Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis, harbors two separate T6SSs on chromosomes 1 and 2, i.e., T6SS1 (VP1386-1420) and T6SS2 (VPA1025-1046). T6SS1 contains at least 7 putative operons: VP1386-1387, VP1388-1390, VP1392-1391, VP1393-1406, VP1400-1406, VP1409-1407, and VP1410-1420. V. parahaemolyticus AphA and OpaR are the two master regulators of quorum sensing (QS) system that are highly expressed at low cell density and high cell density, respectively. ToxR is a membrane-bound virulence regulatory protein conserved across the Vibrio family. In the present work, we show that ToxR coordinates with AphA and OpaR to repress T6SS1 expression in V. parahaemolyticus. OpaR binds to the promoters of VP1388-1390, VP1400-1406, and VP1409-1407 to repress their transcription, but it appears to negatively regulate VP1393-1406 transcription in an indirect manner. By contrast, AphA negatively regulated the above four T6SS1 operons in an indirect manner. In addition, ToxR binds to the promoters of VP1400-1406 and VP1409-1407 to inhibit their transcription, but it presents an indirect interaction with VP1388-1390 and VP1393-1406 promoters. Notably, the expression of ToxR also manifested in a QS-dependent manner and the highest expression occurred at LCD. Meanwhile, the highest expression of T6SS1 occurred at an OD600 value of 0.6 to 0.8 due to the tight regulation of ToxR and QS, suggesting T6SS1 functions only during the mid-logarithmic growth phase. These observations provide significant insight into the molecular mechanism of T6SS1 gene regulation by QS and ToxR in V. parahaemolyticus. Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is mostly disseminated in marine and estuarine surroundings , TDH related hemolysin (trh), two type VI secretion systems (T6SS1 and T6SS2) as well as two type III secretion systems (T3SS1 and T3SS2) (oundings . Virulen seafood . In rareen wound . V. parad T3SS2) . These fV. parahaemolyticus strain RIMD2210633 harbors two different T6SSs on chromosomes 1 and 2, named as T6SS1 (VP1386-1420) and T6SS2 (VPA1025-1046), respectively . The T6SS1 has been thought that predominantly present in clinical isolates of V. parahaemolyticus, but a recent study demonstrated that all of the environmental acute hepatopancreatic necrosis disease (AHPND) strains, but none of the non-AHPND strains, contain the T6SS1  is the process of cell-cell signaling that mediates communal behavior and gene regulation in response to the presence of chemical signals known as autoinducers (AIs) . QS was bacteria . QS is ibacteria . QS regubacteria . AphA anolyticus . The higolyticus . AphA anolyticus . The asyVibrio species  expression compared to the parental strain under non-optimal inducing conditions  in this study  were described in our previous studies . For construction of toxR deletion mutant (ΔtoxR), the 428 and 455 bp DNA regions upstream and downstream of toxR were amplified by PCR, purified, and used as the templates to create an 879 bp deletion construct that was subsequently inserted between the Pst I and Sph I sites of pDS132  at 30°C with shaking at 200 rpm. The glyceric stock of bacterial cells were inoculated into 5 ml of M broth and incubated overnight for at least 12 h. The overnight cell cultures were diluted 1:50 into 15 ml of fresh M broth, and grown to reach at OD600≈1.0, and then diluted 1:1000 into 15 ml of marine broth for the third-round growth, and were harvested at required cell densities. The culture medium was supplemented with 50 μg/ml gentamicin, 5 μg/ml chloramphenicol, or 0.1% arabinose where necessary. V. parahaemolyticus is a biosafety level 2 (BSL-2) pathogen, and thus all the experimental operations involving in live bacteria were done in the BSL-2 lab.TM Kit according to the manufacturer’s instructions. cDNAs were generated by using 3 ∼ 8 μg of total RNAs and 3 μg of random hexamer primers. The SYBR Green qRT-PCR assay was performed and analyzed as previously described . The contaminated genome DNA in the total RNAs was removed by using the Ambion’s DNA-freeescribed . The rel32P-end labeled reverse oligonucleotide primer to generate cDNAs using a Primer Extension System . The same labeled primer was used for sequencing with the AccuPower and Top DNA Sequencing Kit . The primer extension products and sequencing materials were concentrated and analyzed in an 8 M urea-6% polyacrylamide gel electrophoresis, and the results were detected by autoradiography with the Fuji Medical X-ray film .For the primer extension assay , an oligExTaqTM DNA polymerase  using the genomic DNA as the template. PCR amplicons were cloned into the corresponding restriction endonuclease sites of pHRP309 plasmid harboring a promoterless lacZ reporter gene and a gentamicin resistance gene  according to the manufacturer’s instructions.For the LacZ fusion and β-galactosidase assay , 2014, tnce gene . After baphA, opaR, and the truncated toxR  of the strain RIMD 2210633 were amplified, purified, and cloned into plasmid pET28a , respectively. The recombinant plasmid encoding His-tagged protein was transformed into E. coli BL21bbbDE3 cells for protein expression , labeled DNA (1000–2000 CPM/μl), and increasing amounts of His-tagged protein. Three controls were included in each EMSA experiment: (1) cold probe as specific DNA competitor , (2) negative probe as non-specific DNA competitor (the unlabeled coding region of the 16S rRNA gene), and (3) non-specific protein competitor . After incubation at room temperature for 30 min, the products were loaded onto a native 4% (w/v) polyacrylamide gel, and electrophoresed in 0.5× TBE buffer for about 50 min at 200 V. Radioactive species were detected by autoradiography after exposure to Fuji Medical X-ray film at -20°C.For EMSA , the 5′-32P-labeled end were PCR amplified with either sense or antisense primer being end-labeled. The PCR products were purified using the QiaQuick columns . Increasing amounts of His-tagged protein were incubated with the purified, labeled DNA fragment (2–5 pmol) for 30 min at room temperature, in a final 10 μl reaction volume containing the binding buffer used in EMSA. Before DNA digestion, 10 μl of Ca2+/Mg2+ solution (5 mM CaCl2 and 10 mM MgCl2) was added, followed by incubation for 1 min at room temperature. The optimized RQ1 RNase-Free DNase I  was then added to the reaction mixture, and the mixture was incubated at room temperature for 40–90 s. The reaction was quenched by adding 9 μl of stop solution , followed by incubation for 1 min at room temperature. The partially digested DNA samples were extracted with phenol/chloroform, precipitated with ethanol, and analyzed in 6% polyacrylamide/8 M urea gel. Protected regions were identified by comparison with the sequence ladders. The templates for DNA sequencing were the same as the DNA fragments for DNase I footprinting assay. Radioactive species were detected by autoradiography after exposure to Fuji Medical X-ray film at -20°C.For DNase I footprinting , the tart-test was used to calculate statistically significant differences, p < 0.01 was considered to indicate statistical significance. The presented data of primer extension, EMSA, and DNase I footprinting assays were done with at least two independent biological replicates.The LacZ fusion assay and qRT-PCR were performed with at least three independent bacterial cultures and the values were expressed as mean ± standard deviation. Paired Student’s Supplementary Figure S1). Thus, the 400 bp upstream regions of VP1386-1387, VP1388-1390, VP1393-1406, VP1400-1406, and VP1409-1407 in the T6SS1 gene cluster were retrieved from the genome sequence of RIMD 2210633 with the ‘retrieve-sequence’1. Subsequently, the DNA binding boxes of AphA . However, the AphA box-like sequences were not found in all of the upstream regions tested. Thus, the first genes of VP1388-1390, VP1393-1406, VP1400-1406 and VP1409-1407 were selected for the following gene regulation studies.VP1393-1406 and VP1392-1391  are adjacent but are transcribed in the opposite direction and they share the same intergenic DNA region  were measured in WT grown at different cell densities by the primer extension assay . The aphA and toxR mRNA levels decreased considerably with the increasing of cell density, and the highest transcription appeared at an OD600 value of 0.05 to 0.2; when the OD600 value was higher than 0.4, the mRNAs of both aphA and toxR were undetectable. On the contrary, the opaR mRNA level was increased but then reduced with the increasing of cell density, and the highest transcription occurred at an OD600 value of 0.4 to 0.6; when the OD600 value was lower than 0.2, the opaR mRNA was undetectable. The highest transcription level of hcp1 emerged at an OD600 value of 0.6; when the OD600 value was lower than 0.4 or higher than 0.8, the hcp1 mRNA was undetectable. However, the VP1388 mRNAs were detected at all cell densities, and the highest transcription occurred at an OD600 value of 0.8. Thus, the bacterial cells were harvested at an OD600 value of about 0.15 and 0.4–0.6 for characterizing AphA/ToxR- and OpaR-mediated gene regulation, respectively. In addition, the cell density-dependent transcription of VP1388 and hcp1 suggests T6SS1 expression would be under control of QS.The mRNA levels of Figure 2A), the mRNA level of all the four genes, i.e., VP1388, hcp1, VP1400, and VP1409, was enhanced in ΔaphA relative to WT. The primer extension assay  further indicated that the mRNA levels of the four genes were enhanced in ΔaphA relative to WT. The recombinant lacZ fusion plasmid that contains the indicated promoter-proximal region and promoterless lacZ gene was transformed into ΔaphA and WT, respectively, to test the action of AphA on the promoter activity of the above four genes. The results disclosed a significantly enhanced promoter activity of each of the four genes in ΔaphA relative to WT . The promoter DNA regions of the above four genes were amplified, purified, radioactively labeled, and then subjected to EMSA with the purified His-AphA . The results showed that His-AphA was unable to bind to the upstream DNA fragment of each target promoters, these were consistent with the predict results (Table 2). Taken together, AphA