Caenorhabditis elegans, the KGB-1 JNK signaling pathway regulates the response to heavy metal stress. In this study, we identified FOS-1, a bZIP transcription factor, as a target of KGB-1-mediated phosphorylation. We further identified two transcriptional targets of the KGB-1 pathway, kreg-1 and kreg-2/lys-3, which are required for the defense against heavy metal stress. FOS-1 plays a critical role in the transcriptional repression of the kreg-1 gene by recruiting histone deacetylase (HDAC) to its promoter. KGB-1 phosphorylation prevents FOS-1 dimerization and promoter binding, resulting in promoter derepression. Thus, HDAC behaves as a co-repressor modulating FOS-1-mediated transcriptional regulation. This study describes the direct link from JNK signaling, Fos phosphorylation, and regulation of kreg gene transcription, which modulates the stress response in C. elegans.MAP kinases are integral to the mechanisms by which cells respond to a wide variety of environmental stresses. In  Caenorhabditis elegans regulates its response to environmental stresses. Previously, we showed that a conserved KGB-1 JNK mitogen-activated protein kinase (MAPK) pathway regulates the response to heavy metal stress in C. elegans, illustrating that these key stress signaling pathways in mammals are also conserved in C. elegans. Various proteins have been identified as potential targets of JNK MAPK in mammals; however, the identification of JNK MAPK physiological substrates has proven more challenging in C. elegans. Here we demonstrate that Fos, a bZIP family transcription factor, and HDAC, a Class I histone deacetylase, are crucial components functioning downstream of KGB-1 in the JNK-mediated stress response pathway. We find that FOS-1 functions as a transcriptional repressor by recruiting HDAC to its target promoters. In response to stress, activated KGB-1 relieves this repression via phosphorylation of FOS-1. Thus, this study shows how JNK signaling induces tolerance to stress at the gene level and describes a novel mechanism of gene regulation by which this is effected.We have investigated the mechanisms by which the soil nematode  Mitogen-activated protein kinase (MAPK) signal transduction pathways are evolutionarily conserved in eukaryotic cells and transduce signals in response to a variety of extracellular stimuli. Each pathway is composed of three classes of protein kinases: MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK) Drosophila melanogaster and Caenorhabditis elegans are useful for understanding the effects and interactions of JNK proteins, especially since they are amenable to the analysis of cytoprotective gene expression and the specific contributions of different tissues C. elegans have revealed that the JNK MAPK signaling components are highly conserved between C. elegans and mammals. One such C. elegans JNK pathway is the KGB-1 pathway, composed of an MLK-type MAPKKK MLK-1, an MKK7-type MAPKK MEK-1 and a JNK-type MAPK KGB-1 Invertebrate model organisms such as Various targets of JNK phosphorylation have been identified in mammalian systems, including members of the basic region leucine zipper (bZIP) family of transcription factors such as ATF2 and Jun C. elegans homolog of Fos, and showed that it functions downstream of KGB-1. In addition, we identified two genes whose expression is induced by copper in a KGB-1-dependent manner: kreg-1 and kreg-2 (genesKGB-1 regulated ). We found that FOS-1 represses transcription via the recruitment of a Class I histone deacetylase HDA-1 to the promoter. Biochemical assays demonstrated that phosphorylation by KGB-1 inhibits FOS-1 self-association and binding to the kreg-1 promoter. These results suggest that FOS-1 and HDA-1 play an inhibitory role in the response to heavy metal stress, and that the KGB-1 pathway confers tolerance to heavy metals by phosphorylating and thereby negatively regulating FOS-1.To understand the role of KGB-1 signaling in the heavy metal stress response, we screened for proteins that may interact with KGB-1 and identified FOS-1, a C. elegans mixed-stage cDNA library by the yeast two-hybrid method to isolate proteins that interact with KGB-1. Generally, kinase-negative (KN) forms of protein kinases constitutively associate with their substrate. Therefore, as bait we used KGB-1(K67R), a KN form in which Lys-67 in the ATP-binding motif has been mutated to arginine. From this screen, we identified 10 proteins that interact with KGB-1  , lane 2.The FOS-1 protein contains six putative MAPK phosphorylation sites (S/TP): Ser-151, Thr-263, Thr-278, Thr-304, Thr-316, and Thr-318 . We geneFos family proteins function as dimers that bind DNA and regulate the transcription of target genes fos-1 loss-of-function mutants could not be used to assay for heavy metal toxicity, because they have a sterile phenotype (data not shown). We therefore tested the effect of fos-1 knockdown on the stress response using a feeding RNA interference (RNAi) method. Animals were placed on agar plates containing copper (Cu2+) ions, fed a bacteria strain expressing the double-stranded RNA for fos-1, and their development was monitored for any signs of an altered response to heavy metal stress. As shown fos-1 RNAi had no effect on the sensitivity to Cu2+ ions. Animals treated with fos-1 RNAi exhibited an everted/protruded vulval phenotype in the adult as observed in fos-1a loss-of-function mutants fos-1 RNAi indeed had caused knockdown of fos-1. In contrast to the lack of effect in wild-type animals, fos-1 RNAi suppressed the sensitivity to Cu2+ ions in kgb-1(km21) mutants  in wild-type animals. We found that expression of FOS-1(T304A) resulted in sensitivity to Cu2+ ion compared to expression of wild type FOS-1  mutant from the heat shock promoter  genes whose expression was regulated by KGB-1 (2+ ions (kreg-1 and kreg-2. The protein encoded by kreg-1 (F53A9.2) is a novel 83 amino acids protein with polyhistidine streches, while the kreg-2 gene is identical to lys-3, which encodes a lysozyme. We validated our microarray data by quantitative real-time RT-PCR (qRT-PCR)  mutants induction of both genes was considerably reduced. To determine whether the kreg genes play functionally important roles in the resistance to heavy metal stress in C. elegans in vivo, we used RNAi to inhibit the expression of kreg-1 or kreg-2 and then examined the stress response. RNAis against either kreg-1 or kreg-2 caused a partial sensitivity to Cu2+ ions . Thus, bacteria appear to play no role in the susceptibility to heavy metal stress and it remains unclear how LYS-3 may protect against heavy metal stress.To understand how the KGB-1 pathway modulates gene activity and to define the physiological processes in which the heavy metal stress response may be involved, we examined gene expression changes in wild-type and ysis see   . In wildu2+ ions . The krekreg-1 expression patterns and to develop tools for further analysis, we generated a Pkreg-1::venus reporter, consisting of the kreg-1 promoter driving expression of venus. Wild-type animals harboring the Pkreg-1::venus reporter exhibited weak Venus expression in the absence of Cu2+  mutants  animals  mutants. We observed that while expression of the Pkreg-1::venus reporter gene was diminished in kgb-1(km21) mutants, treatment with fos-1 RNAi was epistatic to this and resulted in increased kreg-1 reporter activity  deletion mutant carrying Pelt-2::mek-1 exhibited resistance to heavy metal stress  mutation resulted in enhanced heavy metal stress sensitivity in wild-type animals or suppression of the stress-sensitive phenotype of kgb-1 mutants  had no effect on the expression pattern of the transgene (Pkreg-1Δtre2::venus) resulted in constitutive expression in both wild-type and kgb-1(km21) mutant animals TCA, called the TPA-responsive element (TRE) and TRE2 . To detereporter . Deletioransgene . In cont animals . Furtherransgene . Thus, tkreg-1 promoter via TRE2, we conducted chromatin immunoprecipitation (ChIP) assays. Human embryonic kidney (HEK) 293 cells were co-transfected with the Pkreg-1::venus reporter together with either T7-FOS-1 or the negative control T7-hGrhl2. Lysates were immunoprecipitated with anti-T7 antibodies, and quantitative PCR analysis was performed to amplify DNA fragments contained in the immunoprecipitated complexes. PCR analysis showed that FOS-1 bound efficiently to the kreg-1 promoter, whereas the negative control human Grhl2 protein did not (Pkreg-1Δtre1::venus transgene (data not shown), but not to the Pkreg-1Δtre2::venus transgene  mutants  mutants. Thus, HDA-1 negatively regulates the heavy metal stress response, consistent with the observation that kreg-1 expression is repressed by HDA-1.Finally, we examined whether HDA-1 contributes to the response to heavy metal stress. Knockdown of u2+ ions . In contC. elegans KGB-1 JNK pathway regulates the stress response to heavy metals JNK MAPK cascades are pivotal signaling modules controlling diverse signal transduction pathways in eukaryotes. The kgb-1 mutants has led to the identification of two targets of the KGB-1 pathway, namely kreg-1 and kreg-2/lys-3. Both targets are transcriptionally induced by stress, both require the KGB-1 pathway for their full induction, and both are required for protection of the animal against heavy metal stress. These data suggest that activation of the KGB-1 pathway leads to increased production of these proteins that this in turn leads to protection and defense against heavy metal stress.A key step in understanding the KGB-1 JNK pathway is the identification of downstream targets that are activated by KGB-1 and that perform the actual protective function. Analysis of gene expression comparing wild type and kreg-1 contains polyhistidine stretches, which are well known to bind metal ions  and widely used as an affinity tag Helicobacter pylori, preferentially binds Cu2+ ion and is able to confer copper resistance when expressed in Escherichia coli2+ stress by chelating this ion through these polyhistidine stretches.The identity of one of these genes is particularly revealing: The protein encoded by C. elegans FOS-1 acts as an activator of spermathecal-specific plc-1 gene expression by forming heterodimers with JUN-1 fos-1 and jun-1 as genes important for the KGB-1-mediated defense pathway against pore-forming toxins made by soil bacterium C. elegans FOS-1 binding partner determines whether FOS-1 functions as a repressor or activator.In this study, we identified the FOS-1 bZIP transcription factor as a downstream component of the KGB-1 pathway. FOS-1 was isolated as a protein that binds to KGB-1 and we showed that KGB-1 phosphorylates FOS-1 in the C-terminal regulatory region. Fos and Jun of bZIP transcription factors form part of the AP-1 transcription factor complexes C. elegans ATF-7 and the yeast Sko1p resulting from MAPK activation kgb-1 mutants, but had no effect on the heavy metal sensitivity in wild-type animals. These results strongly suggest that FOS-1 simply acts as a transcriptional repressor of the heavy metal stress response mediated by the KGB-1 pathway. Thus, FOS-1 regulation of the heavy metal stress response does not appear to involve switching of its transcriptional regulation activity.It has been proposed that bZIP transcription factors can switch between repressor and activator mode, as illustrated by the transcriptional regulation of Our analysis showing FOS-1 phosphorylation by KGB-1 and its biological consequences has provided some novel molecular insights into the regulation of FOS-1. We found that phosphorylation blocks FOS-1 dimer formation and that this results in reduced binding to the promoter of target genes. We imagine that dimeric FOS-1 binds DNA with a higher affinity than the monomeric form. Based on these data, we propose that activation of the KGB-1 pathway in response to heavy metal stress results in FOS-1 phosphorylation, leading to a switch of FOS-1 from dimer to monomer and consequent loss of promoter binding .kreg-1 transcription? Our results suggest that the HDA-1 histone deacetylase co-operates with FOS-1 to repress transcription of the kreg-1 gene . The cDNAs for hda-1 and human Grhl2 were amplified by PCR from C. elegans and human cDNA libraries, respectively, and