and control individuals, age range was between 3 months and 3 years.et al.The extraction of ENS progenitor cells from ganglionic bowels in mice was carried out in the same way as in humans, as previously described in Torroglosa Written informed consent for surgery, clinical and molecular genetic studies was obtained from all participants. The study was approved by the Ethics Committee for clinical research of the University Hospital Virgen del Rocío  and complies with the tenets of the declaration of Helsinki.All procedures involving mice were performed in accordance with European Union guidelines (2010/63/EU) and Spanish law  concerning the care and use of laboratory animals and were approved by the Animal Experimentation Ethics Committee (EAEC/IEC) of University Hospital Virgen del Rocío / Institute of Biomedicine of Seville (IBIS).A differential expression study in cultures of NLBs has been performed in a set of transcription factors implicated in the regulatory networks of embryonic stem cells  online. GAPDH or B-ACTIN was used as endogenous control respectively. Following the software recommendations, the upper limit of the cycle threshold (Ct) was set at 32 for the TaqMan Array Gene Signature Plates and 35 for the SYBR Green assay. Positive expression was exclusively considered when Ct values were lower than values described above.Analysis was performed using the RQ Manager Software  based on the comparative Ct (ΔΔCt) method. p value < 0.05.Data are presented as the mean ± SEM (Standard Error Mean) of values obtained from at least three experiments. Comparisons between values obtained in Control-NLBs and HSCR-NLBs were analyzed using the Student´s t test. Differences were considered significant when PAX6  and anti-NESTIN  . The secondary antibodies used were anti-rabbit IgG labeled with Cy2 (1:200) and anti-goat IgG labeled with Cy5 (1:200) . The nuclei of cells were counterstained with DAPI. For the quantification, ten images were taken per condition in which at least a number of 700 cells were counted.For immunocytochemical studies, cells derived from NLBs were seeded onto coverslips fibronectin-poly D lysine coated and fixed with 4% (wt/vol) paraformaldehyde in 0.1 M PBS. Then, they were incubated for one hour in 2.5% (wt/vol) bovine serum albumin (BSA) in PBS and with primary and secondary antibodies. After washing, the coverslips were mounted on slides with Fluoro-Gel  and fluorescent signals were detected using a Leica Spectra confocal microscope. The primary antibodies used were anti-For molecular studies we included a total of 196 Spanish HSCR patients  comprising sporadic and apparently isolated cases. A total of 158 cases were short segment forms (S-HSCR), 31 were long segment forms (L-HSCR) and 7 presented with total colonic aganglionosis (TCA). In addition, we also analyzed a group of 150 normal control individuals comprising unselected, unrelated, race-, age-, and sex-matched individuals. This study was approved by our Ethics Committee for clinical research and conformed to the tenets of the declaration of Helsinki. Fully written informed consent for molecular genetic studies was obtained from all the participants. Genomic DNA was extracted using standard protocols from peripheral blood leukocytes of all individuals included in the study.PAX6 (NM-001127612) was performed by direct sequencing using an ABI Prism 3730 Genetic Analyzer and SeqScape v2.5 software .The mutational screening of the complete coding sequence of PAX6, was performed by fluorescent PCR and subsequent analysis in the ABI Prism 3730 Genetic Analyzer with the Genemapper software . Statistical analysis of the allelic and genotypic distribution in HSCR patients versus controls was performed using Pearson or Chi Squared analysis, with statistical significance set at p < 0.05. Data were analyzed employing the Statistical Package for Social Sciences (SPSS) Version 14.0 for Windows.On the other hand, genotyping of the (AC)m(AG)n STR, located approximately 1 kb upstream of the transcription initiation site and associated with promoter P1 of in silico predictions of transcription factors binding sites we have used TFSEARCH (http://diyhpl.us/~bryan/irc/protocol-online/protocol-cache/TFSEARCH.html). ALGGEN-PROMO , was used to predict transcription factor binding sites in DNA sequences. Putative interactions between different genes and their participation in signaling pathways were analyzed with both Gene-Mania (http://www.genemania.org) and Var-Elect (http://www.genecards.org/?path =  /Search/VarElect).To perform PAX6 promoter and four different regions that cover the full gene   and sonicated in a Bioruptor® system . Immunoprecipitation was performed with P300 and Dnmt3b antibody  following the instructions of ChIP Assay Kit  in two different assays. Amplification of the region (AC)m(AG)n STR within the _013627)  online wHow to cite this article: Enguix-Riego, M. V. et al. Identification of different mechanisms leading to PAX6 down-regulation as potential events contributing to the onset of Hirschsprung disease. Sci. Rep.6, 21160; doi: 10.1038/srep21160 (2016).