appears to negatively regulate the transcription of VP1388-1390, VP1393-1406, VP1400-1406, and VP1409-1407 in an indirect manner.As determined by the qRT-PCR assay . The lacZ fusion results showed that the promoter activity of each of the four operons in ΔopaR was much higher relative to that in WT . The EMSA results showed that His-OpaR was able to bind to the upstream DNA fragment of VP1388, VP1400, and VP1409 in a dose dependent manner, but a negative EMSA result was observed for hcp1 . His-OpaR at all amounts used could not bind to the 16S rDNA fragment as the negative control . As further determined by DNA footprinting , His-OpaR protected two different DNA regions upstream of VP1388 and VP1409 against DNase I digestion that were considered as the OpaR sites, while only a single OpaR site was detected for VP1400. Taken together, OpaR represses the transcription of VP1388-1390, VP1400-1406, and VP1409-1407 in a direct manner, but it appears to negatively regulate the transcription of VP1393-1406 in an indirect manner.The qRT-PCR and primer extension assays indicated that the transcription of all the four target genes VP1388, Figure 4A), the mRNA level of each target gene was greatly increased in ΔtoxR relative to WT. The primer extension assay detected a single ToxR-repressed transcription start site for each of the four target operons . The lacZ fusion results showed that the promoter activity of each of the four operons in ΔtoxR was significantly enhanced than that in WT . The EMSA results showed that His-ToxR was able to bind to the upstream DNA fragment of VP1400 and VP1409 in a dose dependent manner, but negative EMSA results were observed for VP1388 and hcp1 . As further determined by DNA footprinting , His-ToxR protected one or more DNA regions upstream of VP1409 or VP1400 against DNase I digestion that were considered as the ToxR sites. Taken together, ToxR represses the transcription of VP1400-1406 and VP1409-1407 in a direct manner, but it appears to negatively regulate the transcription of VP1388-1390 and VP1393-1406 in an indirect manner.As determined by the qRT-PCR assay .Collection of data of translation/transcription start sites, promoter -10 and -35 (or -12 and -24) elements, AphA/OpaR/ToxR sites, AphA/OpaR/ToxR box-like sequences, Shine-Dalgarno (SD) sequences  enabled us to depict the organization of VP1388, V. parahaemolyticus is under the negative control of both QS and the virulence regulator ToxR . At HCD (OD600 = 0.4–0.6), OpaR binds to the three promoters of VP1388-1390, VP1400-1406, and VP1409-1407 to repress their transcription, but it appears to negatively regulate VP1393-1406 transcription in an indirect manner. At LCD (OD600 = 0.05–0.2), AphA negatively regulated the above four T6SS1 operons in an indirect manner; while ToxR binds to the promoters of VP1400-1406 and VP1409-1407 to inhibit their transcription, but it presents an indirect interaction with VP1388-1390 and VP1393-1406 promoters. The highest transcription of T6SS1 genes occurred at an OD600 value of 0.6 to 0.8 due to the tight regulation of ToxR and QS, suggesting T6SS1 may function at the later stages of HCD.Many bacterial genomes harbor more than one T6SS loci that are involved in different cellular functions , suggestV. cholerae O1 strain A1552. V. cholerae O1 strain C6707 HapR directly binds to the promoter regions of the T6SS genes hcp1 and hcp2 to induce their expression. The T6SS in Aeromonas hydrophila is also under positive control of AhyR, an N-acyl homoserine lactone (s) -mediated quorum regulator, the ahyR and ahyI double knockout mutant is unable to secrete T6SS-associated effectors  as parental strain , suggesting V. parahaemolyticus T6SS1 functions only during the mid-logarithmic growth phase. Similar observations have been reported in other species, including Yersinia pseudotuberculosis  the binding site overlaps with the corresponding OpaR site, indicating the repression mechanisms by ToxR would be similar to that of by OpaR. However, we didn’t detect the ToxR box-like sequence within all the ToxR sites, suggesting the consensus sequence of TNAAA-N5-TNAAA identified in V. cholerae  when grown the bacteria cells in M broth. Thus, T6SS1 genes are assigned as the members of QS and ToxR regulons in V. parahaemolyticus.Collectively, this work reported that ToxR coordinates with QS regulators AphA and OpaR to repress T6SS1 in DZ and XH conceived the study and designed experimental procedures. YiqZ, HG, GO-A, YinZ, WY, HY, and ZY performed the experiments and carried out data analysis. YiqZ, GO-A, DZ, and XH wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To confirm this speculation, the fads2 candidate promoter (2646 bp) was cloned from carnivorous marine teleost Epinephelus coioides, and 330 bp core regulatory region was identified. Several binding sites for transcriptional factors such as nuclear factor 1, nuclear factor Y, sterol regulatory element and hepatocyte nuclear factor 4γ were identified, while that for Sp1 was shown to be absent in the promoter by both bioinformatic analysis and site-directed mutation. Moreover, after the Sp1-binding site from the fads2 promoter of herbivorous Siganus canaliculatus, the first marine teleost demonstrated to have LC-PUFA biosynthetic ability, was inserted into the corresponding region of E. coioides fads2 promoter, activity was significantly increased. The results provided direct data for the importance of the Sp1-binding site in determining fads2 promoter activity, and indicated that its lack may be a reason for low expression of fads2 and poor LC-PUFA biosynthetic ability in E. coioides.Δ6 fatty acyl desaturase (Fads2) is a rate-limiting enzyme in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. Comparative analysis of gene promoters of Fads2 between salmonids and carnivorous marine fish suggested that the lack of binding site for stimulatory protein 1 (Sp1) was responsible for the low expression of  In particular, eicosapentaenoic acid  and docosahexaenoic acid  play important roles in maintaining normal development of the nervous system and sensory organs2, promoting cardiovascular health and immune function4, and involving in the regulation of lipid metabolism6. Fish, especially marine species, are the primary source of LC-PUFA in the human diet. However, with overfishing and the degradation of the marine environment, natural wild fishery stocks have reduced sharply. Thus, the declining capture fisheries has turned attention to farmed marine fish as the major source of LC-PUFA. Fish oil (FO), which is rich in LC-PUFA, was traditionally utilised for meeting the requirement of EFA in farmed marine fish systems. However, the limited availability of FO is a major limitation for the development of marine fish aquaculture7. Therefore, much attention has been given to seeking the sustainable alternatives, especially vegetable oils (VO), which are rich in linolenic acid  and linoleic acid , but devoid of LC-PUFA. Unfortunately, due to most farmed marine fish having only limited capability or inability for converting LNA and LA to LC-PUFA, dietary VO usually have negative effects on the contents of n–3 LC-PUFA of farmed fish8. Thus, much attention has been focused on elucidating the regulatory mechanisms of LC-PUFA biosynthesis, in order to maximize endogenous production in marine fish.Long-chain  is the rate-limiting enzyme, which is responsible for catalyzing the first step in the LC-PUFA biosynthetic pathway, and also involved in DHA synthesis from EPA, and has been commonly regarded as an indicator of LC-PUFA biosynthetic ability in fish11. Although the activity and nutritional regulation of Fads2 have been investigated in several fish species16, the molecular mechanisms underlying their regulation remains largely unknown. The comparative analysis of fads2 gene promoter between Salmo salar (with LC-PUFA biosynthetic ability) and Gadus morhua (a carnivorous marine teleost with very limited LC-PUFA biosynthesis) suggested that low expression of fads2 in the latter could be attributed, at least partly, to the lack of a binding site of stimulatory protein 1 (Sp1) in the fads2 promoter17. Similarly, the Sp1 binding site was not found in the fads2 promoter of the carnivorous marine teleost Dicentrarchus labrax either, and its fads2 promoter activity was weaker than that in Oncorhynchus mykiss11. Therefore, it was speculated that the Sp1 site may play an important role in determining the fads2 promoter activity and LC-PUFA biosynthetic ability in fish17. However, there was no direct evidence to support such a deduction.The LC-PUFA biosynthesis pathway involves consecutive desaturation and elongation steps from LNA and LA catalyzed by fatty acyl desaturase (Fads) and elongation of very long-chain fatty acids (Elovl) enzymesSiganus canaliculatus is a herbivorous marine teleost widespread along the Indo-West Pacific coast and cultured in southeastern Asia including China due to its popularity in markets. It is noteworthy that S. canaliculatus was the first marine teleost demonstrated to have the ability of LC-PUFA biosynthesis, with key enzymes including Fads2, and Elovl4 and Elovl5 elongases required for LC-PUFA biosynthesis being identified and characterized in this species20. Moreover, sequence analysis identified an Sp1 binding site in the Δ6/Δ5 Fad promoter of S. canaliculatus (unpublished data). So, S. canaliculatus provides us a favourable model for investigating the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. On the other hand, the grouper Epinephelus coioides is a typical carnivorous marine teleost, which has been widely cultured in coastal areas of southeastern Asia including China for its fast growth performance and huge economic value12. Moreover, the genes encoding Fads2 of this species have been cloned and functionally characterized in an exogenous yeast system12, which showed that the Fads2 had low enzymatic activity in converting LNA and LA to 18:4n–3 and 18:3n–6, respectively. The results were consistent with a recent feeding trial, which showed that juvenile E. coioides has poor LC-PUFA biosynthetic capacity21. In order to investigate the underlying reasons, the present study