completely sequenced. The mammalian expression constructs for T7-FOS-1, GFP-FLAG-FOS-1, FLAG-HDA-1 and T7-hGrhl2 were constructed by inserting each coding sequence into a vector expressing epitope-tagged protein under the control of the cytomegalovirus (CMV) promoter. Each coding sequence was amplified by PCR using primer sets to create restriction sites immediately before the first codon and after the stop codon. Mutated forms of FOS-1 were made by oligonucleotide-directed PCR and the mutations were verified by DNA sequencing. To construct the Phsp-16::t7::fos-1 plasmids, each t7::fos-1 fragment from the mammalian expression vectors for T7-FOS-1 was subcloned into the pPD49.78 vector. Gateway cloning technology (Invitrogen) was used to construct the Pkreg-1::venus plasmid for expression in animals. The Pkreg-1::venus plasmid was constructed by fusion of the venus coding sequence to a 2.8 kbp genomic fragment containing the kreg-1 promoter. Deletions of Pkreg-1::venus were made by oligonucleotide-directed PCR and the deletions were verified by DNA sequencing. The Pelt-2::mek-1::venus plasmid was constructed by fusing three DNA fragments in the following order: a 2.9 kbp genomic fragment containing the elt-2 promoter, the mek-1 coding sequence, and the venus coding sequence. The Pmek-1::mek-1::venus, Pttx-3::gfp and sur-5::gfp plasmids were described previously The yeast expression vector for the LexA DNA-binding domain (DBD)-fused KGB-1(K67R) was constructed by inserting each coding sequence into pBTM116. The mammalian expression vectors for HA epitope-tagged KGB-1 (HA-KGB-1) and FLAG epitope-tagged MEK-1 (FLAG-MEK-1) were described previously Anti-phospho-FOS-1 rabbit polyclonal antibody was raised against a synthetic phospho-polypeptide, CSNTGL(P)TPSGQP , which corresponds to the C-terminal portion of FOS-1 and affinity purified. Anti-HA monoclonal antibody 16B12 (Covance), anti-FLAG monoclonal antibody M2 (Sigma), anti-T7 monoclonal antibody (Novagen) and anti-GFP polyclonal antibody (Clontech) were used.kgb-1(km21), mek-1(ks54), atf-7(qd22), and eri-1(mg366). Strains carrying the Phsp-16::t7::fos-1 transgene were generated by injecting this DNA together with the sur-5::gfp plasmid, which expresses GFP in the nuclei of most somatic cells from embryogenesis, into the gonads of young adult N2 animals as described Pkreg-1::venus transgene were generated by injecting this DNA together with the Pttx-3::gfp plasmid, which expresses GFP in a pair of AIY interneurons, into the gonads of young adult N2 animals.All strains were maintained on nematode growth medium (NGM) plates at 20°C and fed with bacteria of the OP50 strain, as described fos-1 transgenes on heavy metal toxicity were carried out as follows. Animals were grown and allowed to lay eggs on NGM plates seeded with bacteria of the OP50 strain. Embryos expressing GFP were transferred to NGM plates containing the indicated concentrations of copper sulfate. After incubation for 1 day at 20°C, the numbers of hatched embryos were determined by counting unhatched embryos. After additional incubation for 3 days either at 20°C or 33°C for 1 hour twice a day, the animals that developed into adulthood were counted. The percentage of adults was calculated by multiplying the number of adults by 100 and dividing by the number of hatched animals. The relative viability was estimated by dividing the percentage of adults in the presence of heavy metals by the percentage of adults in the absence of heavy metals.Assays for the effect of fos-1, kreg-1, kreg-2, jun-1 or hda-1. After incubation for 1 day at 20°C, the numbers of hatched embryos were determined by counting unhatched embryos. The animals that developed into adulthood were counted 4 days after egg laying. The relative viability was estimated as described above.Assays for the effect of RNAi on heavy metal toxicity were performed as follows. Animals were grown and allowed to lay eggs on NGM plates seeded with bacteria of the OP50 strain. Embryos were transferred to NGM plates containing the indicated concentrations of copper sulfate and seeded with bacteria of the HT115 strain carrying plasmids expressing the respective double-stranded RNAs for 2O or 1 mM copper sulfate for 1 hour. Total RNA was then prepared using Trizol reagent (Invitrogen), followed by DNase I treatment, phenol/chloroform extraction and ethanol precipitation. RNA was dissolved in water and used as a template for a genome-wide microarray analysis and real-time qRT-PCR. Affymetrix GeneChip microarray processing was performed once by Takara Bio Inc. according to the manufacturer's protocol (Affymetrix). Briefly, total RNA was prepared from wild-type and kgb-1 mutant animals subjected to Cu2+ ion exposure or left untreated (control). Biotinylated cRNA was hybridized to Affymetrix Genechips containing probes against 22,500 transcripts. qRT-PCR was performed with a 7300 real-time RT-PCR system (Applied Biosystems) using SYBR Premix Ex Taq (Takara). A standard curve was generated from diluted RNA derived from wild-type animals, and levels of gene expression were normalized to act-1 expression.Adult worms of each strain were incubated with H2+ ions and in a manner dependent on KGB-1. We selected target genes by the following process  for 1 hour. These animals were then transferred to NGM plates and incubated for 3 hours. The percentages of animals in each expression category are listed. “Weak” refers to animals in which intestinal Venus was present at low levels. “Strong” indicates that Venus was present at high levels in most of the intestine.Wild-type and 8 HEK293 cells, and immunoprecipitated with anti-T7 monoclonal antibodies and protein G magnetic beads (VERITAS) overnight. The immunoprecipitated DNA-histone complexes were incubated overnight at 65°C to reverse cross-linking and then treated with RNase A and protease K. Purified DNA fragments were subjected to quantitative PCR.For phosphatase treatment, cell lysates were incubated with or without calf intestinal alkaline phosphatase (NEB) at 36°C for 5 minutes. Immunoprecipitation from COS-7 cells was carried out as described previously 2, 2 mM EGTA, 10 mM NaF, 2 mM dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 100 units/ml aprotinin, 0.5% Triton X-100. Binding reactions were performed at room temperature for 30 minutes by incubating cell extracts and Cy5.5-labeled retardation probes in binding buffer containing 25 mM Tris (pH7.9), 250 mM KCl, 1 mM EDTA, 5% glycerol, 1 mM dithiothreitol, 0.25 mg/ml BSA, 0.1% Triton X-100 and 0.1 µg/ml of poly(dI)•poly(dC). The samples were analyzed on 3–12% polyacrylamide gels. For supershift experiments, anti-T7 antibodies or normal mouse IgG (Santa Cruz) (1 µg per lane) were added in the binding reactions. The sequences of the gel retardation probes are as follows: TRE2 probe, TGAGTCACAGACAT-3′5′-AATTGC; mutated TRE2 probe, AAGCTTACAGACAT-3′5′-AATTGC; probe deleting the core 6 bases of TRE2, 5′-AAATAATTGCCAGACATTAC-3′. TRE2 and mutated TRE2 are underlined.Transfected COS-7 cells were lysed in lysis buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 12.5 mM β-glycerophosphate, 1.5 mM MgClCaenorhabditis elegans cDNA library in pACTII Saccharomyces cerevisiae reporter strain L40 . Yeast cells were plated onto a synthetic medium plate lacking histidine and containing 3-amino triazole, and allowed to grow at 30°C. Transformants grown on selective medium plates were then streaked on selective medium plates again. Plasmids were collected from colonies that grew on selective medium plates and subjected to DNA sequencing.The LexA DBD-KGB-1 (K67R) plasmid was used as bait to screen the Figure S1FOS-1 is phosphorylated by KGB-1. (A) Phosphorylation of FOS-1 by KGB-1. COS-7 cells were co-transfected with expression vectors encoding T7-FOS-1, HA-KGB-1, and FLAG-MEK-1 as indicated. Whole cell extracts were analyzed by Western blot. (B) Effect of the T304E mutation on FOS-1. COS-7 cells were transfected with expression vectors encoding T7-FOS-1 WT and FOS-1(T304E) as indicated. Whole cell extracts were analyzed by Western blot.(TIF)Click here for additional data file.Figure S2Sites of FOS-1 phosphorylation by KGB-1.  COS-7 cells were co-transfected with expression vectors encoding T7-FOS-1 variants, HA-KGB-1 WT, HA-KGB-1 KN, and FLAG-MEK-1 as indicated. Whole cell extracts were analyzed by Western blot. In the FOS-1 variants, each Ser or Thr residue was replaced with Ala.(TIF)Click here for additional data file.Figure S3fos-1 inhibition on stress sensitivity. Each animal was cultured from embryogenesis on normal plates containing copper sulfate (40 µM) and seeded with a bacteria strain expressing the double-stranded RNA for fos-1. The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors. Error bars indicate 95% confidence interval. **P<0.01 as determined by Student's t test. NS, not significant.Effect of (TIF)Click here for additional data file.Figure S4kreg genes. (A) Flow chart for microarray screening. Comparisons among groups subjected to different treatments are presented. There were 334 genes whose expression was up-regulated >2-fold between Cu2+-treated/non-treated wild-type animals. Of these 334 genes, 66 genes showed >2-fold up-regulation in Cu2+-treated wild-type animals/Cu2+-treated kgb-1 animals. Of these 66 genes, 50 showed increase or no change in non-treated wild-type animals/non-treated kgb-1 animals. (B) qRT-PCR analysis of genes isolated from microarray screen. Wild-type and kgb-1 mutant animals were cultured on plates seeded with a bacteria strain. At 3 days after hatching, animals were treated with copper sulfate (1 mM) for 1 hour and total RNA was isolated. Expression of genes was analyzed by qRT-PCR and six genes were identified as kreg (KGB-1-regulated gene). Data are compared using a one-way ANOVA. *P<0.05, **P<0.01. NS, not significant.Identification of (TIF)Click here for additional data file.Figure S5kreg genes. The eri-1 mutant animals were cultured from embryogenesis on normal plates containing copper sulfate (100 µM) and seeded with bacteria strains expressing the indicated double-stranded RNA. The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors. Error bars indicate 95% confidence interval. **P<0.01 as determined by Student's t test. NS, not significant.Heavy metal sensitivity caused by inhibition of (TIF)Click here for additional data file.Figure S6kreg expression. Wild-type and kgb-1 mutant animals were cultured on plates seeded with a bacteria strain expressing the double-stranded RNA for fos-1. Total RNA was isolated and expression of kreg-1 (A) and kreg-2 (B) was analyzed by qRT-PCR. Data are compared using a one-way ANOVA. **P<0.01.FOS-1 represses (TIF)Click here for additional data file.Figure S7mek-1 in the intestine determines resistance to heavy metal stress. Each animal was cultured from embryogenesis on normal plates containing copper sulfate (100 µM). The relative viability is shown with standard errors. Error bars indicate 95% confidence interval. **P<0.01 as determined by Student's t test.Expression of (TIF)Click here for additional data file.Figure S8kreg-1 expression. Wild-type animals harboring the Pkreg-1::venus transgene as an extrachromosomal array were cultured on plates seeded with a bacteria strain expressing the double-stranded RNA for fos-1, jun-1 or atf-7. “Weak” refers to animals in which intestinal Venus was present at low levels. “Strong” indicates that Venus was present at high levels in most of the intestine. Percentages of animals in each expression category are listed. The numbers (n) of animals examined are shown. (B) Effect of JUN-1 on heavy metal sensitivity. Each animal was cultured from embryogenesis on normal plates containing copper sulfate (40 µM) and seeded with a bacteria strain expressing the double-stranded RNA for jun-1. The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors. Error bars indicate 95% confidence interval. **P<0.01 as determined by Student's t test. NS, not significant. (C) Effect of ATF-7 on heavy metal sensitivity. Each animal was cultured from embryogenesis on normal plates containing copper sulfate (40 µM). The percentages of worms reaching adulthood 4 days after egg laying are shown with standard errors.Effects of JUN-1 and ATF-7 on the KGB-1 pathway. (A) Effect of JUN-1 and ATF-7 on (TIF)Click here for additional data file.Figure S9hda-1 depletion on expression of kreg-1 and kreg-2 genes. Wild-type and kgb-1 mutant animals were cultured on plates seeded with a bacteria strain expressing the double-stranded RNA for hda-1. Total RNA was isolated and expression of kreg-1 (A) and kreg-2 (B) was analyzed by qRT-PCR. Data are compared using a one-way ANOVA. **P<0.01.Effect of (TIF)Click here for additional data file.Table S1C. elegans mixed-stage cDNA library were screened by the yeast two-hybrid method to isolate proteins that interact with KGB-1. From this screen, we identified 10 proteins that interact with KGB-1.List of KGB-1 interacting proteins isolated by yeast two-hybrid screening. A (XLS)Click here for additional data file.Table S22+ treatment. A microarray analysis was carried out to examine gene expression changes in wild-type animals subjected to heavy metal stress. Transcript expression levels were compared between wild-type animals with or without Cu2+ treatment.Comparison of transcript expression levels between wild-type animals with or without Cu(XLSX)Click here for additional data file.Table S3kgb-1 mutant animals with or without Cu2+ treatment. A microarray analysis was carried out to examine gene expression changes in kgb-1 mutant animals subjected to heavy metal stress. Transcript expression levels were compared between kgb-1 mutant animals with or without Cu2+ treatment.Comparison of transcript expression levels between (XLSX)Click here for additional data file.Table S42+ in wild-type animals. A microarray analysis was carried out to examine gene expression changes in wild-type and kgb-1 mutant animals subjected to heavy metal stress. 