Vibrio cholerae. SXT/R391 ICEs propagate by conjugation and integrate site-specifically into the chromosome of a wide range of environmental and clinical Gammaproteobacteria. SXT/R391 ICEs bear setC and setD, two conserved genes coding for a transcriptional activator complex that is essential for activation of conjugative transfer. We used chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) to characterize the SetCD regulon of three representative members of the SXT/R391 family. We also identified the DNA sequences bound by SetCD in MGIVflInd1, a mobilizable genomic island phylogenetically unrelated to SXT/R391 ICEs that hijacks the conjugative machinery of these ICEs to drive its own transfer. SetCD was found to bind a 19-bp sequence that is consistently located near the promoter −35 element of SetCD-activated genes, a position typical of class II transcriptional activators. Furthermore, we refined our understanding of the regulation of excision from and integration into the chromosome for SXT/R391 ICEs and demonstrated that de novo expression of SetCD is crucial to allow integration of the incoming ICE DNA into a naive host following conjugative transfer.Integrative and conjugative elements (ICEs) of the SXT/R391 family have been recognized as key drivers of antibiotic resistance dissemination in the seventh-pandemic lineage of  Vibrio cholerae, the etiologic agent of cholera, which remains a major cause of mortality and morbidity on a global scale , mob1–2 (DNA processing), mpf1–3  and reg (regulation)  have recently been shown to be the most abundant conjugative elements in practically all prokaryotic clades ,2. As sual scale –9. Most  cholera ,11. Sincd Africa ,8. SXT/Rd Africa –15, are bacteria ,16–18 anboratory . The SXTboratory . Highly boratory . These c) Figure  (6).Gammaproteobacteria  of SXT/R391 ICEs , and the integration of MGIs into the 3′ end of yicC (protein of unknown function), respectively , which facilitate the integrase-mediated excision from the chromosome of SXT/R391 ICEs and MGIs as circular molecules that serve as substrates for the conjugative transfer machinery (Mobilizable genomic islands (MGIs) are small <33 kb) genomic islands found in several species of marine bacteria . SXT/R39391 ICEs . Unlike 3 kb geno391 ICEs –23. The ectively ,24. xis achinery .attR attachment site . se. seattR and setD ,27. Agen391 ICEs   and RNA sequencing (RNA-seq). From this analysis, we identified and validated sequences of the SetCD operators. Finally, we investigated the dynamics of integration and excision of SXT to address the regulation of expression of E. coli strains used in this study, all derivatives of CAG18439 or BW25113  and β-galactosidase assays, Southern blotting, contour-clamped homogeneous electric field pulsed field gel electrophoresis (CHEF-PFGE) and sequencing were performed using standard molecular biology techniques. Details are provided in Text S1.Genomic and plasmid DNA preparation, PCR products amplification and purification, electro-transformation of VflInd1 were constructed using the one-step chromosomal gene inactivation technique using pKD3, pKD13 and pVI36 as templates  experiments were conducted as described in Carraro et al. . AdditioVflInd1/MGIVflInd1 ChIP-exo, SRX708086 and SRR1583199 for SXT RNA-seq as well as SRX708115 and SRR1583202 for SXT 5′-RACE. Complete data from aligned reads for ChIP-exo, RNA-seq and 5′-RACE can also be visualized using the UCSC genome browser at http://bioinfo.ccs.usherbrooke.ca/setCD.html.Fastq files for each experiment were deposited at the NCBI Sequence Read Archive under accession numbers SRX708080 and SRR1583172 for SXT ChIP-exo, SRX708425 and SRR1583516 for R391 ChIP-exo, SRX708426 and SRR1583532 for ICEV. cholerae O139 , were used in these experiments . The 3xFLAG tag did not affect the function of the SetC orthologs based on the similar transfer frequency of each ICE relative to its wild-type counterpart .The exact target genes and sequence motif recognized by the SetCD complex, which plays an essential role in ICE conjugative transfer activation, has yet to be determined. Using RNA-seq and ChIP-exo ,36, we urae O139  and R391rettgeri  as well luvialis . E. colitraL , traV , dsbC  and traF . One peak was also present inside the 3′ end of the predicted relaxase gene traI, upstream of the gene traD . Two additional peaks were observed, one upstream of xis (peak I) and one (peak II) in the intergenic region between mobI (auxiliary component of the relaxosome) and s003, which is part of the operon containing int. The strongest peak (peak VIII) was detected in the