was focused on clarifying the importance of the Sp1 binding site in determining the transcriptional activity of the fads2 promoter, as speculated in marine carnivorous G. morhua and D. labrax. First, the candidate promoter of E. coioides fads2 gene was cloned and functionally analyzed. Then, targeted mutation of potential transcriptional factor (TF) binding sites was performed to identify key elements in the fad promoter. Moreover, the role of the Sp1 binding site in the fads2 promoter was clarified. The results will be helpful for identifying the reasons underpinning the low LC-PUFA biosynthetic ability of E. coioides, and will provide novel insights into the regulatory mechanisms of LC-PUFA biosynthesis in vertebrates.Rabbitfish E. coioides fads2 were determined by qPCR. The highest expression of fads2 were detected in the brain, followed by the eyes, liver, muscle and gill, and relatively low expression of fads2 were observed in other tissues  were included in the construct (SD2). The activity of construct SD3 decreased but showed no significant difference with SD2. However, when 5′ truncation came to 17 bp upstream of TSS (SD4), the activity of promoter decreased to the level as negative control. The results indicated that the region between −347 to −17 may contain some important regulatory elements  was used for further functional analysis.To determine the core regulatory region of the E. coioides fads2 promoter was subjected to further in silico analysis with TRANSFAC®, MatInspector® and JASPAR®, and several potential binding sites for TF such as TBP, YY1, NF-Y, NF1, HNF4γ and RXR::VDR were predicted , but absent in carnivorous marine teleost such as E. coioides, D. labrax and cod G. morhua  promoter of S. canaliculatus. Dual luciferase assay showed that the activity of E. coioides fads2 promoter with Sp1 binding site was significantly increased as showed in constructs SD2 and SD3  and large yellow croaker (Larimichthys crocea), respectively38. Those results indicated that the conserved SRE element was also essential in driving fads2 promoter activity in fish.Studies on mammalian fads2 core promoters of the different fish species. For example, the C/EBPα element was predicted in the promoter region of G. morhua and S. canaliculatu32, PPARα binding element was discovered in D. rerio fads2 promoter33, HNF4α site was discovered in S. canaliculatu fads2 (Δ4 Fad) promoter, and the HNF4γ binding sites influenced the promoter activity of E. coioides fads2. Whether HNF4γ is involved in the regulation of fads2 expression has not been clarified fully so far, but it can combine with HNF4α, a regulator in stimulating the fads2 gene transcription, to regulate gene expression39, and thus HNF4γ is likely to be involved in the regulation of the fads2 gene in fish. Interestingly, Sp1 elements only appear in the fads2 promoter regions of some fish species with LC-PUFA biosynthetic ability, such as S. salar17, D. rerio and S. canaliculatu32. However, the carnivorous marine fish species, like G. morhua, D. labrax, L. japonicus, L. crocea and E. coioides, which were shown to lack of the Sp1 elements38.Apart from these conserved elements above, like NF-Y and SRE, there were several different cis-elements in the 36. The lack of an Sp1 binding site in carnivorous marine fish may, to some extent, account for their low fads2 gene expression. In comparison to salmonids, G. morhua, and D. labrax fads2 gene promoters without an Sp1 site showed lower activity, and thus it is speculated that the low expression of fads2 can be attributed to their incomplete and less active promoter17. Recently, the Sp1 binding site was detected in mammalian FADS and ELOVL7 promoters, and Sp1 up-regulated the expression of Fads2 and Elovl741. These results suggested that the lack of Sp1 binding site maybe a reason leading to the low promoter activity of fads2, and thus result in low hepatic fads2 expression in carnivorous marine teleost, like E. coioides.Sp1 has been shown to contribute with NF-Y transcription factors to the regulation by SREBP of the expression of several genes involved in cholesterogenic and lipogenic pathwaysE. coioides fads2 promoter with the Sp1 binding site mutations was significantly increased. The result provided direct evidence that Sp1 plays an important role in determining fads2 promoter activity. This was consistent with the hypothesis that the lack of a Sp1 binding site in the E. coioides fads2 promoter is a reason leading to low gene expression of fads2, and thus result in poor LC-PUFA biosynthetic ability in E. coioides. Interestingly, the addition of the Sp1 binding site significantly increased the activity of SD2 and SD3, while the activity of SD1 did not show a significant increase upon the same treatment. This may be attribute to the existence of some inhibitory elements located between SD1 and SD2, which requires to be investigated further.In the present study, the activity of E. coioides fads2 was detected in brain, followed by eyes, liver, muscle and gill. A sequence of 2646 bp candidate promoter of fads2 gene was cloned and functionally characterized, and its core regulatory region ranging from −347 bp to −17 bp identified. Binding sites for NF1, NF-Y, HNF4γ and RXR::VDR were identified as key elements in the E. coioides fads2 promoter. The lack of an Sp1 binding site in fads2 promoter was proved to be at least partly responsible for the low expression of hepatic fads2 mRNA and poor LC-PUFA biosynthetic capacity of E. coioides.In summary, high expression of In present study, all animal experiments were done in accordance to the National Institutes of Health guide for the care and use of Laboratory animals  and approved by the Animal Care and Use Committee of South China Agricultural University . All surgery was performed under 0.01% 2-phenoxyethanol  anesthesia, and all efforts were made to minimize suffering of fish.E. coioides juveniles were bought from a local aquafarm in Fujian, China. After they were reared in floating cages (1 m × 1 m × 1.5 m) at Nan Ao Marine biology station (NAMBS) of Shantou University and fed a formula feed with 50% crude protein and 10% crude lipid for two weeks, six individuals (body weight ~24 g) were anaesthetized with 0.1% 3-amino benzoate methane sulphonate . Tissue samples including brain, eye, gill, liver, intestine, kidney, heart, muscle, spleen, stomach, pyloric caecum and fat (adipose) were collected and immediately frozen in liquid nitrogen, then stored at −80 °C until further use for the analysis of fads2 transcripts distribution, or genomic DNA isolation.fads2 mRNA was determined by quantitative real time PCR (qPCR). Total RNA was respectively extracted from the above tissue samples using TRIzol® Reagent  according to manufacturer’s protocol, and 1 μg of total RNA was reverse-transcribed into cDNA using random hexamers . Gene-specific primers  were used for qPCR analysis. qPCR was carried out with an initial activation step at 95 °C for 5 min, followed by 40 cycles: 10 s at 95 °C, 20 s at 60 °C and 20 s at 72 °C. After the amplification phase, a dissociation curve of 0.5 °C increments from 65 °C to 95 °C was performed, enabling confirmation of the amplification of a single product in each reaction. The relative expression of fads2 were normalized with β-actin expression calculated by the 2−ΔΔCt method42.Tissue distribution of E. coioides as described previously32 and used as template for candidate promoter cloning. The sequence upstream of fads2 gene was obtained from the genomic sequencing data of E. coioides. For identifying the core region within 5′ flanking sequence of the E. coioides fads2, one of the forward primers , augmented with a 5′ KpnI site  according to the manufacturer’s protocol, and were confirmed by sequencing. The construct SD3 including the core promoter region was used as wildtype for mutations experiments. TRANSFAC®, MatInspector® and JASPAR® were used to predict potential TF binding sites on E. coioides fads2 promoter. Ten TF binding sites were found  promoter were mutated into “G”, and thus E. coioides fads2 promoter  acquired a Sp1 binding site (−165 bp to TSS). The influence of TF binding site mutations or Sp1 mutation on promoter activity of E. coioides fads2 were measured by Dual luciferase assay as below.Mutations of und Fig. , and thu® LTX Reagent with PLUSTM reagent  according to the manufacturer’s instructions. Briefly, 100 ng of promoter reporter plasmid were co-transfected with 10 ng of pGL4.75 into the cells. Each plasmid complex was transfected in triplicate in three independent experiments. Firefly and Renilla luciferase activities were measured using Dual-Glo Luciferase Assay system E2940  according to manufacturer’s instructions. Specifically, 24 h after transfection, 75 μL of Dual-Glo Luciferase Assay Reagent was added into each well, the plate was incubated at room temperature for 10 mins. Then the firefly luminescence was measured on a Tecon microplate reader , followed by the addition of Dual-Glo Stop & Glo Reagent into the plate. Finally, after incubation at room temperature for 10 mins, Renilla luminescence were measured and the ratio of firefly/renilla luminescence for each well was calculated. The promoter activity was calculated from the chemical luminescence intensity ratio of firefly: renilla luciferase for each construct, and then compared with the activity of vector pGL4.10 luciferase17.Tilapia liver cell line HepaT were cultured in Leibovitz’s L15 Medium  with 10% fetal bovine serum  at 28 °C in a humidified incubator. For DNA transfection, HepaT cells were seeded in 96-well cell culture plates and grown for 24 h to 90% confluent. Transfection were conducted using Lipofectaminefads2 mRNA and n = 3 for evaluating the effects of progressive deletions, TF binding site mutations, Sp1 replacement on promoter activity. Data were analyzed suing One Way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test or Student’s t-test with Origin 7.0. A significance of P < 0.05 was applied to all statistical tests performed.All data were presented as mean ± SEM, n = 6 for tissue-specific distribution of Supplementary Fig. S1. Sequence of candidate promoter of fads2 in Epinephelus coioides.


Toxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue cyst formation is still poorly understood. Significant changes in gene expression are associated with tissue cyst development, and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 complexes and the operating principles of ApiAP2 mechanisms are not well defined. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway. Toxoplasma biology that underlies human chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite stage. We investigated the roles of two alkaline-stress-induced ApiAP2 transcription factors, AP2IV-3 and AP2IX-9, in bradyzoite development. These factors were expressed in two overlapping waves during bradyzoite development, with AP2IX-9 increasing expression earlier than AP2IV-3, which peaked as AP2IX-9 expression was declining. Disruption of the AP2IX-9 gene enhanced, while deletion of AP2IV-3 gene decreased, tissue cyst formation, demonstrating that these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified AP2IX-9 or AP2IV-3 with the small molecule Shield 1 had a reciprocal effect on tissue cyst formation, confirming the conclusions of the knockout experiments. The AP2IX-9 repressor and AP2IV-3 activator tissue cyst phenotypes were borne out in gene expression studies that determined that many of the same bradyzoite genes were regulated in an opposite manner by these transcription factors. A common gene target was the canonical bradyzoite marker BAG1, and mechanistic experiments determined that, like AP2IX-9, AP2IV-3 regulates a BAG1 promoter-luciferase reporter and specifically binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest that the AP2IX-9 transcriptional repressor and the AP2IV-3 transcriptional activator likely compete to control bradyzoite gene expression, which may permit Toxoplasma to better adapt to different tissue environments and select a suitable host cell for long-term survival of the dormant tissue cyst.The IMPORTANCEToxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue cyst formation is still poorly understood. Significant changes in gene expression are associated with tissue cyst development, and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 complexes and the operating principles of ApiAP2 mechanisms are not well defined. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway. Toxoplasma is heteroxenous, with a sexual reproductive cycle exclusive in the felid host and a second clonal reproductive phase (intermediate life cycle) that is possible in virtually any endothermic animal. There are two competing demands of the Toxoplasma intermediate life cycle, (i) expansion of tachyzoite numbers to ensure survival and (ii) production of the bradyzoite-tissue cyst required to transmit the infection to the next host. Our previous studies demonstrated that Toxoplasma bradyzoite differentiation is a multistep process requiring slowing of the tachyzoite cell cycle combined with key changes in gene expression and exit from the cell cycle  that are weakly similar to a group of transcription factors found in plants. The plant-like transcription factors in the Apicomplexa harbor one to six Apetala2 (AP2) DNA binding domains   –13. In oium spp. ), which complexa . Thereafromerids . These glciparum  have yetToxoplasma life cycle, including the feline sexual cycle, have permitted the reanalysis of ApiAP2 factors (http://toxodb.org/toxo/) representing type II strain transcriptomes from in vitro tachyzoites, early and mature bradyzoites, merozoites, and sporozoites to assess how all 67 ApiAP2 mRNAs are developmentally expressed . For 22 ApiAP2 genes, the mRNA expression data did not identify a specific developmental profile. Many nondevelopmental ApiAP2 factors are expressed at low levels , while others showed variable moderate-to-higher expression across the stages. Notably, the cell cycle-regulated tachyzoite factor AP2VI-1  were strongly upregulated in low-passage-number type II ME49 parasites by 2 days following alkaline stress induction of tachyzoites, and four of these factors  were downregulated in mature bradyzoites (21-day murine brain tissue cysts), indicating that their role is restricted to the transition of the tachyzoite to the bradyzoite. The expression of three of these ApiAP2 factors during early bradyzoite development has been confirmed at the protein level . AP2IV-3 mRNA expression in 21-day bradyzoites from mouse brain tissue  by genetic knock-in revealed a nuclear factor that was induced by alkaline stress during the early stages of bradyzoite switching while largely absent from tachyzoites  that reflect the characteristic asynchronous nature of bradyzoite development   (ap2IX-9 parasites to form tissue cysts (68%) at more than twice the rate of the Pru-Δap2IV-3 strain in this medium (29%). Complementation of the Pru-Δap2IV-3 strain with a cosmid carrying a single copy of the AP2IV-3 gene fully restored tissue cyst formation to levels that matched or exceeded that of the Pru parent or Pru-Δap2IX-9 parasites   and detee medium . We compap2IX-9)  and B. Pap2IX-9) , but herap2IX-9)  similar ap2IX-9) . In contap2IX-9) . Thus, cue cysts 8% at mor10.1128/mSphere.00347-16.1FIG S1 ap2IV-3 clones grown in pH 8.2 medium for 72 h. The statistical significance of differences in tissue cyst formation between parent and knockout parasites is indicated . Download FIG S1, TIF file, 0.2 MB.Disruption of AP2IV-3 by genetic knockout in the type III CTG strain also shows a decrease in the formation of tissue cysts. IFA quantification in triplicate of tissue cyst numbers (DBA positive) formed by CTG parent parasites versus CTG-ΔCopyright © 2017 Hong et al.2017Hong et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the DDAP2IV-3 isolate was exposed to increasing alkaline stress plus or minus Shield 1 (100 nM), and the tissue cyst number was determined after 72 h  tissue cysts similar to the images shown in in vitro. In contrast, previous experiments where AP2IX-9 was overexpressed clearly demonstrated a strong repressive effect on tissue cyst formation in type II and III strains /Shield 1 conditional expression model –24. The –ter 72 h . As expet manner . Combina strains .DDAP2IV-3 and CTG-Δap2IV-3 transgenic strains grown under different medium conditions plus 100 nM Shield 1   and LDH2 (up ~4-fold), as well as a bradyzoite-specific rhoptry protein  increased by overexpression of DDAP2IV-3 in tachyzoites failed to be induced by strong alkaline stress when AP2IV-3 was absent   were aff of 320)  and B. A of 320) . Many of of 320) , includi protein , BRP1  grown in pH 7.0 to 8.2 medium as indicated. The complete microarray data for this experiment have been deposited in GEO (GSE89469). Download DATA SET S1, XLSX file, 0.04 MB.Analysis of 320 previously published alkaline-stress-responsive genes  in CTG pCopyright © 2017 Hong et al.2017Hong et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00347-16.4DATA SET S2 DATA SET S2, XLSX file, 0.1 MB.Expression profiles of 42 mRNAs altered by AP2IV-3 Shield 1 overexpression in ME49 parasites grown in pH 7.0 and 8.2 media and M4 strain merozoites and sporozoites. Download Copyright © 2017 Hong et al.2017Hong et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the DDAP2IV-3 and a second gene encoding firefly luciferase driven by the BAG1 promoter (BAG1-Luc). In previous studies, the BAG1 promoter conferred alkaline-stress-inducible luciferase expression on developmentally competent strains (DDAP2IV-3::BAG1-Luc parasites (yellow bars) at all medium pHs (plus Shield 1) than in the CTG::BAG-Luc control strain (blue bars). This difference was strongly amplified by increasing alkaline stress; CTG-DDAP2IV-3::BAG1-Luc parasites cultured in pH 8.2 medium plus 100 nM Shield 1 expressed luciferase at ~20-fold higher levels than the CTG::BAG1-Luc control strain . AP2IV-3 binding appeared to be stronger and to have a stronger preference for the −950 to −636 region than AP2IX-9 binding. To examine this binding preference further, we generated new stable CTG-DDAP2IV-3 transgenic clones expressing either the full-length BAG1 promoter  or a truncated promoter (457 bp) missing the AP2IV-3 binding region as defined by the ChIP-qPCR experiments. The full-length and truncated BAG1-Luc reporters were both induced by alkaline stress or AP2IV-3 overexpression, which, when combined, gave the highest luciferase expression for each promoter construct , followed by quantitative PCR (qPCR) . The bineviously  and showonstruct . HoweverToxoplasma ApiAP2 factors (AP2IX-9 and AP2IV-3) induced early in bradyzoite development have important roles in the regulation of bradyzoite gene expression. AP2IX-9 is a transcriptional repressor that prevents the induction of bradyzoite gene expression and reduces tissue cyst formation ; 5% fetal bovine serum (Sigma); and 1% penicillin-streptomycin-amphotericin B (HyClone) at 37°C with 5% CO2.Parasite cultures were maintained in primary human foreskin fibroblasts (HFF) as previously described . All of 10.1128/mSphere.00347-16.5DATA SET S3 DATA SET S3, XLSX file, 0.01 MB.Transgenic strains produced in this study and key primer sequences. Download Copyright © 2017 Hong et al.2017Hong et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the DDAP2IV-3). The plasmid pCTDDHA3x-AP2IV-3 was introduced by electroporation into the type III CTG strain. Transgenic parasites were selected with