334 genes were chosen that were up-regulated greater than 2-fold by Cu2+ in wild-type animals.List of genes that were up-regulated by Cu(XLSX)Click here for additional data file.Table S52+ in kgb-1 mutants was <50% of the induction seen in wild-type animals. Among the 334 genes listed in 2+ in kgb-1 mutants was <50% of the induction seen in wild-type animals.List of genes whose induction by Cu(XLSX)Click here for additional data file.Table S6kgb-1 mutants versus wild-type animals. Among the 66 genes listed in kgb-1 mutants versus wild-type animals.List of genes whose basal expression was decreased or not changed in (XLSX)Click here for additional data file.Table S72+ in wild-type animals. Among the 50 genes listed in 2+ in wild-type animals.List of the top thirteen genes whose expression was significantly induced by Cu(XLSX)Click here for additional data file.Table S8kreg genes. We analyzed the 13 genes listed in kreg genes (KGB-1-regulated gene).List of six (XLSX)Click here for additional data file.


Brachyury, which encodes for a sequence-specific transcription factor. Known for decades as a main regulator of notochord formation, Brachyury has recently gained interest as a biomarker and causative agent of chordoma, and therefore as a promising therapeutic target. Here, we review the main characteristics of chordoma, the molecular markers, and the clinical approaches currently available for the early detection and possible treatment of this cancer. In particular, we report on the current knowledge of the role of Brachyury and of its possible mechanisms of action in both notochord formation and chordoma etiogenesis.Chordoma is a rare, but often malignant, bone cancer that preferentially affects the axial skeleton and the skull base. These tumors are both sporadic and hereditary and appear to occur more frequently after the fourth decade of life; however, modern technologies have increased the detection of pediatric chordomas. Chordomas originate from remnants of the notochord, the main embryonic axial structure that precedes the backbone, and share with notochord cells both histological features and the expression of characteristic genes. One such gene is  Chordomas are very rare tumors that affect roughly one in a million individuals; the incidence in the US is ~300 new cases per year . Yet, thChordomas are classified on the basis of their location along the spine and their histological type. Depending on their location, chordomas are predominantly subdivided into clival (or skull-base), sacrococcygeal, cervical, thoracic, and lumbar. Even though historically the sacrococcygeal region was regarded as the most frequently occurring site for the formation of these tumors , recentChordomas rarely occur in people below 40 years old; however, numerous cases of pediatric chordomas have been reported  and genBrachyury gene as a novel diagnostic marker that distinguishes chordomas from similar lesions, such as myoepitheliomas and chondrosarcomas [In addition to the more frequent axial tumors, extra-axial chordomas have also been reported. The location of extra-axial chordomas ranges from wrist  to feet sarcomas , 12. Histologically, chordomas are categorized as classical , chondroid, and dedifferentiated . The fiChondroid chordomas show histological features resembling both chordoma and chondrosarcoma, a malignant tumor of the bone and soft tissue . This hen bloc surgical removal [Since the late 1970s, treatment of chordoma has traditionally relied on  removal ; this te removal –28. Adva removal , 30. Sin removal  and, to  removal , 33.Unfortunately, as slow-growing tumors, chordomas are resistant to conventional chemotherapies , and theBrachyury in the cell line JHC7, derived from primary sacral chordoma, has shown that the downregulation of this transcriptional regulator leads to growth arrest [Brachyury  locus, seen in the MUG-Chor1 cell line, is found in a subset of familial chordomas [The development of chordoma cell lines in the past decade has also greatly widened the knowledge of the chordoma-specific molecular profile and has accelerated the identification of novel therapeutic targets. The first human chordoma cell line, U-CH1, was developed in 2001  and has h arrest . Chromoshordomas  and is dBrachyury in >90% of the chordomas analyzed, and validated its use as a specific marker of this tumor [Brachyury is overexpressed in a number of tumor types, including lung cancer, and is a potent mediator of epithelial-mesenchymal transition (EMT) [As described above, chordomas were classically identified on the basis of their histological features and their immunoreactivity for S-100 and markers such as epithelial membrane antigen (MUC1) and cytokeratins. However, the immunoreactivity for S-100, which is shared among chordomas and chondrosarcomas, made the assignment of tumors to either category quite challenging. This was particularly true when only small biopsies could be obtained, as in the case of clival chordomas . Anotheis tumor , 53, 56.on (EMT) , 58, oneon (EMT) ).RIZ locus, encoding a zinc-finger protein; [HPC1, [TGF-alpha  locus), and 7q33 , 1q25 , 2p13  displayductase; . Other fectively . Of noteectively .Brachyury [Some of the most relevant advancements in the search for the molecular bases of chordoma are the result of systematic studies of families affected with this cancer. The first of these analyses showed genetic linkage to chromosome 7q33 , which wrachyury , 69.Brachyury amplifications in 181 sporadic chordoma samples: 7% of the tumors displayed amplification of the Brachyury locus, 39% of the tumors were polysomic for chromosome 6, and 4.5% of primary tumors showed a minor allelic gain of Brachyury [Brachyury locus was identified in non-neoplastic tissue from 40 patients, indicating that the copy number gain is somatic [Subsequent work analyzed the frequency of rachyury . No relarachyury . Of note somatic .Brachyury are found in both sporadic [in vitro, as shown by the results of the silencing experiments reported above [Brachyury coding region [In sum, these studies demonstrated that chromosomal aberrations resulting in copy number gain of sporadic  and heresporadic . Consisted above . Furtherg region . The resg region . This muBrachyury was already well known as a major regulator of notochord formation. The notochord is the main defining feature of the chordate phylum, which includes humans and all other vertebrates. This embryonic structure provides crucial support to the developing embryo, patterns the central nervous system, and ensures the proper development of a variety of organs, from heart and aorta to liver and pancreas [nuclei pulposi (NP) of the intervertebral discs. The histological identity of these cells remained controversial, since cells of the NP were described as either “chordoid,” that is, notochordal, or as “chondroid,” that is, as chondrocytes [Long before its identification as a marker and a possible cause of chordoma, pancreas . As the drocytes . It has drocytes . Brachyury (Greek for “short tail”) mutation was first identified in 1927 by Nadine Dobrovolskaïa-Zavadskaïa through the observation of short-tailed mice, which were later found to be heterozygous for a mutation in the eponymous locus. Subsequent studies confirmed that the mutation was dominant, attributable to a single factor, and accompanied by abnormalities of the posterior skeleton [Brachyury embryos die in utero by gestation day 11 due to severe defects in the formation of the allantois [The skeleton , particuskeleton . It was llantois . Interesllantois .  Brachyury , which in mouse is part of the t-complex that spans 40 cM on chromosome 17 [Brachyury orthologs in Xenopus, zebrafish and chick [The identification of the geneosome 17 , did notosome 17  and founosome 17 . Three yosome 17 . Parallend chick –86. Of nnd chick , 87.Brachyury orthologs in basal chordates, such as the ascidians Halocynthia and Ciona [Branchiostoma [Brachyury genes predates that of the notochord by several million years. In fact, although Brachyury orthologs have not been found in plants, genes related to Brachyury have been identified recently in the fungus Spizellomyces punctatus and in the amoeba Capsaspora owczarzaki [Brachyury and its related genes as metazoan-specific innovations. Rather, it has become evident that the expansion of the repertoire of genes encoding transcription factors related to Brachyury accompanied the evolution of multicellularity in the animal kingdom. Indeed, Brachyury is the founding member of a family of transcription factors that share related DNA-binding domains and are therefore collectively designated as T-box (Tbx) proteins [Brachyury and other Tbx genes throughout different phyla. For example, single-copy Brachyury and Tbx2/3 orthologs have been reported in the placozoan Trichoplax adhaerens [Brachyury and other Tbx genes have been described in sponges , one of the first metazoan phyla [The discovery of nd Ciona , 89 and hiostoma , and theczarzaki , shiftinproteins . The avadhaerens  and Bracs . Likewian phyla .Brachyury is expressed in scattered individual cells that do not coincide with known anatomical structures [Suberites domuncula, Brachyury is expressed at the time when cell-cell and cell-matrix interactions are being established, suggesting that it might regulate cell adhesion and migration. The ctenophore Mnemiopsis leidyi expressed Brachyury (MlBra) in ectodermal cells around the site of gastrulation as well as in cells deriving from the blastopore; morpholino oligonucleotide-mediated knockdown supports its involvement in gastrulation movements [Nematostella vectensis, a basal cnidarian, expression of Brachyury is restricted to a circle of cells surrounding the blastopore [Placozoan ructures , and itsructures . Consistovements . In the astopore . Drosophila Brachyury, called brachyenteron, is required for the formation of the hindgut and of the midgut constrictions, and for the elongation of Malpighian tubules [Tribolium and the grasshopper Locusta [Drosophila, a remarkable parallelism with the roles played in mesoderm formation by chordate Brachyury and Forkhead (Fox) orthologs [In protostomes, a large division of animals including arthropods, mollusks, annelids, and other less-known taxa, the blastopore is the embryonic region that gives rise to the mouth.  