intergenic region between the two divergent genes s063 and s089. s089 is the first gene of a large operon coding for a RecA-independent homologous recombination system (rumA (UmuD-like protein) and s024. Transcriptional activity measured by RNA-seq in E. coli HW220 (wild-type SXT) and DPL3 (SXT ΔsetCD) correlates with the presence of a SetCD-binding site as the expression of 29 out of 52 core genes in SXT, including genes located downstream of SetCD-binding sites, is significantly increased upon mitomycin C induction compared to a ΔsetCD mutant under the same conditions .The ChIP-exo and RNA-seq assays were carried out after induction of the cell cultures using mitomycin C to trigger expression of SetCD from its native promoter. ChIP-exo data analyses revealed nine major SetCD enrichment peaks located upstream of the same genes and operons—most of which playing a key role in conjugative transfer—in the conserved core sequence shared by the three ICEs  and xisP (DPL393) that are responsible for the expression of int and xis, respectively. For each promoter, two mutants were also generated. A first variant, Δ-53, lacked the sequence immediately upstream of the promoter-proximal SetCD motif (DPL400 and DPL394) while another variant, Δ-36, lacked the entire region upstream of the −35 promoter box, thus removing the first 17 bp of the proximal SetCD box (DPL490 and DPL489) . When the putative SetCD boxes were missing, the β-galactosidase activity dropped below the detection limit of our assay .We validated that the proposed SetCD-binding motifs promote the observed binding of SetCD and transcriptional activation by fusing the ) Figure  and B. βcis, was sufficient to confer SetCD-dependent induction of gene expression, we substituted the native-binding site of the cAMP receptor protein (CRP) of the lacP promoter upstream of lacZYA in E. coli MG1655 by the SetCD box of s003P. Two chimeric promoters containing the operator mutations lacZoCD1 and lacZoCD2 were constructed  retained the −35 element of lacP, whereas it was substituted by the −35 element of s003P in the lacZoCD2 mutant (DPL501). The three operator sites o1, o2 and o3 of the LacI repressor remained unaffected in both constructs. setCD was expressed under the control of PBAD from pGG2B in the strains containing the constructions and in wild-type MG1655. The absence of significant β-galactosidase activity observed on M9 glycerol medium supplemented with glucose for both lacZoCD1 or lacZoCD2 mutations confirmed the inability of the hybrid promoters to respond to the activation by CRP bound to cAMP regardless of the alleviation of LacI repression by IPTG and of the presence of repressed setCD  is very distant from the σ70 consensus, while lacZoCD1 harbors the more canonical −35 of Plac (TTTACA). Taken together, these experiments confirm that the ChIP-exo derived SetCD motif alone is sufficient to confer SetCD-dependent activation of gene expression.To confirm that this motif alone, and not a hypothetical factor acting in d Figure . The lacD Figure . In cont2 Figure  and E. S2 Figure  and E. Tt Figure . This phVflInd1, an MGI originally detected in the same V. fluvialis strain that contains ICEVflInd1 , SXT ΔsetCD (AD133) or pGG2B, the plasmid expressing SetCD from PBAD (AD132). While expression of cds4 was induced by mitomycin C in the strain containing wild-type SXT, it was nearly abolished in the absence of setCD regardless of the presence of mitomycin C  analysis on MGIVflInd1 sequence revealed the presence of SetCD-binding motifs upstream of cds4 and rdfM  suggests that like in SXT, SetCD acts as a class II activator in MGIs  is unable to integrate in recipient cells lacking setCD , indicating that SXT integration into the recipient's chromosome is not a rate-limiting step. This conclusion is also supported by setCD overexpression in the donor cell, which resulted in a ∼3-log increase of transfer . Consequently, mating pore assembly or DNA translocation is likely the rate-limiting step of SXT conjugative transfer. As expected, transfer of the ΔsetCD mutant was abolished, even upon expression of int in recipient cells .To clearly address the role of SetCD in the establishment of SXT in the recipient cells, we conducted mating assays using combinations of D Figure . OverexpT Figure a and b,r Figure a and c.s Figure d and f.setCD was only partially restored when setCD was overexpressed in donor cells, at a rate of only one-fifth of wild-type SXT and ∼4 logs lower than wild-type SXT upon setCD overexpression . Because in such a context, expression of the transfer genes is not compromised in donor