chloramphenicol (20 μM), and clones were isolated by limiting dilution. Overexpression of AP2IV-3 was achieved by adding 100 nM Shield 1 synthesized in house to the culture medium.For conditional overexpression, the AP2IV-3 single exon coding region was PCR amplified from genomic DNA to incorporate in-frame Xma1/Sbf1 sites, which were used to clone the purified PCR fragment into the pCTDDHA3x plasmid. This cloning results in the fusion of the FKBP peptide (11.2 kDa) and three copies of the HA epitope for detection (4.4 kDa) in frame with the N terminus of the AP2IV-3 protein with a final fusion protein mass of ~162 kDa . Briefly, both the 5′ and 3′ untranslated regions (UTRs) of ApiAP2 genomic coding regions were PCR amplified from type I genomic DNA and used to create Gateway entry clones. The 5′ UTR was cloned into the pDONR_P4-P1r vector, and the 3′ UTR was cloned into the pDONR_P2r-P3 vector, by the BP recombination reaction. After isolation of the entry clones, pDONR_P4-P1r_AP2 and pDONR_P2r-P3_AP2 were combined in an LR recombination reaction with the p221-DHFR and pDEST_R4-R3 vectors to create the pDEST_AP2_DHFR knockout plasmid. The final plasmids were introduced into Pru and CTG strains, and transgenic parasites were selected by using pyrimethamine, followed by clonal isolation by limiting dilution. Thirty micrograms each of the gRNA plasmid and the pDEST_AP2_DHFR knockout plasmid was transfected into 3 × 107 parasites by electroporation. For complementation of AP2IV-3, purified cosmid PSBLJ40 carrying a single copy of the AP2IV-3 gene was transfected into Pru-Δap2IV-3 parasites. To quantify genetic rescue, established drug-resistant populations were tested for tissue cyst formation in alkaline medium (pH 8.2) and AP2IV-3 mRNA and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were analyzed by reverse transcription (RT)-qPCR.For disruption of ApiAP2 factors, we used the multi-guide-RNA (multi-gRNA) CRISPR-Cas9 system. The gRNA for each gene was generated by the CRISPR gRNA design tool supported by DNA2.0 . The CRISPR-Cas9 gRNA plasmid (pSAG1::Cas9-U6::sgUPRT) was provided by David Sibley . The single gRNA in this plasmid was replaced with a short oligonucleotide matching each targeted gene see  by Q5 DNToxoplasma parasites. Purified parasites were lysed in SDS-PAGE sample buffer with Laemmli loading dye, heated at 95°C for 10 min, and briefly sonicated. After separation on the gel, proteins were transferred onto nitrocellulose membrane, detected with MAbs and horseradish peroxidase-conjugated secondary antibodies , and visualized by enhanced-chemiluminescence detection (PerkinElmer).Parasites were inoculated onto confluent HFF coverslips and incubated for the times indicated, and IFA was performed with the following primary reagents: anti-HA antibody , anti-myc antibody , biotin-labeled DBA , and 4',6-diamidino-2-phenylindole . All Alexa - and streptavidin -conjugated secondary antibodies were used at 1:1,000. Image acquisition was performed with a Zeiss Axiovert microscope equipped with a 100× objective. Western blotting with a specific antibody monitored the protein expression in DDAP2IV-3, and CTG-Δap2IV-3 parasites maintained in pH 7.0 and 7.8 media (32 to 36 h postinfection) or following 48 h of exposure to pH 8.2 medium with the RNeasy minikit (Qiagen) according to the manufacturer’s instructions. Two biological replicates were prepared for each parasite line under each experimental condition, and RNA quality was assessed with the Agilent Bioanalyzer 2100 (Agilent Technology). Following fragmentation, 10 μg of cRNA was hybridized for 16 h at 45°C to Toxoplasma Affymetrix U133 Plus 2.0 arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. Hybridization data were analyzed with the GeneSpring GX software package .Total RNA was purified from intracellular CTG, CTG-2 flasks were inoculated with CTG-BAG1-Luc and CTG-DDAP2IV-3::BAG1-Luc transgenic parasites at a multiplicity of infection (MOI) of 3:1, parasites were allowed to invade cells for 1 h, and the culture medium was changed to either pH 8.2 or pH 7.0 medium plus or minus 100 nM Shield 1. The alkaline-stress-shifted cultures were grown under non-CO2 conditions for 48 h and then harvested for whole-cell lysates according to the manufacturer’s protocols. Each experimental condition was assessed in three independent cultures.Luciferase assays were performed according to manufacturer’s protocols  with modifications as previously described . Briefly2 flasks, allowed to invade cells for 1 h, and rinsed three times with Hanks balanced salt solution to remove free-floating parasites, and the medium was replaced with pH 8.2 medium supplemented with 100 nM Shield 1. ChIP and qPCR were performed by previously published methods (Parasites were inoculated at an MOI of 3:1 into T175 175-cm methods .GSE89469.All of the microarray data from this study have been deposited at NCBI GEO under accession no.


Schizosaccharomyces pombe is controlled by lncRNA transcription upstream of the +tgp1 permease gene. Here we demonstrate that transcriptional interference of +tgp1 involves several transcription-coupled chromatin changes mediated by conserved elongation factors Set2, Clr6CII, Spt6 and FACT. These factors are known to travel with RNAPII and establish repressive chromatin in order to limit aberrant transcription initiation from cryptic promoters present in gene bodies. We therefore conclude that conserved RNAPII-associated mechanisms exist to both suppress intragenic cryptic promoters during genic transcription and to repress gene promoters by transcriptional interference. Our analyses also demonstrate that key mechanistic features of transcriptional interference are shared between S. pombe and the highly divergent budding yeast Saccharomyces cerevisiae. Thus, transcriptional interference is an ancient, conserved mechanism for tightly controlling gene expression. Our mechanistic insights allowed us to predict and validate a second example of transcriptional interference involving the +S. pombe pho1 gene. Given that eukaryotic genomes are pervasively transcribed, transcriptional interference likely represents a more general feature of gene regulation than is currently appreciated.Long non-coding RNA (lncRNA) transcription into a downstream promoter frequently results in transcriptional interference. However, the mechanism of this repression is not fully understood. We recently showed that drug tolerance in fission yeast  The organization of DNA into chromatin poses a significant physical challenge to eukaryotic transcription. In order to activate gene expression, chromatin must be opened in manner that allows transcription factors, co-activators, and RNA polymerase II (RNAPII) access to the DNA template . Active Numerous chromatin remodelers, histone chaperones, and chromatin-modifying activities cooperate to reorganize nucleosomes and permit transcription into gene bodies . These fAs RNAPII travels away from the initiation site, the Rpb1 CTD gradually loses Ser5P and acquires Ser2P ,11. The Over the last decade, the location of complexes such as RNAPII and nucleosomes, including their variant modified forms, have been mapped genome wide in various organisms and cell types . Such sttrans, lncRNAs more frequently influence adjacent gene expression in cis. Notably, antisense transcription can compete with transcription on the sense strand to regulate gene expression (+fbp1 gene in fission yeast (Schizosaccharomyces pombe) occurs in response to glucose starvation and requires upstream lncRNA transcription to evict promoter-associated repressors and permit transcription factor binding  pathway or heterochromatin components, little is known about the mechanism that mediates this repression. Here we show that numerous conserved transcriptional elongation factors  are required to establish a repressive chromatin environment over the +tgp1 promoter in the wake of upstream initiating lncRNA transcription. Furthermore, the identification of key factors associated with transcriptional interference at the +tgp1 locus allowed us to demonstrate that a second phosphate-response gene (+pho1) is regulated by a similar mechanism. We conclude that the mechanism of transcriptional interference appears to be well conserved between S. pombe, which retains active RNAi, and the distantly related budding yeast S. cerevisiae, which lacks the RNAi pathway. Thus, RNAi does not play a role in the mechanisms that repress genes by transcriptional interference. Rather, upstream lncRNA transcription-coupled changes in promoter chromatin status underpin gene silencing by transcriptional interference in two evolutionarily distant eukaryotes.Recently we reported that the e levels . While lS. pombe strains used in this study are listed in Supplementary Table S1. Standard methods were used for fission yeast growth, genetics and manipulations (Yeast extract plus supplements) at 32°C, unless otherwise indicated. For phosphate starvation experiments, cells were grown to mid-log phase in EMMS (Edinburgh minimum medium with supplements) (Formedium), washed twice in dH2O, and then grown for indicated times in EMMS without phosphates (Formedium). nmt1-nc-tgp1 cells were grown in phosphate-rich PMG  medium in the presence or absence of thiamine (15 μM). Endogenous genetic manipulations were carried out by lithium acetate