tubules . This fu Locusta . Notablyrthologs , 102.Brachyury orthologs have also been studied in the two main phyla of non-chordate deuterostome animals, echinoderms  and hemichordates . In the sea urchin Lytechinus variegatus, microinjection of a dominant-negative form of its Brachyury ortholog caused a block in gastrulation [Brachyury in hemichordates, it has been shown that in the acorn worm Ptychodera flava, Brachyury is expressed in the blastopore at the time of gastrulation, and later in the stomodeum, the precursor of the mouth and anterior pituitary gland [Lastly, rulation . While try gland .Brachyury was the promotion of cell movement and adhesion, which are fundamental for both morphogenesis and tumorigenesis. As the evolution of multicellular organisms proceeded, this gene became involved in the specification of an area of the blastopore with distinctive properties in axis formation, and later on turned into a driver of mesoderm specification . EventuXenopus Brachyury (Xbra) bound to a 24-nucleotide palindrome and revealed the peculiar interactions between the C-terminus of the Xbra protein and the minor groove of DNA [Drosophila Brachyenteron based on its interaction with the orthopedia gene [orthopedia regulatory region contains multiple copies of this site, which are bound cooperatively by Brachyenteron and are also recognized in vitro by mouse and Xenopus Brachyury [Brachyury has been reported to act as a transcriptional activator, although it possesses two repression domains in its C-terminal region . Crystale of DNA . Subseque of DNA . In facte of DNA , have bee of DNA –111). A dia gene . The ortrachyury .Xenopus and the invertebrate chordate Ciona intestinalis and, more recently, in zebrafish, mouse stem cells, and chordoma. As the knowledge of Brachyury binding preferences continued to accumulate, the quest to identify its downstream genes gained momentum and led to the discovery of numerous Brachyury targets in chordates, but also in nonchordate model systems, such as the sea urchin . In chorXenopus led to the identification of five putative targets of Xbra: the signaling molecule Xwnt11, the zinc-finger transcription factor Xegr-1, a member of the BTG/Tob family of antiproliferative proteins, Xbtg1, a protein of unknown function, BIG3/1A11, and the homeodomain transcription factor Bix1 [eFGF to regulate its expression [Studies in tor Bix1 , 115. Fotor Bix1 . Additiopression , 117. ToCiona intestinalis, since in this and other tunicates the expression of Brachyury (Ci-Bra) is confined to the notochord [Xenopus, most of the validated notochord Ci-Bra targets encode various structural proteins, including a non-muscle tropomyosin, fibrillar collagen, laminins, and thrombospondin [Ciona embryos is close to 2,000 [NFAT5 was found to be expressed in the NP, where it is required for the survival of the notochord cells in response to hyperosmotic stress [The identity of numerous notochord-specific targets of Brachyury was later revealed by studies in otochord . A subtrotochord , 118–121ospondin , 123; hoospondin , 122 andto 2,000 . In addito 2,000 . Of notec stress . Brachyury ortholog, no tail (ntl), has been found to control the expression of numerous target genes, including transcription factors as well as genes that participate in signaling and differentiation pathways [wnt11, snail1a, connexin 43.3, and tbx16), muscle specification , and generation of posterior identity  [floating head, the ortholog of the mammalian homeobox transcription factor gene Noto, which is required for the formation of the posterior notochord [In zebrafish, a pathways . These gx genes) . Among totochord . Putative target genes of mouse Brachyury, 396 in total, have been recently identified in mouse differentiating stem cells . SimilarCiona, zebrafish and mouse, is the gene Snail  [Snail  encodes a transcriptional repressor that, along with transcription factors of the ZEB, Slug, and Twist families, is a mediator of EMT [Xenopus and zebrafish [FGF8 promoter region [H. roretzi, the FGF/MEK/MAPK/Ets signaling pathway is required for the initial expression of Hr-Bra through the binding of Ets to the 5′-flanking region of this gene [Ciona embryos, FGF9/16/20 promotes the activation of Ci-Bra expression, while subsequently FGF8/17/18, together with FGF9/16/20, ensures its maintenance [It is noteworthy that one of the recurring targets of Brachyury in different animals, including e Snail  , 129. Snr of EMT , 131. Thebrafish , 133 andhis gene . Similarntenance .laminin alpha2, collagen type VI alpha3, and olfactomedin 4 [The identity of Brachyury target genes in humans has been recently revealed by transcriptome and ChIP-Seq analyses in the chordoma cell line U-CH1 . This stomedin 4 . Lastly,omedin 4 . Somewhat surprisingly, a preliminary comparison of the Brachyury targets identified in mouse differentiating stem cells with those found in chordoma does not reveal numerous commonalities. Among the shared Brachyury targets are the Ets-family transcription factor ETV1, which is translocated in certain sarcomas , CIT  and forkhead (fkh) cooperatively specify the caudal visceral mesoderm, which is absent in byn/fkh double mutant flies and is formed ectopically only when both genes are over-expressed [Brachyury and Foxa2  have been reported to be upstream of the homeobox gene Noto, based upon the evidence that this gene is down-regulated in mutant embryos for either gene [cis-regulatory module whose activity depends on both Ci-Bra and Ci-FoxA2 binding sites, thus providing the first report of a synergistic interaction between these factors within a compact regulatory region [Even though a physical interaction between Brachyury and members of the Fox subfamily of winged-helix transcription factors has not been reported, it is noteworthy that these transcription factors have been found to work synergistically in different model systems. For example, in otochord . In Drosxpressed . In the her gene . Finallyy region .Together these studies suggest that the various interactions of Brachyury with other proteins exert a considerable influence on its function, suggesting that their impairment could be both a cause and a consequence of tumorigenesis.Brachyury locus as a hallmark of hereditary chordoma. It is important to note, however, that duplications of Brachyury have occurred in various species along the phylogenetic tree without negative consequences. For example, two Brachyury genes have been identified in the cnidarian Hydra vulgaris. Both genes are expressed in the hypostome, although HyBra1 is expressed predominantly in endoderm, and HyBra2 in ectoderm [Xenopus animal cap assays, only HyBra1 is capable of inducing mesoderm formation, similarly to vertebrate Brachyury proteins [Brachyury in Ciona and other tunicates [Brachyury orthologs have been found in zebrafish, the well-characterized no tail, and its paralog Brachyury ; combined inactivation of these genes exacerbates the ntl-a mesodermal phenotype, indicating that these factors act synergistically [Brachyury gene, Brachyury the Second (T2), has been found and mapped to the proximal region of chromosome 17 near Brachyury; a mutation in this gene reportedly causes defects in notochord formation, although not as severe as those seen in Brachyury mutants [T2 does not seem to be present in the human genome.We have previously discussed the duplication of the ectoderm . In Xenoproteins . In basaunicates , 135, whunicates ; both amunicates . In vertstically . Also, i mutants . T2 doesBrachyury genes can become functional paralogs, and either segregate into different embryonic territories or continue to be expressed within the same tissue; in the latter case, they may act synergistically and likely control shared target genes. Nevertheless, this partial or complete functional redundancy is expected to require strict tissue-specific regulation in order to prevent the damage that an excessive level of Brachyury protein could cause. In fact, the increased dosage of Brachyury has been shown to exert strong effects on development, as seen in transgenic mouse embryos where three copies of this gene resulted in an extension of the anterior-posterior axis [cis-regulatory regions of its target genes. An example of this mechanism has been documented in the case of the Drosophila gene orthopedia that, as mentioned above, is controlled in a dose-dependent fashion by Brachyenteron through multiple binding sites [Therefore, it appears that duplicated copies of ior axis . The maing sites . It has ng sites . In concBrachyury itself using different model systems. The emerging consensus is that the essential signaling pathways that govern body plan formation and mesoderm induction, Wnt/β-catenin, TGF-β/Nodal/activin, BMP, and FGF, also act upstream of Brachyury in concert with different combinations of early-onset transcription factors. The involvement and cross-interactions of each of these pathways vary among different animals. In zebrafish, a Nodal morphogen gradient activates expression of both Brachyury orthologs, ntl-a and ntl-b; however, transcription of ntl-a is additionally regulated by both Wnt and BMP signaling, while transcription of ntl-b is not responsive to these pathways [Xbra in Xenopus [Brachyury transcription, being able to induce its expression in hES cells; the same study showed that activin-A and Wnt3a also exhibited minor inducing ability [While the identities of Brachyury-downstream genes continue to be elucidated, in parallel, several groups have studied the regulation of the expression of pathways . The BMP Xenopus , 164. In ability .Brachyury expression are observed between different regions of the notochord in the same animal. For example, in the ascidian Ciona, the first step leading to the activation of Brachyury expression in the main notochord lineage  is the translocation of cytoplasmic β-catenin to the nuclei in vegetal blastomeres, an event that activates transcription of FGF9/16/20 and of the transcription factor FoxD [Brachyury notochord CRM and activates transcription [Brachyury expression is instead regulated by Nodal and Delta-like [In some instances, differences in the regulation of tor FoxD . In turncription . Howeverlta-like . Brachyury overall appear to be conserved across the phylogenetic tree, the transcription factors that control expression of this gene vary among different organisms. In Ciona, Ci-FoxA2 has also been reported to activate Brachyury expression [Foxa2 precedes that of Brachyury genes. The finding that the mouse Foxa2 promoter region is bound by Brachyury suggests that the two genes might be regulating each other [ntl-a in zebrafish and of Xbra in Xenopus [Although the signaling pathways upstream of pression , and thich other . In addi Xenopus . Further Xenopus , and ChI Xenopus , althoug Xenopus . Brachyury is also modulated by repressive interactions. In Ciona, expression of Ci-Bra is excluded from muscle cells, and thus confined to the notochord, by the zinc-finger transcriptional repressor Snail [Xenopus, Xbra is actively silenced by another zinc-finger repressor, Smad-interacting protein (SIP1) [Xenopus, Xbra and the homeodomain protein goosecoid repress each other, while transcription of both genes is stimulated by activin [Brachyury is repressed in goosecoid-overexpressing mouse ES cells [Brachyury promoter and causes histone deacetylation, thus impeding transcription [Transcription of or Snail ; in Xenon (SIP1) . Also in activin . This inES cells . Represscription .Brachyury, along with the ability of these cells to undergo EMT [Brachyury as a possible therapeutic target. Within this context, the discovery of the immunogenic properties of Brachyury and the subsequent development of specific CD8+ cytotoxic T-cells able to destroy Brachyury-expressing tumor cells from various cells lines in vitro [From the studies that we have summarized above, it seems that Brachyury-expressing cells have the potential to somehow give rise to chordomas, possibly through one or more of the mechanisms that we have outlined. If this is the case, it remains to be determined whether and how normal Brachyury-expressing notochord cells turn into the Brachyury-expressing cells found in chordomas. Possible clues to the solution of this problem might come from a recent gene expression study showing that chordomas contain cancer stem-like cells . In thisergo EMT . Taken tin vitro , 180 repin vitro . Brachyuin vitro . Lastly,