cells, the low rate of transfer of mating e suggests that integration of SXT ΔsetCD into the recipient's chromosome became the rate-limiting step of transfer. Assuming that this phenotype was attributable to weak or lack of expression of int in the recipient cells, we can rule out that the SetCD protein complex is translocated, at least in significant amounts, from the donor to recipient cells during SXT transfer. Indeed, stimulation of int expression mediated by translocated SetCD should have enabled normal integration of SXT ΔsetCD at a wild-type rate despite the lack of setCD genes in the recipient cells. In fact, transfer of SXT ΔsetCD was fully restored to wild-type level only upon concomitant overexpression of setCD in the donor and int in the recipient . Altogether, these results suggest that the SetCD complex is not translocated from the donor to recipient cells during conjugation. Instead, setCD is expressed de novo upon entry of SXT in the recipient cells, allowing int expression to mediate SXT integration.Intriguingly, transfer of SXT ΔsetCD mutation seemed to hinder the expression of int in the recipient cells, eventually leading to loss of the incoming SXT. Suboptimal int expression could reduce integration or promote maintenance of the ICE by other means. We submitted a sample of seven ΔsetCD exconjugants randomly picked from mating e  . At least two possible mechanisms could explain the formation of such anomalous exconjugants. First, SXT ΔsetCD could have integrated by homologous recombination or transposition potentially through one of the insertion sequence present in the variable region VRIII  Figure . All exhI Figure . Alterna0 Figure . Insteadfragment  suggestisetCD suggests that expression of int strictly depends upon activation by SetCD. To test this hypothesis, we monitored the expression of int by qRT-PCR on cDNA derived from E. coli strains containing SXT (HW220) or its ΔsetCD derivative (DPL3). We also monitored the expression of xis in the same conditions as it is expected to be expressed only upon activation of SXT transfer to promote the site-specific excision reaction over integration. The DNA-damaging agent mitomycin C increased int and xis mRNA transcript levels by 31- and 39-fold, respectively, in a setCD-dependent fashion . In the same conditions, expression of both int and xis dropped below the detection level for SXT ΔsetCD regardless of the presence or absence of mitomycin C , assembly of the mating apparatus , initiation of ICE DNA transfer (mobI/oriT and traIDJ) and finally integration into the recipient cell's chromosome (int) . Suc. Suc916,) Figure .xis, s003, mobI, traI, traD, traL, traV, dsbC, s063, s089 and traF  and close examination of the ChIP-exo signal in the region upstream of traI suggests the presence of a potential weaker promoter footprint as well as a degenerate SetCD-binding motif for all three ICEs . This raises interesting questions about the selective pressure operating on sequences inserting near the −35 promoter element of traI for the conservation of a functional SetCD operator. Second, traD expression is driven from a SetCD-dependent promoter located within traI  sequence is located 75 bp downstream of the original traV annotation. As a consequence, we propose to redefine the start codon of traV at this new location . This suggests that the reported increased expression of mosAT likely resulted from read-through of the mRNA transcript initiated by SetCD at the promoter traVP.Our data allowed us to deepen our understanding of the regulation of ICEs of the SXT/R391 family. To date both ectively ,20. To oI Figure  and G. F Figures  and G. Cn Figure  and G. Wn Figure  reportedrumAB of R391 was reported to contain a strong match to known LexA-binding sites at positions −44 to −25 relative to the rumA initiation codon . Our data rather support the idea that rumAB is part of the LexA regulon, not of the SetCD regulon   Figure . This isR391 ICE . This stR391 ICE . Our datint and xis requires the presence of SetCD in both donor and recipient cells. Our results indicate that an SXT ΔsetCD mutant complemented in trans by SetCD only in the donor cells is incapable of integrating site-specifically into the 5′ end of prfC. This observation led us to conclude that neither SetCD nor Int are translocated into the recipient strain during conjugation. Instead, setCD is expressed de novo in the recipient, allowing int expression to mediate SXT integration into prfC in a site-specific fashion (Figure int and setCD are physically linked on SXT/R391 ICEs, these mobile elements do not need to rely on a dual