transformation. Selections were performed on PMG/agar plates with according auxotrophy or on YES/agar plates with appropriate antibiotic(s) and grown at 32°C. Colony PCR confirmed all endogenous genetic modifications and crosses. For spotting assays, serial (1:4) dilutions of equal numbers of cells were spotted onto YES/agar and grown at 32°C. For drug-sensitivity experiments, cells were spotted onto YES/agar with vehicle (DMSO) or TBZ (20 μg/ml), HU (10 mM), caffeine (15 mM).ulations . All str2O before being grown in EMMS without phosphates (-PO4) for 6 h. For temperature sensitive strains, cells were shifted from 32°C to the restrictive temperature of 36°C for 1 h. ChIP was performed essentially as described . For quantitative reverse transcriptase PCR (RT-qPCR) experiments, first strand cDNA synthesis was performed on Turbo DNase (Life Technologies) treated RNA using random hexamers and Superscript IV (Invitrogen) as per manufacturer's instructions. Negative controls lacking the reverse transcriptase enzyme (-RT) were performed alongside all RT-qPCR experiments. Quantitative analysis was performed by qPCR. To make RNA probes for northern analysis, DNA fragments specific to target transcripts were amplified from genomic DNA by PCR and gel-purified using the Wizard® SV Gel and PCR Clean-Up System (Promega). The T7 promoter was equipped at the end of the DNA fragment using an oligonucleotide containing T7 promoter sequence at the 5′-end (TAATACGACTCACTATAGGGAGA). The T7 promoter containing PCR products were transcribed in vitro using the MaxiScript T7 Kit (Ambion) to produce UTP-(α32P)-labelled RNA probes following the manufacturer's instructions. Unincorporated radionucleotides were removed using NucAway Spin columns (Life Technologies) according to manufacturer's instructions. The UTP-(α32P)-labelled RNA probes were hybridized to membranes overnight in church buffer  at 68°C in a rotating oven. Hybridized membranes were washed twice in a pre-warmed buffer containing 2× SSC and 0.1% SDS for 30 min at 68°C followed by two washes in a buffer containing 0.5× SSC and 0.1% SDS for 15 min at 68°C. To detect transcripts, northern blots were analyzed after 1–2 days of exposure on a Phosphor Screen (Molecular Dynamics) using a Typhoon Phosphorimager .RNA was isolated from act1+). Expression levels were expressed relative to levels detected in wild-type cells. ChIP enrichments were calculated as the ratio of product of interest from IP sample normalized to the corresponding input sample and expressed as ‘%IP.’ Error bars represent standard deviation resulting from at least three independent replicates.Primers used in this study are listed in Supplementary Table S2. Quantitative real-time PCR (qPCR) was performed using SYBR Green on a Roche Lightcycler. Data was analyzed with LightCycler 480 Software 1.5.0.39. RT-qPCR levels were calculated by normalizing product of interest to an internal reference gene  were assessed by quantitative ChIP in wild-type cells grown in the presence or absence of phosphate. These analyses revealed the presence of increased levels of Rpb1-Ser2P over the +tgp1 promoter in phosphate-replete cells (i.e. when the repressive lncRNA nc-tgp1 is actively transcribed), compared to phosphate-starved cells  . This manipulation prevented nc-tgp1 transcription in phosphate-rich conditions and results in constitutive +tgp1 expression  brings nc-tgp1 under the control of thiamine, rather than phosphate. In these cells, Rpb1-Ser2P and H3K36me3 levels were significantly higher in the absence of thiamine (i.e. when nmt1-nc-tgp1 is transcribed) than in cells grown in the presence of thiamine (i.e. when nmt1-nc-tgp1 transcription is reduced)  , suggestn Figure . To deten Figure . Consiste Figure . Additionc-tgp1 into the +tgp1 promoter directs the accumulation of H3K36me3, we next sought to determine if the presence of this mark plays a direct role in curbing +tgp1 expression. We measured +tgp1 mRNA levels in wild-type cells grown in the presence or absence of phosphate as well as in phosphate-replete cells lacking H3K36 methylation (i.e. set2Δ cells). As expected, northern analysis detected the +tgp1 mRNA in phosphate-starved cells but not cells grown in the presence of phosphate (repressed condition) , suggesting that other factors must cooperate with Set2 to mediate full repression of +tgp1. Nonetheless, these results suggest that Set2 activity contributes to the efficient silencing of +tgp1 mediated by upstream nc-tgp1 transcription.Having established that the act of transcribing ) Figure . Notablya Figure . Quantit2 Figure . This fi2Δ cells . While ts Figure , or phos+tgp1 expression renders S. pombe cells hypersensitive to growth in the presence of various compounds  were more resistant to growth in the presence of TBZ, HU, and CAF than cells lacking Set2 alone  and that this would result in hypoacetylated chromatin over the region upstream of +tgp1. ChIP analyses revealed that the levels of histone acetylation (H3K9ac and H4K12ac) over the +tgp1 promoter were highest in cells with active +tgp1 expression (phosphate-starved cells), while much lower levels of histone acetylation were observed in wild-type cells grown in the presence of phosphate  exhibited partial +tgp1 induction  travel with RNAPII during transcription elongation and perform two main tasks: (i) disassembly of nucleosomes to permit transcription elongation and (ii) reassembly of nucleosomes in a manner that helps suppress intragenic transcription initiation  lacking the activity of Spt6 (spt6-1) or FACT (spt16-18) are substantially reduced over the +tgp1 promoter . We conclude that the histone chaperones Spt6 and FACT are instrumental in mediating differences in nucleosome density to repress +tgp1 activation in response to nc-tgp1 transcription. However, we note that similar to results obtained in mutants defective in Set2 and Clr6CII, loss of either Spt6 or FACT leads to only the partial activation of +tgp1 expression. It is likely that these and other elongation factors cooperate to mediate complete transcriptional interference.Active promoters display nucleosome-depleted regions, whereas repressed gene promoters exhibit increased nucleosome density . Indeed,cription . Histoneitiation . Thus, tr Figure . DecreasA Figure  and C. C+tgp1 regulation by upstream lncRNA transcription requires several conserved transcription elongation factors to direct the assembly of repressive chromatin over the +tgp1 promoter. To identify other genes that may be similarly regulated we surveyed other nutritionally regulated genes in S. pombe that display similar hallmarks of transcriptional interference . A prime candidate was the +pho1 gene, which exhibits exosome-sensitive lncRNA transcription that initiates upstream and overlaps the phosphate-regulated +pho1 gene  . Im. Imtgp1+o1+ gene . Neverthnditions –54. In cgure S2) . Therefo+pho1 regulation by transcriptional interference, our analyses demonstrate that +pho1 expression was induced in phosphate-replete cells lacking Set2 , Spt6 (spt6-1) and FACT (spt16-18 or pob3Δ) . Notably, +pho1 is not induced in cells lacking factors involved in heterochromatin formation, such as RNAi (Ago1) or H3K9 HMT (Clr4) –defective cells  . These results suggest that heterochromatin accumulation might be a secondary mechanism contributing to +pho1 regulation in conditions that reduce or impair exosome function.Consistent with 2 Figure . We note2Δ cells . In addigure S2) ,49,51. T+pho1 regulation by transcriptional interference since the levels of Rpb1-Ser2P, histone H3, and H3K36me3 were all substantially reduced over the +pho1 promoter in wild-type cells following phosphate starvation , the removal of Pho7 from these cells reduced the level of tgp1+ induction and completely prevented pho1+ expression from exceeding wild-type repressed levels . This difference in dependence of tgp1+ and pho1+ activation on Pho7 is expected as partial +tgp1 induction has previously been observed following phosphate-starvation in pho7Δ cells, whereas +pho1 expression is fully dependent on Pho7   to allow the transcriptional elongation phase of RNAPII to deposit sufficient H3K36me3 over the SER3 promoter (+tgp1 and +pho1 repression are each initiated ∼2 kb upstream of their respective target gene (S. pombe along with those in the evolutionarily distant S. cerevisiae demonstrate that transcription elongation-associated chromatin changes are pivotal for mediating transcriptional interference in eukaryotes.Research from diverse organisms suggests that transcription elongation is itself too rapid to mediate strong repression of downstream genes . In bact binding . Here we Figures  and 6. T Figures . Moreove Figures ,72, whic by Set2 , suppresactivity . To explpromoter . This expromoter . Our datget gene ,51. It iS. pombe and S. cerevisiae, two highly divergent yeast species separated by hundreds of millions of years of evolution, will also be conserved in metazoans. Indeed, Spt6 and FACT have already been implicated in transcriptional interference of the Ubx gene in Drosophila (Since numerous elongation factors that are known to prevent intragenic transcription initiation also contribute to the effectiveness of gene silencing by transcriptional interference, we suggest that gene promoters occluded by upstream initiating transcripts are in essence themselves ‘cryptic promoters’ residing within the interfering transcription unit. Importantly, elongation factors are widely conserved in eukaryotes. A prediction therefore is that the basic mechanistic features of transcriptional interference revealed in both osophila , while Hosophila ,64. Exteosophila , should osophila –65 imply