The third international conference on the genomic impact of eukaryotic transposable elements (TEs) was held 24 to 28 February 2012 at the Asilomar Conference Center, Pacific Grove, CA, USA. Sponsored in part by the National Institutes of Health grant 5 P41 LM006252, the goal of the conference was to bring together researchers from around the world who study the impact and mechanisms of TEs using multiple computational and experimental approaches. The meeting drew close to 170 attendees and included invited floor presentations on the biology of TEs and their genomic impact, as well as numerous talks contributed by young scientists. The workshop talks were devoted to computational analysis of TEs with additional time for discussion of unresolved issues. Also, there was ample opportunity for poster presentations and informal evening discussions. The success of the meeting reflects the important role of Repbase in comparative genomic studies, and emphasizes the need for close interactions between experimental and computational biologists in the years to come. The diversity of topics and focal areas presented at the 2012 Asilomar conference on the “Genomic Impact of Eukaryotic Transposable Elements” was remarkable. Overall the conference had a variety of foci ranging from the biology of transposable elements (TEs) to how the host responds to their impact upon the genome. In addition, a number of valuable new tools were presented that will aid in the detection and characterization of transposons within sequenced genomes. The organizer’s decision to give much stage presence to junior scientists was commendable. These talks impressed the audience and were considered among the best and most exciting contributions at the meeting.It is impossible to begin this conference report without paying homage to the towering figure of Roy John Britten, who passed away at the age of 92 on 21 January 2012. Indeed, the conference started with a personal homage to Roy Britten’s life by the organizer, Jerzy Jurka. Roy’s life and scientific contributions inspired many generations of scientists and his work transcended many fields. Roy was trained as a physicist and began his post-doctoral work as a staff member in the Department of Terrestrial Magnetism at the Carnegie Institution of Washington. There he exploited DNA hybridization to make the groundbreaking discovery of eukaryotic repetitive sequences [As DNA sequences became more available in the 1980s, Roy began systematic sequence studies of repetitive DNA and served as an inspiration to generations of younger scientists. The relationship between repetitive sequences and TEs, originally discovered by Barbara McClintock, was not yet clear. Notably, Roy contributed to the discovery of human Alu subfamilies, which led to the concept of source/master genes equivalent to active TEs .The role of TEs in evolution remained Roy’s lifelong interest until his last paper in 2010 documenting more recent activity of Alu elements in the human genome when compared to other primate genomes . A year Escherichia coli and of McClintock on maize ‘controlling elements’. One of the pivotal ideas in the Britten-Davidson model was that interspersed repeats could distribute the same cis-regulatory elements to a ‘battery’ of genes scattered throughout the genome, allowing for the coordinated control of gene expression.In 1969, Roy Britten and Eric Davidson proposed an influential hypothesis for gene regulation in ‘higher cells’. The model merged concepts drawn from the seminal discovery that diverse eukaryotic genomes are replete with interspersed repetitive DNA  with thecis-regulatory elements, at least in mammalian genomes. The role of mobile elements in regulatory evolution is less clear in other organisms, but recent studies in Drosophila presented by Nikolai Tchurikov  and by Josefa Gonzales  suggest that mobile elements have contributed adaptive regulatory mechanisms; however, these impacts may be harder to pinpoint because of the volatile nature of mobile element sequences in these genomes.More than 40 years have elapsed since the formulation of the Britten-Davidson hypothesis and several talks at the meeting presented data in support of this prescient model. David Haussler  presented data suggesting that the co-option of mobile elements for regulatory function is an ancient and pervasive phenomenon in the history of vertebrate genomes that has contributed innovations at various levels of cell signaling at different times during vertebrate evolution. Nori Okada  discussed how a series of ancient exaptation events of multiple members of the AmnSINE1 family have contributed to the emergence of novel enhancers that control the expression of genes implicated in the development of brain, sensory, and craniofacial features unique to mammals. Okada argued that these exaptations were key to mammalian survival in a well-documented mass extinction at the Permian-Triassic boundary. Intriguingly, recent studies by Gill Bejerano  also pointed to a dramatic enrichment of exapted AmnSINE1 family members near genes involved in mammalian corticogenesis. Petra Schwalie  also presented data consistent with the Britten-Davidson model. She showed that several waves of SINE amplification at different times during mammalian evolution have dispersed thousands of binding sites for the insulator protein CTCF, a major organizer of genome architecture and regulation. Likewise, King Jordan  presented evidence that MIRs, an ancient class of mammalian SINEs, commonly function as insulator elements in the human genome through a mechanism that likely is independent of CTCF binding. These exciting studies yield growing support to the visionary idea of Britten and Davidson that mobile elements have been a profuse source of new Another facet of mobile element exaptation is the ‘domestication’ of their gene product(s). Several classic and novel examples of this type of exaptation were discussed at the meeting. Jürgen Brosius  described the startling behavioral phenotypes of mice knockouts for BC1, a tRNA-derived retrogene, which is unusual because it has continued to give rise to new retroposition events long after its exaptation in a rodent ancestor. Lucas Gray  presented new functional data on the primate-specific PGBD3-CSB protein, which arose by fusion of a transposase to the Cockayne Syndrome B gene. He showed that the fusion protein has transcriptional activator properties and is tethered to hundreds of binding sites in the human genome derived from a related transposon family.Not only retroposition-derived elements (mostly parts thereof) but also DNA transposons propagating in the “cut and paste” mode can contribute to exaptations. One of the most notable examples is the transposase-derived RAG recombinase, which was instrumental in the evolution of the adaptive immune system in jawed vertebrates . Nancy CAs documented above, TEs also can contribute to the proteome of a cell. Casimir Bamberger  used mass spectrometry to examine which of the many TEs contribute to the proteome. For example, he demonstrated that loss of the Piwi-like protein nvPiwil1 and UV irradiation in the starlet sea anemone induces translation of open reading frames with sequence similarity to TE sequences. Certain forms of stress also recruited TE proteins to the chromatin-associated proteome in human cells. Clearly, how TEs contribute to the proteome is an area for further exploration in the future.Despite these interesting cases of exaptations, we need to be mindful that TEs are first and foremost a tremendous mutational force. Several talks at the meeting illustrated the dazzling array of effects that TE insertions can have on the structure and expression of the genome and the deleterious consequences TE insertions can have on host genomes.David Symer  used targeted deep sequencing to map endogenous retrovirus (ERV) insertions in the genomes of highly divergent mouse strains. Polymorphic insertions of young ERVs are relatively depleted from gene introns and especially from genes that are highly expressed in stem cells or are involved in development. These findings suggest that the ERVs are likely to have a deleterious impact on gene expression. Indeed, Symer described several cases (and perhaps at up to 100 or more genes across the genome) where intronic ERVs strongly trigger premature transcriptional termination at distances up to >12.5 kb upstream of the ERV elements. In heterozygous animals, the parent of origin of the ERV influences expression levels of the non-terminated gene transcripts, suggesting that the impact of ERVs upon certain transcripts can be regulated by epigenetic controls.The subsequent talk by Dixie Mager  examined the distribution of ERVs and other TEs within human and mouse introns. These analyses allowed predictions of what types of intronic TE insertions are most likely to be deleterious. Mager showed the following factors influence the mutational spectrum of TE insertions: a bias against proximity to splice sites, full-length versus truncated TEs, and TE orientation. Genes with extremely low or high TE densities were also highlighted in the presentation, and evidence was presented that actively transcribed genes in embryonic stem cells are more likely to be rich in TEs. These data suggest that the signature of initial insertion site preference may still exist for mammalian TEs.Another major contribution discussed how mobile elements generate structural variation post-insertionally, through homologous recombination. Prescott Deininger  presented analyses from his laboratory based upon cell culture assays demonstrating the potential that mobile elements have to serve as scaffolds for homologous recombination events. Deininger emphasized the roles that sequence identity and cellular factors play in modulating the rate of recombination. For example, longer regions of sequence identity tend to promote more efficient recombination events between Alu elements. The combination of insertional mutagenesis by mobile elements, as well as the potential for post-insertion recombination, represents a powerful force acting to alter the genomic landscape of eukaryotic genomes.Ben Koop  presented analyses of the repeat element content of Salmonids. Salmonids represent rapidly evolving species complexes that are thought to be pseudotetraploid, though they may be in the process of reverting to a stable diploid state. The pseudotetraploid state presents a number of opportunities for evaluating the contribution of various repeat families to the overall structure and instability of the genome, as well as for the determination of the contribution of repeat sequences to speciation within the Salmonid species complex. The pseudotetraploid genome state, along with relatively high levels of repeated sequences, presents significant challenges for the accurate sequence assembly of the salmon genome sequence.Due to the potentially harmful effects of TE acquisition and expansion, hosts have evolved mechanisms such as DNA methylation of TEs to control their “virulence” in the genome. Apparently, modes other than DNA methylation, such as covalent histone modification, play a key role in TE silencing. Matthew Lorincz  reported that several subclasses of ERVs are maintained in a silent state in mouse embryonic stem cells by histone H3 lysine 9 methyltransferase Setdb1/Eset. Interestingly, this transcriptional silencing is independent of DNA methylation status. In addition, Lorincz discussed RNAseq-based transcriptome analyses in mouse embryonic stem cells to identify the novel “chimaeric” genic transcripts that initiate in long terminal repeat (LTR) promoters and splice to genic exons.To document the interplay between host defense mechanisms and the activity of retroposons, Michael Wilson  studied a model in which human chromosome 21 had been introduced into the mouse. This chromosome contains TEs that arose and spread in primates after they diverged from a common ancestor with rodents. Many repetitive elements on human chromosome 21 were hypomethylated at the DNA level and were aberrantly associated with histones harboring activating modifications in somatic and germline tissues. Hence, many