promoter regulation of their integrase gene to ensure their survival and dissemination.Unlike in MGIs, we demonstrated here that expression of SXT n Figure . This resetCD and setR likely allows buildup of the SetCD and SetR pools. Entry by conjugation of the ICE as single-stranded DNA is known to activate the SOS response (xis and int in the presence of SetCD whereas the presence of the RDF Xis in the recipient cells reduces SXT transfer, likely by interfering during the recombination between attP and attB (916, the RDF xis and integrase int genes are part of a single long tetracycline-inducible mRNA transcript reading through the attP attachment site and extending to all of the transfer genes (xis and int in SXT/R391 ICEs are not organized as an operon. Instead they are two convergent genes suggesting that other factors besides SetCD regulate their expression.Upon entry of an ICE of the SXT/R391 family into a naive host, the repressor SetR is initially absent, and concomitant expression of response , which cresponse . Howeverand attB . Therefoer genes . Unlike V. cholerae populations and in other Gammaproteobacteria.In summary, our study establishes the SetCD-dependent regulation of excision, transfer and integration the ICEs of the SXT/R931 family as well as the genomic islands they mobilize, and highlights the importance of SetCD in triggering the spread of antibiotic resistance conferring mobile elements in Supplementary Data are available at NAR Online.


TPI gene promoter is complete within 10 s and isdescribed by a single-stage kinetic model. The complex formation of TBP withthe wild-type TATA box occurs 5.5 times faster and the complex dissociationoccurs 31 times slower compared with the SNPcontaining TATA box. Within thefirst seconds of the interaction, TBP binds to and simultaneously bends theTATA box. Importantly, the TATA box of the wild-type TPI genepromoter requires lower TBP concentrations compared to the TATA box containingthe -24T → G SNP, which is associated with neurological and musculardisorders, cardiomyopathy, and other diseases.The TATA-binding protein (TBP) is a key part of the transcription complex ofRNA polymerase II. Alone or as a part of the basal transcription factor TFIID,TBP binds the TATA box located in the core region of the TATA-containingpromoters of class II genes. Previously, we studied the effects of singlenucleotide polymorphisms (SNPs) on TBP/TATA-box interactions using gelretardation assay. It was demonstrated that most SNPs in the TATA boxes of somehuman gene promoters cause a 2- to 4-fold decrease in TBP/TATA affinity, whichis associated with an increased risk of hereditary diseases, such as βthalassemias of diverse severity, hemophilia B Leyden, myocardial infarction,thrombophlebitis, lung cancer, etc. In this work, the process of TBP/TATAcomplex formation has been studied in real time by a stopped-flow techniqueusing recombinant human TBP and duplexes, which were identical to the TATA boxof the wild-type and a SNP-containing triosephosphate isomerase gene promoterand were fluorescently labeled by the Cy3/Cy5 FRET pair. It has beendemonstrated for the first time that real-time binding of TBP to the TATA boxof the  The specific nucleotide sequences of a promoter and around it serve as a codethat determines when, where, and how efficiently certain genes are transcribed.This code consists of sequences of three types: the core promoter, the proximalpromoter region, and distal sequences more remote from the promoter. The corepromoter is a region situated at a distance of about 100 nucleotides upstream(in the 5’-region) and downstream (in the 3’-region) of thetranscription start site, which comprises such regulatory elements as the TATAbox, TFIIB-recognition element (BRE ), initiator (Inr), motif ten element (MTE), downstream promoter element (DPE), downstream core element (DCE ), X corepromoter element 1 (XCPE1), and others; their amounts may vary . In the presence ofSNP, the rate constant of the TBP/TATA complex formation(kon) decreases by 35 times and the dissociationrate constant (koff) reduces by 30%.Thus, it was demonstrated [14] that the -24T → G SNP in the TATA box ofthis gene strongly reduces the TBP/TATA affinity. The equilibrium dissociationconstant of the complexes, TPI gene promoter in themillisecond and second ranges. Therefore, binding of TBP to the TATA-box of theTPI gene promoter was studied using the“stopped-flow” method. The method is based on fast, within ~ 1 ms,mixing of the reactants and registration of the FRET (Forster Resonance EnergyTransfer) signal. Recombinant full-length human