Open chromatin profiling integrates information across diverse regulatory elements to reveal the transcriptionally active genome. Tn5 transposase and DNase I sequencing-based methods prefer native or high cell numbers. Here, we describe NicE-seq (nicking enzyme assisted sequencing) for high-resolution open chromatin profiling on both native and formaldehyde-fixed cells. NicE-seq captures and reveals open chromatin sites (OCSs) and transcription factor occupancy at single nucleotide resolution, coincident with DNase hypersensitive and ATAC-seq sites at a low sequencing burden. OCSs correlate with RNA polymerase II occupancy and active chromatin marks, while displaying a contrasting pattern to CpG methylation. Decitabine-mediated hypomethylation of HCT116 displays higher numbers of OCSs.The online version of this article (doi:10.1186/s13059-017-1247-6) contains supplementary material, which is available to authorized users. The mammalian genome is largely packaged into chromatin consisting primarily of DNA, proteins, and RNA. This macromolecular structure is further condensed into larger folded structures, such as chromosomes, during cell division. The cell cycle phase and the transcriptional status of the cell influences the state of the chromatin. It often undergoes remodeling events for switching between closed and open conformations to provide accessibility to DNA-binding proteins, including transcription factors –3. In adA series of genome-wide methods and approaches for mapping chromatin accessibility (open chromatin), nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding have been established to decipher the epigenetic information encoded in the chromatin –14. EarlTwo other well-adapted methods for the identification of open chromatin and regulatory sites across the genome are FAIRE-seq  and ATAC-seq (assay for transposase-accessible chromatin using sequencing) –23. FAIRHere we report a robust and sensitive method, nicking enzyme assisted sequencing (NicE-seq), for epigenetic profiling of the mammalian chromatin that can provide in-depth open versus closed chromatin sequence information from limited amounts of either native or fixed cells. We used NicE-seq to identify regions of transcription factor occupancy and compared these to DHSs identified by DNAse-seq and ATAC-seq identified sites. Finally, we studied the dynamic open chromatin sites (OCSs) of human colorectal cancer HCT116 cells before and after chemotherapeutic treatment with the DNA demethylating drug decitabine.Escherichia coli DNA polymerase I plus dNTP mix supplemented with TexasRed-dATP to demonstrate the labeling of OCSs. The cells were scored for TexasRed-dATP incorporation and compared to DAPI for open chromatin index (OCI) measurement. Indeed, the cells without Nt.CviPII and E. coli DNA polymerase I (control) displayed no TexasRed signal . We hypothesized that Nt.CviPII would be able to travel across the nuclear membrane and nick open chromatin regions inside the nucleus. We used formaldehyde-fixed cell nuclei in buffer containing various concentrations of Nt.CviPII for a fixed time, extracted DNA, and analyzed the product on a 1% agarose gel. When sufficient enzyme was added, high molecular weight genomic DNA was digested and nucleosome banding patterns were observed, confirming the activity of Nt.CviPII on chromatin inside the nucleus. To avoid false positives in open chromatin nicking and to facilitate random and optimal nicking by the enzyme, we chose a median concentration of 2.5 unit of Nt.CviPII/μg of chromatin for all downstream experiments Fig. . The nicnal Fig. , althougeus Fig. . In the eus Fig. . Thus, bThe genomic DNA from the biotin labeling reaction was purified, fragmented, and captured using Streptavidin beads for library construction. Streptavidin-captured DNA from putative open chromatin regions was used for high-throughput sequencing. The sequence information was analyzed by various peak-calling packages, such as MACS2, SICER, ZINBA, and PeaKDEcK, and about half of the peaks were common to all methods  cells and obtained 79,000 OCSs, compared to 87,000 for HCT116 cells. Further comparison between the OCSs of MCF7 and HCT116 cells showed an overlap of 24,000 peaks, suggesting the presence of both common and unique OCSs specific to the two cell types Fig. . A genomTo further our understanding of the distribution of open chromatin in the genome, we annotated the genomic features of open chromatin regions identified by NicE-seq. The open chromatin peaks were mostly concentrated at gene promoters, specifically transcription start sites. In the genic region both introns and coding exons displayed varying degrees of open chromatin status Fig. .ENCODE datasets for various active chromatin marks and DNA-binding protein factors were investigated using OCSs identified by NicE-seq for further validation of the technique. We determined the distribution of tag densities for various ChIP-seq experiments  in a ±3-kb window around the OCSs identified by NicE-seq and generated heat maps. NicE-seq heat maps positively correlated with RNA pol II binding, suggesting that most of the OCSs may be transcriptionally active. Furthermore, the signature transcriptional activation marks H3K27ac and H3K4me3 displayed strong enrichment around OCSs in the heat map. Also, H3K4me1, which is mainly enriched in the enhancer regions, was more depleted at the OCS peaks and showed a bimodal distribution around OCSs Fig. . InteresTranscription of mammalian genes is regulated by promoter elements and transcription factors. Transcription start sites (TSS) are speculated to be devoid of nucleosomes for transcription factor accessibility and have characteristic histone marks. NicE-seq tags were highly enriched at the TSS region of genes in HCT116 cells Fig. . We analp value of 2.2e−16. This suggested a high level of correlation between the two methods , exhibiting strong correlation between OCSs and DHSs as seen in the heatmap  were unique to CTCF and showed no enrichment for RNA pol II occupancy, suggesting binding of CTCF is associated with intergenic or closed chromatin status Fig. . We furtFor validation of transcriptional status, we analyzed the distribution of the active chromatin marks H3K4me3 and H3K27ac in peaks common to ChIP, NicE-seq, and DNase-seq (bin II), peaks common to ChIP and NicE-seq (bin III), and peaks common to ChIP and DNase-seq (bin IV). Indeed, peaks from bin II displayed high enrichment of H3K4me3 and H3K27ac marks, corresponding to transcriptional activation  and DNase-seq  for GM12878 cells displayed an overlap of 57,697 peaks (48% of DNase-seq peaks), suggesting half of the open chromatin regions are method specific  and 60,640 peaks (70% of NicE-seq peaks for HCT116 cells)—overlapped between ATAC-seq and NicE-seq by at least a single base . We hypothesized that genome demethylation would lead to open chromatin and transcriptional activation of previously silenced genes. To test our hypothesis, we treated HCT116 cells with decitabine and measured levels of genomic 5mC. As expected, we observed a gradual demethylation of the genome and by the sixth day of treatment 5mC was reduced by 75% Fig. . Mean meGenome-wide studies using DNase-seq, FAIRE-seq, and ATAC-seq have provided crucial clues to regions of transcriptional regulation. Humans have approximately 1700 to 1900 transcription factors, which bind different types of DNA elements, including promoters, enhancers, silencers, insulators, and locus control regions. By comparing data from ChIP-seq, microarray, and genome-wide studies to detect open chromatin, one would be able to decipher crucial clues about gene regulation, particularly transcription. Classically, DHSs according to cell type specificity have been classified as common or unique. In one study, 900,000 DHSs were identified in 29 cell lines, accounting for almost 8.3% of the human genome . CategorWe have compared the random nicking activity of DNase I with a sequence-specific nicking enzyme, Nt-CviPII, and its impact on open chromatin identification. Nt-CviPII recognizes the CCD trinucleotide sequence and these sites are densely dispersed throughout the human genome. We have also demonstrated that the frequency of nicking sites has no bearing on open chromatin enrichment; rather, accessibility to the open chromatin by the nicking enzyme is the major determinant of tag reads of OCSs. However, when DHS and OCS reads were compared for HCT116 cells, about a third from either method remain unique. Additional transcriptional and histone mark analysis and comparison of these unique sequence tags revealed that the OCS peaks are more enriched in H3K4me3 and H3K27ac, suggesting Nt-CviPII has a higher specificity than DNase I, resulting in more accurate open-chromatin region determination. Furthermore, OCSs between two different cancer-derived cell lines demonstrated cell type-specific and common open chromatin regions, suggesting conservation of OCSs in mammalian cells. Comparison between NicE-seq, ATAC-seq, and DNase-seq demonstrated both unique and common open chromatin