dormant promoters, transcription factor binding sites, and insulators that are silenced in the human genomic background, are activated in the new genomic environment. It will be interesting to see, in long-term studies, whether some of the elements (in the case that master copies reside on human chromosome 21) spread to other mouse chromosomes and whether these elements ultimately become silenced in the mouse genome.Erez Levanon  demonstrated that certain retrotransposons are likely to be frequent targets of editing by the APOBEC3 family of cytidine deaminases. The APOBEC3 protein family, by introducing C-to-U mutations in (-) strand cDNA  strand of the retrotransposed cDNA), is thought to inactivate viruses and virus-like sequences by hyper-mutation. Thus, he posited that APOBEC3-dependent “DNA editing” may provide a potential mechanism for retrotransposon domestication.Drosophila virilis. Contrary to earlier findings, he concluded that all chromosomes could possibly contribute to maternal protection against TE-mediated hybrid sterility. He also highlighted certain aspects of evolution of piRNA machinery in members of the genus Drosophila with varying TE content, reporting that these fast-evolving genes can develop codon biases that favor the increase in translational efficiency with the increasing transposon load, thereby avoiding titration of the silencing machinery by increasing piRNA quantities. Erin Kelleher  compared piRNA abundance in D. melanogaster, D. simulans, and their respective inter-specific hybrids. While the difference between piRNA pools in these species was evident, there was no correlation between maternal deposition and TE silencing in hybrid offspring. Rather, she observed global de-repression of TEs originating from either parent, presumably due to the failure of species-specific piRNA machinery to adapt to novel environments. Fernando Rodriguez  reported an increase in relative abundance of pi-like RNAs in bdelloid rotifers subjected to a dose of ionizing radiation that would cause hundreds of DNA double strand breaks per genome. These small RNAs predominantly mapped to low copy number TEs, were mostly recovered in anti-sense orientation, and exhibited strong 5’-uridine bias.Several reports focused on the role of small RNAs as central players in TE silencing. Andrea Schorn  investigated the role of small RNA-induced TE repression in embryonic and extra-embryonic tissues of mice. In embryonic and trophectoderm stem cells, 17-31nt RNA profiling revealed an increase in small RNA production for relatively young TE families, such as IAP, ERV-K, and ERV-L endogenous retroviruses. Small RNA levels were correlated with an abundance of bidirectional transcripts, but not with TE methylation status. Such small RNA-mediated post-transcriptional silencing may be driven by the necessity to suppress expression of active TEs in embryos, and to avoid embryo re-infection with retrovirus-like elements from the surrounding somatic tissues. Justin Blumenstiel  presented an overview of the contribution of maternally transmitted piRNA to hybrid dysgenesis in MuDR DNA transposon can be mediated by small RNAs. When SGS3, a key component of a silencing pathway required for the production of trans-acting RNAs (tasiRNAs), is downregulated, it leads to loss of transcriptional silencing due to absence of DNA methylation. In maize, SGS3 downregulation occurs in transition leaves just as the plant is preparing to enter reproductive growth, suggesting a connection between vegetative phase transition in plants and epigenetic regulation of TEs. Lisch has also demonstrated that maize genes can be co-opted into the tasiRNA-silencing pathway following insertion of TEs, resulting in a high level of plasticity with respect to gene regulation.In plants, Damon Lisch  demonstrated that silencing of the autonomous maize Penelope-like elements (PLEs) and may play a role in telomere maintenance, and another that belongs to a previously unknown class named rvt genes, found in a wide but patchy collection of organisms. Eugene Gladyshev  described his initial biochemical and genetic characterization of the RVT protein of Neurospora crassa, an active representative of this mysterious group of reverse transcriptase-related cellular genes. The N. crassa RVT protein is non-essential for growth, but is dramatically upregulated when protein synthesis is inhibited in vivo. The RVT protein also exhibits robust terminal transferase activity in vitro. Ken Kraaijeveld  further explored the theme of asexuality and its influence on TE content. He used next-generation Illumina sequencing to compare TE abundance in genetically distinct sexual and asexual lineages of the parasitoid wasp Leptopilina clavipes. Asexuality in this wasp was recently induced by Wolbachia infection, which may have led to an increase in copy numbers for several DNA TEs and one gypsy-like LTR TE. However, despite the presence of the Dnmt1 gene in L. clavipes, gypsy methylation was not observed in either lineage. Jens Bast  used a similar approach for comparisons between four taxa of oribatid mites, two of which are sexual and two are long-term parthenogens. In this case, long-term asexuals appeared to display reduced TE loads in comparison with sexuals. Finally, Sarah Schaack  argued that there are several opposing forces that may cause either decreases or increases in TE copy numbers in sexually and asexually reproducing populations of the cyclically parthenogenetic crustacean, Daphnia pulex. She further stressed that it may often be difficult to discriminate between forces exerting the most influence. On balance, recently developed asexuality may not allow sufficient time to affect TE content significantly, while long-term asexuality may be expected to leave its imprint on TE diversity and abundance.It has long been thought that the reproductive mode of an organism can influence its TE content, which has stimulated comparisons between sexually and asexually reproducing species. Irina Arkhipova  described a multilayered system of genome defense against TEs in asexual bdelloid rotifers. She demonstrated that these defenses include, but are not limited to, small RNA-induced TE silencing, deletion via homologous recombination and microhomology-mediated end-joining, and insertion of asparagine-rich segments within coding sequences of certain TEs. At the same time, bdelloid genomes harbor at least two types of single-copy reverse transcriptase-related genes, one that belongs to D. melanogaster and D. simulans, which are present in the latter species in much smaller copy numbers. She argued that the levels of variation in piRNA machinery could contribute substantially to the observed variation in TE copy number. The accelerating speed of accumulation of next-generation sequencing data will undoubtedly dominate population genetic studies in the near future.The population genetics session was opened by Dmitri Petrov  who presented an overview of Drosophila TE dynamics in natural populations and the major forces governing the distribution and behavior of 1824 diverse TEs in genomic sequences of 158 individual or pooled Drosophila strains. In agreement with earlier findings, most TEs were found at low population frequencies and were largely subject to purifying selection against deleterious products of ectopic recombination, rather than against deleterious effects on neighboring genes or against over-expression of TE-encoded products. Cristina Vieira  addressed the problem of TE control in natural populations by studying germline piRNA-mediated silencing of endogenous Tirant retroviruses in One of the major themes of the meeting was in the characterization of the various ways that transposons impact the genome. Individual transposons serve as insertional mutagens in both the germline and somatic tissues, but they can also serve as additional sources to generate structural genetic variation through post-insertion recombination based processes. Kyle Upton  presented a new approach to identify newly integrated somatic retrotransposable element insertions called “retrotransposon capture sequencing”. Using this approach, thousands of somatic L1 and Alu element insertions have been identified in neuronal tissues. These data suggest the overall importance of transposons in creating somatic structural variation. Scott Devine  discussed the amount of TE-related variation that can be obtained using high throughput second generation sequencing approaches. He reported on the movement of transposons in certain lung tumors, suggestive once again of the contribution that these elements make to somatic mutagenesis. In addition, Devine discussed ongoing efforts to make risk estimates for L1-based mutagenesis on the basis of the number of “hot” L1 drivers, expanding upon some of the initial pioneering research on allelic variation in retrotransposition performed in the Kazazian and Moran laboratories. Adam Ewing  reported the use of sequence comparisons involving normal human tissue with a large number of different tumors to detect hundreds of novel TE integrations in somatic cells. To this end, existing algorithms were supplemented with novel computational approaches and applied to The Cancer Genome Atlas whole-genome datasets. Hopefully, this information will not only yield insights with respect to TE expansion in somatic tissue, but also might be relevant for understanding insertional changes leading to, and accompanying, the clonal evolution of tumor cell populations. Mark Batzer  also discussed the overall contribution of mobile elements to genomic diversity in the human 1000 genomes project. Thousands of new mobile element insertion polymorphisms have been identified in the pilot phase of these studies. Sue Wessler  presented the results from second-generation analyses of rice genomes. She reported on the burst of MITE movement that occurred and the levels of associated insertion polymorphism.All the above studies present new high throughput approaches for the detection of newly inserted transposons. As more of the studies are completed, the mode and tempo of transposon proliferation in different tissues within an individual and in different eukaryotes will be elucidated. These studies truly represent insights into TE mobilization on a previously unprecedented scale.A series of talks presented the next frontier for the computational identification of TEs. At the core are still pipelines or networks that reliably recognize and characterize transposed elements provided by well-founded tools such as RepeatMasker and RepBase. However, it is important to note that these tools are not static entities, but rather are adaptive components in need of regular improvement to complement the emerging requirements of growing genome information. Arian Smit  and Travis Wheeler  presented a novel approach to detect highly diverged ancient TEs using hidden Markov models (HMMs) implemented in the software package HMMER3. This probabilistic inference technology was originally introduced for protein searches. The reconstruction of ancestral genomes and the consideration of genomes with low substitution rates, such as the crocodile genome , are ideal additional strategies for digging even deeper into the past of retroposed elements. As a predictor, HMMER surpasses the performance of the RepeatMasker-implemented, single-sequence homology search tools cross_match and blastn. Smit demonstrated the potential utility of this approach by detecting more and more complete highly diverged test “ghost” repeats. David Pollock  then briefly discussed an alternative approach for the identification of all repeated DNA sequences using “P-clouds”, and presented a refined alternative to the “master gene” model to explain the expansion of some families of transposons. A prediction with the aid of P-clouds suggested that more than two thirds of the human genome is derived from TEs! Overall, these talks provide exciting new computational approaches for detection and analysis of repeated DNA sequences.All known “cut-and-paste” DNA transposons encode only one to two conserved proteins. On the other hand, “self-synthesizing” Polintons encode up to eight conserved proteins. It is believed that such complexity