TBP and 15 bp oligonucleotidesidentical to the TATA box with flanking nucleotides of the wild-typeTPI promoter and the SNP-containing TATA box promoter andlabeled with fluorescent Cy3 and Cy5 dyes were used in the study. This methodenables one to determine the rate constant for the recognition of the wild-typeTATA box by the TATA-binding protein and to reveal the structural features ofthe TBP/TATA complex in real time, under both normal and pathologicalconditions.The objective of the present work was to measure and analyze the kineticparameters of the real-time TBP/ TATA interaction. The EMSA classical method,which was used to explore the thermodynamic and kinetic parameters of TBP/TATAcomplexes, does not allow for studying the interaction dynamics of TBPmolecules and the TATA-box of the Escherichia coli cells transformed with the pAR3038- hTBP plasmid. BL21 (DE3) E. coli transformation wasperformed according to [Only recombinant full-length human TBP containing the naturally occurring aminoacid sequences was used in the study. TBP was expressed in BL21 (DE3)rding to . Expressrding to  using thrding to .15 bp oligodeoxynucleotides (ODNs) labeled at the 5’-ends of the chainswith cyanine fluorophores Cy3 and Cy5 were synthesized and purified at“NanoTekh- S”, Novosibirsk, Russia.TPI gene promoter , was analyzed by the“stopped-flow” method on an SX20 spectrometer . The fluorescence excitation wavelength of the Cy3 dye was550 nm; the voltage on the detector was 575 V. Cy5 fluorescence was recorded atwavelengths longer than 645 nm using the RG-645 filter .Binding to the DNA duplexes was studied using the following TBP concentrations:the T allele – 1 × 10-7, 2 × 10-7, 4× 10-7, 6 × 10-7, 8 ×10-7, 10 × 10-7, and 20 × 10-7M; the G allele – 4 × 10-7, 6 × 10-7,8 × 10-7, 10 × 10-7, 20 ×10-7, 30 × 10-7, and 40 × 10-7M. The DNA duplex concentration was 1 × 10-7 M in all cases,the measurement time was 50 s; the total number of points per curve was 6000.Experiments were conducted at 25°C.TBP binding to the DNA duplex, which corresponds to the wild-type TATA box ofthe To determine a kinetic model for the interaction of TBP with the DNA duplexesand to calculate the rate constants of all elementary reaction steps, theDynafit software   wasusedStudying pre-steady-state kinetics allows one to conduct a detailed analysis ofthe reaction mechanism. The advantage of the “stopped-flow” methodis the opportunity it affords to observe transient reactions and to record theconformational transitions of a protein and DNA during a real-time interaction.Although this approach is technically more complex and its use requires a morelabor-intensive mathematical analysis, studying the binding of the TATA bindingprotein to TATA boxes under pre-steady-state conditions enables one to deepengreatly knowledge about the mechanism of their interaction.Fig. 1) were used whichcontained a donor (Cy3)–acceptor (Cy5) pair at the duplex ends, while thecentral part of the duplex was the TATA box of the wild-type TPIgene promoter or that comprising the SNP.In this study, FRET substrates.The kinetics of the binding of the DNA duplexes to TBP, presented inTPI gene and to theSNP-containing TATA box, which is described by a one-stepScheme:Based on these data, we have suggested a kinetic mechanism for TBP binding tothe wild-type TATA box of the Table.It is seen that the complex formation of TBP with thewild-type TATA box occurs 5.5 times faster (1.1 × 106M-1s-1) than with the G allele (0.2 ×106 M-1s-1) and the dissociation of TBP/TATAcomplexes occurs 31 times slower . It should be noted that this difference in the rate constants of theTBP/TATA complex formation and decomposition leads to a difference in thevalues of the equilibrium dissociation constants by 150 times (2.7 ×10-9 M in the norm and 0.4 × 10-6 M in thepresence of the mutation). The difference in the dissociation constant(KD) values between the wild-type andSNP-containing TATA box means a sharp decrease in the TBP affinity foroligonucleotides with an altered TATA box.The rate constants for the forward and reverse reactions are given inin vivo transcribed and expressed lessefficiently. These results have been confirmed clinically [The obtained data indicate that the G/C-pair occurring in the TATA box makesthe DNA structure more rigid, which complicates the TATA box binding to TBP andthe formation of a functional complex possessing the optimal conformation. Itimplies that the triosephosphate isomerase gene containing the -24T → GSNP in the TATA box is inically .TPI promoter increases the risk of development of somediseases associated with the