is due to evolution of Polintons from DNA viruses. Vladimir Kapitonov , reported two groups of fungal DNA transposons, Entons and Intons, that encode five to six conserved proteins and evolved from their En/Spm and IS3EU “cut-and-paste” ancestors >100 million years ago. Therefore, some complex DNA viruses might have evolved from simple DNA transposons through explosion of their complexity. He also presented diverse DNA transposons from different super-families that harbor conserved proteins acquired from a wide range of host proteins, suggesting that the latter are involved in regulation of transposition.Retrotransposed elements accumulate over millions or hundreds of millions of years and encrypt valuable information about the evolution of their organism. Some eventually might influence the fitness of the species harboring them, but most are neutral hitchhikers throughout the persisting stream of life.Symphalangus syndactylus) at the base of the gibbon phylogeny.Mark Batzer  reported on several thousand such young, polymorphic hitchhikers his group discovered at the species level within the framework of the human 1000 Genomes Project and compared these insertion polymorphisms to other mobile element insertion polymorphisms previously uncovered from PCR display studies. From steps deeper along the tree of primates, he used mobile element insertions as clade markers to understand the early evolution of gibbons, surprisingly placing the Siamang  and Annette Damert  discussed their studies of a newly identified family of composite non-autonomous non-LTR retrotransposons in the gibbon genome termed LAVA elements. Carbone demonstrated that LAVA elements are involved in centromere related remodeling in Hoolock gibbons. The expansion of LAVA elements within the gibbon genome is a hallmark of the increased genomic plasticity in the gibbon lineage and contributes to the enhanced structural variation within the gibbon lineage. Damert discussed her studies of LAVA, PVA, and FVA elements. These elements share structural features with SVA elements . Interestingly, PVA and FVA have acquired additional sequences at their 3’ ends by RNA splicing (derived from exon 4 and part of intron 4 of PTGR2 and from a free right Alu monomer (FRAM)). It will be interesting to develop a cell-based assay to assess whether LAVA elements retrotranspose in cultured cells and how the variant 3’ ends of PVA and FVA elements influence retrotransposition.Jürgen Schmitz  presented retroposon-based reconstructions of 160 million-year-old phylogenies that are close to the boundary of resolution. He illustrated a range of phylogenetically informative retrotransposon presence/absence patterns in mammals and birds as well as the advantage of such a marker system to distinguish sequence divergences and ancient polymorphisms. Retrotransposons not only encrypt information about species phylogeny, but also indirectly convey information about their biogeography, sex determination, or brain evolution. He discussed how using transposed elements as phylogenetic markers and for other reconstructions requires very careful comparative evaluations of presence/absence loci, multidirectional screening strategies, and stringent statistical evaluations of the results.Andrew Shedlock  provided the first glimpse of new genomic information and molecular evolution of TEs in the Anolis lizards and painted turtle genomes compared to other large-scale reptilian genome sequences and their mammalian sister groups. He demonstrated that having such comparative reptile genome information is essential to understand the retrotransposon landscape and distribution signals in amniotes and in vertebrates generally.In the age of genomics, reliable automated tools to explore the depths of species evolution are indispensable. Wojciech Makalowski  presented new automated approaches, including the transposition in transposition (TinT) strategy to reliably derive the historical succession and activity patterns of retrotransposon families and the TEclass program to classify such elements. He also discussed different approaches for the detection and biological analyses of TEs with emphasis on pipelines that enable a streamlined evaluation of the historical content of retrotransposable elements.With the exception of monotremes, the retrotransposon landscape of mammals is significantly shaped by LINE-1 and associated co-mobilized elements. Holly Wichman  illustrated the importance of LINE-1 retroposition and described its potential functional role in genome integrity. Interestingly, a screen of over 200 mammalian species from almost all eutherian and marsupial clades revealed the extinction of LINE-1 elements in only a few cases. Such examples are exciting scenarios for understanding a future without active LINE-1 elements.Several talks discussed mechanistic aspects of TE mobility. John Moran  built on previously published findings, which demonstrated that endonuclease-deficient LINE-1 elements could use dysfunctional telomeres as integration substrates in Chinese hamster ovary cells lacking a protein important for the non-homologous end-joining pathway of DNA repair ). He demonstrated that LINE-1 ribonucleoprotein (RNP) preparations could use oligonucleotides that mimic the G-rich overhang present at telomeric ends as substrates to initiate LINE-1 mRNA reverse transcription. These RNP preparations also contain a nuclease activity that could process oligonucleotide adapters before being used to prime LINE-1 mRNA reverse transcription. Whether the nuclease activity is attributed to the LINE-1 encoded proteins or an associated cellular protein remains an open question. However, these findings further highlight mechanistic similarities between an alternative endonuclease-independent pathway of LINE-1 retrotransposition and telomerase action.Haig Kazazian  discussed recent efforts, which use photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP), to identify cellular RNAs that interact with LINE-1 ORF1p in HEK293T cells. He found that ORF1p binds unstructured regions in non-autonomous retrotransposons (SVA and Alu RNA), as well as the 3’-UTR and last exons of a wide variety of cellular mRNAs. It will be interesting to examine whether low concentrations of ORF1p binding are required for Alu and/or SVA retrotransposition, and if ORF1p binding to cellular mRNAs is correlated with processed pseudogene formation.trans to promote the faithful retrotransposition of genetically tagged (SINE-VNTR-Alu) SVA elements in cultured human HeLa cells. Similar work also was reported from the Kazazian lab. Interestingly, members of the human-specific SVAF1 subfamily, which acquired a 5’ MAST2 sequence by RNA splicing and a 3’ AluSp sequence by 3’ transduction, are trans-mobilized by the L1 protein machinery approximately 25-fold more efficiently than the parental element lacking the AluSp sequence. Clearly, this retrotransposition assay should allow the rapid identification of cis-acting sequences within SVA elements that are important for its transcription and retrotransposition.Gerald Schumann  showed that the human LINE-1 encoded proteins (ORF1p and ORF2p) could act in cis-preferential binding of ORF1p to LINE-1 mRNA.Anthony Furano  discussed recent biochemical experiments using recombinant human LINE-1 ORF1p. First, he showed that, in the absence of nucleic acids, ORF1p polymerizes in the very low salt conditions that are optimal for nucleic acid binding. The ORF1p C-terminus mediates polymerization, and the addition of nucleic acid rapidly allows the ORF1p polymer to resolve into homotrimers. Second, he demonstrated that ORF1p has a biphasic nucleic acid chaperone activity. ORF1p initially recognizes mismatched strands in a duplex as single stranded DNA and protects the duplex from melting. However, when present in the polymerized form, ORF1p subsequently promotes melting of the same DNA substrate. These data have important implications in understanding how ORF1p interacts with nucleic acids. Moreover, the ability of ORF1p to polymerize may prevent it from diffusing into the cytoplasm, providing a plausible mechanism for the Finally, Zuzsanna Izsvak  reported that the heat shock (HS) response machinery of the host is regulating transposition of the DNA transposon Sleeping Beauty (SB). SB harbors conserved transcription factor binding sites for HS factor 1 and 2 (Hsf1/2), which induce transcription of the transposase gene. SB is a vehicle for distribution of HS factor binding sites in the host genome. Furthermore, the transposase protein product, albeit stable, is sensitive to mis-folding; hence, the HS response might activate latent SB integrations and thus trigger transposition.In early evolution, the Darwinian Threshold  was not in place yet, with rampant exchange of genetic material . Even toA similar scenario was also borne out in the talk of Cedric Feschotte , who stressed that horizontal gene transfer not only plays a role in bacterial evolution, but also plays a role in eukaryotic evolution. Two vehicles of horizontal transfer are significant. Transposons are not only moving between loci in individual genomes and then are propagated vertically, but also are crossing species barriers, spawning huge waves of lineage-specific invasions in “naive” hosts. Another route of horizontal transfer is the viral one. Not only the well-known endogenous retroviruses, but almost every major type of virus can be integrated into host genomes. Thus, when jumping species barriers, viral genes offer potential fodder for exaptation.in silico findings. We thank the organizer, Jerzy Jurka, for bringing this community of scientists together to interact and discuss their findings. The meeting was incredibly successful and we eagerly await the next meeting of this highly interactive group of scientists.The series of the Asilomar meetings “Genomic Impact of Eukaryotic Transposable Elements” has earned a distinctive place among other meetings in the field of mobile DNA, such as the FASEB Summer Research Conference “Mobile elements in mammalian genomes”, the ASM Conference on Mobile DNA, and the International Congress on Transposable Elements in France. It attracts researchers from all over the world and brings together computational biologists working on genome annotations and experimental scientists investigating various aspects of transposon biology. Such interactions serve as a catalyst for new ideas and lead to new discoveries both by theoreticians analyzing vast amounts of experimental data and by experimentalists seeking to uncover and validate novel biological phenomena emerging from ERV: endogenous retrovirus; HMM: hidden Markov model; HS: heat shock; LTR: long terminal repeat; RNP: ribonucleoprotein; SB: Sleeping Beauty; tasiRNA: trans-acting RNA; TE: transposable element.The authors declare that they have no competing interests.IA, MB, JB, CF, JVM, JS, and JJ all wrote an approximately equal number of meeting presentation summaries. All authors read and approved the final manuscript.


Gene expression is an immensely complicated process which requires fine tuning regulation involving multiple actors that have to work in a highly coordinated fashion. Indeed, each step of the transcription cycle  is intimately coupled to a specif c step in pre-mRNA processing . Recent advances in the field highlighted the key role of the C-terminal domain (CTD) of the RNA polymerase II largest subunit in coordinating different steps of the transcription cycle and RNA processing. Transcription from the long terminal repeat (LTR) leads to RNAPII pausing and premature termination after synthesis of a short RNA, the transactivation response element (TAR). The HIV-1 transactivator protein, Tat, together with Cyclin T1 binds a bulge-loop within TAR leading to the recruitment of the super elongation complex (SEC) and allowing CDK9 to phosphorylate the RNAPII CTD and the negative transcription elongation factors NELF and the DRB-sensitivity-inducing factor DSIF/Spt4:Spt5 to overcome their negative action, licensing RNAPII for productive elongation. We will discuss how these recent advances might impact our view of how to design an efficient strategy leading to reactivation of transcriptionally silent HIV-1.


Chromatin structure complexity requires the interaction and coordinated work of a multiplicity of factors at different transcriptional regulation stages. Transcription control comprises a set of processes that ensures proper balance in the gene expression under different conditions, such as signals, metabolic states, or development. We could frame those steps from epigenetic marks to mRNA stability to support the holistic view of a fine-tune balance of final mRNA levels through mRNA transcription, export, stability, translation, and degradation. Transport of mRNA from the nucleus to the cytoplasm is a key process in regulated gene expression. Transcriptional elongation and mRNA export are coregulated steps that determine the mature mRNA levels in the cytoplasm. In this paper, recent insights into the coordination of these processes in eukaryotes will be summarised. Gene expression is a coordinated multistep process that begins with transcription and RNA processing in the nucleus, followed by mRNA export to the cytoplasm for translation. In eukaryotes, it depends on the orchestrated action of several multiprotein complexes which regulate the gene expression by RNA polymerase II (RNAP-II) at multiple levels. in vitro and in vivo, and by establishing a bridge between RNAP-II and elongation factors .Interestingly, recent genome-wide profiling has provided a partial picture of the chromatin landscape, including a wide variety of epigenetic information about posttranslational modifications (PTMs), histone variants, DNA methylation patterns, and nucleosome occupancy (reviewed in ). In thiMechanisms to export mRNA are integrated into the biogenesis of messenger ribonucleoparticles (mRNPs). In this context, the nuclear pore complex (NPC) provides a molecular environment that regulates gene tethering and mRNA export. Moreover, a functional connection between transcription and RNA export has been reinforced by human genetic disorders caused by mutation in mRNA export factors and adaptors , 4. mRNAEmerging studies have provided strong evidence for a role of the NPC in gene expression control. For instance, gene positioning is a dynamic process which determines gene regulation . ActuallAs we have seen, the complexity of chromatin structure requires the interaction and coordinated work of a multiplicity of factors at different transcriptional regulation levels. Despite the fact that proteins involved in gene expression are highly conserved from yeast to higher eukaryotes, several processes such as mRNA biogenesis, which are coupled to nuclear export, are more divergent. This paper will pay attention to the factors implicated in gene transcriptional elongation coupled to mRNA export processes, more specifically to complexes which coordinate mRNP biogenesis and whose misregulation causes important diseases.Due to the compartmentalisation of the eukaryotic cell, the transcribed mRNAs need to be transported from the nucleus towards the cytoplasm to be translated into proteins. After mRNAs have been packaged into mRNPs, they can be exported. One of these proteins involved in the formation of mature mRNPs is Npl3 , which is an mRNA-binding protein that shuttles between the nucleus and cytoplasm for rounds of mRNPs export. Npl3 is directly involved in the mRNA export process, since mutations in this gene cause nuclear accumulation of mRNA , 19. IntYRA1 gene encodes an mRNA-binding protein which acts as an adaptor for the ATP-dependent RNA-helicase Sub2 (UAP56) and is involved in splicing and mRNA export [The TREX complex in yeast comprises Yra1, Sub2, Gbp2, Hrb1, and the THO complex . The THO complex integrates the core proteins Tho2, Hpr1, Mft1, and Thp2 . Among tA export , 31. BesA export , 32, 33.The THO complex and the aforementioned RNA export factors provide evidence for a functional link between transcription and mRNA metabolism . Yra1 anhpr1Δ mutant, Thp3 and Csn12, which form a complex that is recruited to transcribed genes, establishing a further link between transcription elongation and mRNA processing [Recently, novel links between transcription elongation and mRNA export have been described. For instance, the Prp19 splicing complex (homologous to human XAB2) also has a novel function in transcription elongation, since it ensures the stabilisation of the recruitment of the TREX complex at the transcribed genes . This reocessing .It is interesting to note how histone chaperones seem to play a key role in transcription elongation by working together with the TREX complex. Nap1 is a histone chaperone recruited to specific transcribing ORFs by the TREX component Yra1, and it is important for chromatin remodelling during transcription elongation. In addition, Nap1 shows a genetic interaction with Mex67. These data suggest a new connection between transcription elongation and mRNPs formation . In humaInteraction of genes with nuclear pores also contributes to the coupling of mRNP biogenesis and export. In accordance with the “gene gating” hypothesis , the linvice versa [After nuclear quality control mechanisms (reviewed in ), mRNPs ce versa . Nucleopce versa , 54. An ce versa . yra1 mutant allele [An early discovery, which suggested a physical connection between the transcription machinery and the NPC, was the identification of Sus1 in yeast as a factor that is part of two complexes involved in transcription and mRNA export . Sus1 wat allele . It is at allele , 57. Sust allele .THP1 was originally identified as a eukaryotic-conserved gene whose null mutations conferred genetic instability, transcription defects and hyperrecombination phenotypes like those on THO mutants [Initially, the role of TREX-2 docking mRNPs to specific nucleoporins at the nuclear entrance of the NPC was reported. Sac3 interacts with Mex67p-Mtr2p for proper mRNA export. Furthermore, Sac3p-Thp1p TREX-2 components connect with the NPC environment through Nup1p . THP1 wa mutants . Recentl mutants , 62.in vivo and in vitro. In vitro, purified RNAP-II engaged directly on an oligonucleotide with a dC-tail [In vivo, transcriptional run-on assays using a pulse of radioactive UTP and further array hybridisation [sus1Δ mutants in vivo, but only slightly in vitro, and they concluded that sus1Δ leads to similar gene expression defects as those of mutants thp1Δ and sac3Δ [Drosophila, have clearly detected the elongating form of RNAP-II in chromatin immunoprecipitations [Most of the mutants in TREX-2 components compromise transcription elongation to different extents. Transcription elongation has been traditionally studied by genetic and biochemical approaches both  dC-tail , or anal dC-tail , have bedisation  and RNA disation , 67 havedisation –70. Howend sac3Δ . We demond sac3Δ  and thatnd sac3Δ . All thend sac3Δ . These dnd sac3Δ , 73. Fornd sac3Δ . It has nd sac3Δ , 76. Othitations . HoweverOnce mRNP has been properly formed and assembled, it is thought that the export is facilitated by a close location to the NPC. TREX-2 mediates the location of the active genes to the NPC through a binding to both the NPC nuclear face and the SAGA complex. The crystal structure of Sus1 and Cdc31 , bound tDrosophila and humans. For simplicity reasons, the mammalian (human) GCN5-containing STAGA, TFTC, and PCAF complexes have been renamed as human SAGA by Pijnappel and Timmers in 2008 [The SAGA complex's function in transcription activation has been largely characterised, mainly its role as a histone-modifying complex (reviewed in –80). SAG in 2008 , 83, 84.sus1Δ cells [GAL1 mRNA export by tethering the gene to the NPC [ GAL1 gene to the NPC [In fact, SAGA-promoted histone acetylation appears to enhance the processivity of RNAP-II during transcription elongation, indicating its direct contribution to this process , 85. Fur1Δ cells . Secondl the NPC . Thirdly the NPC . Finally the NPC  by expanSeveral nuclear steps in mRNPs formation occur in close temporal and physical proximity to the NPC. In this scenario, SAGA and TREX-2 could coordinate the expression of a subset of genes in the vicinity of the NPC. The precise sequence of events is poorly understood, but a consensus model for the mechanism of this process is framed by the Blobel gene-gating hypothesis . AccordiUBP8 and GCN5 genes responsible for SAGA enzymatic activities cause synergistic transcription elongation defects, suggesting that the roles of histone acetyl transferase (HAT) and DUB modules in elongation are functionally distinct [Besides Gcn5-mediated histone acetylation, several studies have demonstrated that K123 at the C-terminus of H2B is ubiquitylated (ubH2B). ubH2B is essential for the trans-tail methylation of histone H3 and is also required for optimal gene activation . Thus, hdistinct . Among tS. cerevisiae. Nevertheless, several pieces of evidence open up the possibility that gene tethering to the nuclear periphery might also occur in some cases in other eukaryotes . Intereiewed in . versus terminally differentiated cells. D'Angelo et al. showed in 2009 that lack of replacement of NPC scaffold components in the somatic cells from C. elegans and rat brain neurons comes with an age-dependent deterioration of the NPC, leading to an aberrant nuclear accumulation of cytoplasmic tubulin [NUP155 which, in turn, can lead to sudden cardiac death [-/+ mice have shown an AF phenotype, suggesting that reduction in the level of NUP155, or its mistargeting, results in a tissue-specific disorder [NUP98 and NUP214 genes, which have been characterised as mutations leading to several types of leukaemia [Nuclear factor export proteins, along with NPC components, play a critical role in the selective transport of proteins, RNA and ribonucleoproteins across the nuclear envelope. Defects in mRNA export, as well as Nups mutations, have been linked to several human diseases  tubulin . In huma tubulin ). One exac death . Experimdisorder . Other eeukaemia .Furthermore, the human TREX complex has been identified as a culprit of aggressive human breast cancer . hTREX84Drosophila and humans. Thus, we could also expect that defects in DUBm composition would affect mRNA export. Therefore, mRNA processing defects could trigger diverse genetic disorders Several lines of experiments in yeast have proposed Sus1 (ENY2 orthologue) to be a mediator between the NPC, Nups, and active genes  and alsoin vivo targets. Likewise, understanding the time-specific interaction of these components during development will unravel the tight-coupled mechanism that regulates the cytoplasmic fate or their target mRNAs. Further studies are needed to shed light on the molecular mechanism underlying the specificity and the biological role of mRNA export factors, TREX components, and NPC coupling transcriptional initiation, elongation, and mRNA export of The latest advances in proteomics have improved knowledge about the composition of protein complexes, and the newly established protein-protein networks show additional levels of plasticity to coordinate mRNA transcription, elongation, and export. However, further studies are needed to comprehend the particular contribution of each component orchestrating the gene expression in eukaryotes. Deciphering the molecular mechanisms that coordinate the transcriptional output and mRNA level in response to cellular signals will be a future determinant to discover new therapeutic targets and new cellular pathways involved in different processes, for example, development and differentiation in response to stress conditions or even in complex diseases such cancer and human genetic disorders.