lack of triosephosphate isomerase. The lack of TPImay be compensated in other ways , whichfollows from differences in the response of patients to TPI deficiency in thebody [ TPI gene promoter is reduced 150-fold, TPIactivity in the erythrocytes of some patients falls to 3–10% of the norm[Comparison of our data and published ones, 20demothebody , 21.Desthe norm. TPI gene TATA box. This resultis consistent with the data obtained previously using full-length human TBP andthe AdMLP TATA box with a consensus sequence5’-CGCTATAAAAGGGC-3’, the 5’-end of which wasattached to the TAMRA fluorophore and the 3’-end was attached tofluorescein [It should be noted that by detecting the real-time interaction of human TBPwith the Cy3 and Cy5 fluorescently labeled TATA-containing duplexes, it hasbeen demonstrated for the first time that TBP rapidly binds to andsimultaneously bends DNA of theorescein , which hIt should be noted that these studies have been conducted around the worldusing different TBP types, full and truncated forms , andprimarily only the model AdML promoter (less often E4) with the TATA boxconsensus sequence. The obtained results improved the concept of the TBP/TATAinteraction, which is the key interaction in the initiation and regulation ofthe transcription and synthesis of proteins in eukaryotic cells.


Synechocystis PCC 6803. In addition to the L03 promoter showing wide dynamic range of transcriptional regulation, we observed the L09 promoter as unique in high leaky gene expression under repressed conditions. In the present study, we attempted to identify the cause of L09 promoter leakage. TetR binding to the promoter was studied by theoretical simulations of DNA breathing dynamics and by surface plasmon resonance (SPR) biosensor technology to analyze the kinetics of the DNA–protein interactions.There is a need for strong and tightly regulated promoters to construct more reliable and predictable genetic modules for synthetic biology and metabolic engineering. For this reason we have previously constructed a TetR regulated L promoter library for the cyanobacterium −4 cannot lead to detectable variations in the DNA–protein interactions. Higher probability at the downstream region of transcription start site of the L09 promoter compared to the L10, L11, and L12 promoters was observed. Having practically the same kinetics of binding to TetR, the leakage problem of the L09 promoter might be due to enhanced RNA Polymerase (RNAP)-promoter interactions in the downstream region.DNA breathing dynamics of a promoter was computed with the extended nonlinear Peyrard–Bishop–Dauxois mesoscopic model to yield a DNA opening probability profile at a single nucleotide resolution. The L09 promoter was compared to the L10, L11, and L12 promoters that were point-mutated and different in repressed promoter strength. The difference between DNA opening probability profiles is trivial on the TetR binding site. Furthermore, the kinetic rate constants of TetR binding, as measured by SPR biosensor technology, to the respective promoters are practically identical. This suggests that a trivial difference in probability as low as 1 × 10Both theoretical and experimental analyses of the L09 promoter’s leakage problem exclude a mechanism of reduced TetR binding but instead suggest enhanced RNAP binding. These results assist in creating more tightly regulated promoters for realizing synthetic biology and metabolic engineering in biotechnological applications. Transcription regulation plays a major role in controlling gene expression and consequently in altering biochemical reactions catalyzed by these gene products. Hence, it has high impact in synthetic biology and metabolic engineering –3. The rSynechocystis PCC 6803 and identified the L03 promoter that exhibits a wide dynamic range of regulation  was calculated according to Eq. MTetR, Mpromoter (g mol−1)], the number of binding sites on the promoter  and the surface density of the immobilized promoter [Rimmob (RU)].The theoretical signal under saturating conditions bind DNA , 23. As  [2 Fig. . The pro [2 Fig. . Additio [2 Fig. , we furtA flanking point mutation in the L09, L10, L11, and L12 promoters neither causes significant difference in the DNA breathing dynamics at the TetR binding sites nor affects the kinetic rate constants of the interactions between TetR and the respective L promoter. The leakage problem of the L09 promoter may be due to enhanced RNAP binding. These results assist in creating more tightly regulated promoters for realizing synthetic biology and metabolic engineering in biotechnological applications.