regions. This may be attributed to sequence and structural bias of DNase I or Nt-CviPII or biased tagging of DNA by the transposase used in ATAC-seq. Therefore, for limited numbers of cells, both NicE-seq and ATAC-seq may be performed to negate sequence bias.When we used NicE-seq to determine the impact of decitabine-mediated hypomethylation on OCSs, we observed large numbers of OCSs compared to the control genome. DNA methylation of the genome attracts methyl CpG binding proteins and associated transcriptional repressors to confer a heterochromatin structure. In the absence of a DNA methylation signature, the heterochromatin structure is destabilized  and presTherefore, NicE-seq is a straightforward method that can be performed on potentially any cell type from any species with a sequenced genome. The protocol described here works for cell numbers between 250 and 25,000. Below 250 or above 25,000 cells the dynamic range of the current NicE-seq protocol is compromised, resulting in a drop in the number of peaks. This could be overcome by optimizing the concentration of nicking enzymes, reaction conditions, and cell numbers. Furthermore, different cell types and cells with different chromatin compaction, such as mitotic (compact chromatin) versus replicating (relaxed chromatin), will offer challenges to nicking enzyme catalysis. This may result in under representation of OCS for compact chromatin regions. The sequence specificity of Nt.CviPII (CCD) may cause it to misidentify AT-rich open chromatin sequences and thus may not be suitable for AT-rich genome studies. However, NicE-seq has similar resolution to ATAC-seq and DNase-seq in the human genome and no prior knowledge is required with regards to histone modifications, transcription factor binding sites, gene annotation, or relative degree of sequence conservation between species. It can aid in the identification of the most active gene regulatory elements in order to understand the chromatin landscape during mammalian development programming and epigenetic drug discovery studies.We demonstrate that the sequence-specific nicking enzyme Nt.CviPII can be used to probe open chromatin landscapes in a variety of cancer cells. NicE-seq captures OCSs in both fixed and native cells and reveals the genomic location of these and transcription factor occupancy at single-nucleotide resolution, with as few as 25 cells. Using NicE-seq we have demonstrated that colorectal cancer cells treated with the anti-cancer drug decitabine displayed time-dependent accumulation of OCSs coinciding with hypomethylation of the genome, particularly in SINEs and satellite repetitive DNA. DMRs were more pronounced in promoters, 5′ UTRs, CpG islands, and exons. Therefore, NicE-seq offers a quick and reliable method for open chromatin analysis in biological and pharmacological studies. We envision that when NicE-seq is coupled with fluorescent-labeled dNTPs it will facilitate visualization of open chromatin in eukaryotic cells.HCT116 cells were cultured in McCoy’s 5A media supplemented with 10% fetal bovine serum (FBS). Cells  were treated with 5 μM 5-aza-2′-deoxycytidine (decitabine) for 6 days . Samples2, 60 mM KCl, 0.5 mM DTT, 15 mM NaCl, 300 mM sucrose, and 1% NP40) for 10 min on ice with occasional agitation. Nuclei were precipitated by spinning at 1000 × g, 4 °C for 5 min and the supernatant was discarded. Open chromatin DNA was labeled with biotin by incubating the nuclei in the presence of 2.5 U of Nt.CviPII (NEB R0626S), 10 U of DNA polymerase I (M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP  and 6 μM of biotin-16-dCTP  in 200 μL of 1× NEB buffer 2. The labeling reaction was carried out at 37 °C in a thermo-mixer at 800 RPM for 2 h. We added 20 μL of 0.5 M EDTA and 2 μg of RNase A to the labeling reaction and incubated it at 37 °C for 0.5 h to stop the reaction and digest RNA. Finally DNA–protein cross-linking was reversed by adding 20 μL of proteinase K  and 20 μL of 20% SDS to the reaction and incubating at 65 °C for overnight. Biotin-labeled genomic DNA was extracted using phenol chloroform. Open chromatin labeling in native cells was performed without the formaldehyde crosslinking step.For library construction, we used HCT116 and MCF7 cells. Cells were cross-linked using 1% formaldehyde for 10 min at room temperature and quenched by using 125 mM glycine. Nuclei were isolated by incubating the cross-linked cells in cytosolic buffer . The cells were fixed using 1% paraformaldehyde for 10 min and then washed thrice with 1× PBS for 5 min followed by incubation with cytosolic buffer for 10 min. The nicking reaction mix was as described earlier but with biotinylated dATP and dCTP replaced by TexasRed-dATP  for 1 h followed by washing the cells with 1× PBS supplemented with 50 mM EDTA and 0.1% TritonX-100 (wash buffer) for 5 min. Nuclear staining was performed by 0.01% Hoechst stain for 5 min. Finally, cells were washed thrice with wash buffer for 10 min each and visualized using a Zeiss LSM880 confocal microscope with 20× objective.Open chromatin labeling efficiency was analyzed by dot blot on genomic DNA and by imaging labeled cells. A serial dilution of genomic DNA was spotted onto positively charged nylon membrane  and cross-linked by UV. Membrane was blocked by 5% non-fat milk and probed using an HRP-conjugated goat anti-biotin antibody . Biotin signal was revealed using LumiGLO reagent .Biotin-labeled genomic DNA from fixed  and native (HCT116 cells) reactions were sonicated into 150-bp fragments (Covaris) and 1 μg of DNA was end-repaired, dA-tailed, and ligated with NEB Illumina adaptor (E7370S). Without further purification, the ligation product was mixed with 50 μL of Streptavidin magnetic beads  in 1 mL of B&W buffer . Biotin-labeled open chromatin DNA was captured by streptavidin magnetic beads at 4 °C for 2 h with end-over-end rotation. The beads were washed four times with B&W buffer plus 0.05% of Triton X-100 followed by one wash with TE plus Triton X-100. The beads were resuspended in 40 μL of nuclease-free water and 4 μL was used for library amplification using PCR . Ten PCR cycles were usually sufficient to generate the amount of library DNA needed for sequencing.For library construction using 250 cells, all procedures remained the same except for the addition of 10 μg of glycogen during genomic DNA extraction. In the case of the 25-cell experiments, 0.25 U of Nt.CviPII and 5 U of DNA polymerase I were used. The entire DNA was sonicated and 10 μL of streptavidin beads were added to capture the biotinylated DNA, which was used as template for library amplification. For input library preparation 1 μg of purified genomic DNA was used instead of the labeled chromatin.ATAC-seq was performed on 25,000 HCT116 cells according to the previously described protocol  with thehttp://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with default settings. Sequencing reads were mapped to reference human genome hg19 with Bowtie2 [Adaptor and low quality sequences were trimmed from paired-end sequencing reads using Trim Galore , we downsized the mapped reads from different experiments to 14.4 million mapped read pairs. Peaks were called using the same parameter with MACS2 as mentioned above.To exclude the possibility that open chromatin DNA enrichment is solely caused by the occurrence of Nt.CviPII sites  in certain genomic regions but not the accessibility of chromatin by labeling enzymes, we did correlation analysis on CCD site density and open chromatin tag density in 100-bp genomic tiles or open chromatin peak regions called by MACS2. Hg19 was scanned for Nt.CviPII sites using an in-house Perl program and the genomic coordinates of Nt.CviPII sites were recorded. Nt.CviPII site density was expressed as sites per kilobase genomic sequence. Pearson linear correlation was implemented on the number of Nt.CviPII sites and the number of sequencing tags or Nt.CviPII site density and log2 fold enrichment of open chromatin tags in MACS2 peak regions. The Pearson product-moment correlation coefficient was used to measure the degree of correlation.We compared NicE-seq, ATAC-seq with DNase I hypersensitivity sequencing (referred throughout as DNase-seq) for sensitivity and specificity. DNase-seq data was downloaded from ENCODE (wgEncodeUwDnaseHct116AlnRep1 and wgEncodeUwDnaseMcf7AlnRep1) . ATAC-seOpen chromatin peaks identified by NicE-seq were correlated with ChIP-seq targeted to H3K4me1, H3K4me3, H3K27ac, RNApolII, and YY1 identified in the HCT116 cell line . The sequencing tag density of chromatin and transcription factor marks on open chromatin peaks was counted using HOMER , and heaFor correlation between CTCF occupancy and chromatin status, peaks for the ChIP-CTCF data set from ENCODE (wgEncodeEH003220) were called using MACS2. Overlap between peaks identified by ChIP-CTCF, NicE-seq, and DNase-seq was determined by plotting a Venn diagram using DiffBind . FurtherBased on the expression level, all of the genes discovered by RNA-seq were divided into four groups, high, medium, low, and no expression. RNA-seq data were from a previous study using the HCT116 cell line  . SequencGenomic DNA of control and decitabine treated (days 2 and 6) was digested with NEB nuclease cocktail for 1 h and nucleoside analysis was performed using liquid chromatography–mass spectrometry for quantification of 5mC levels .Genomic DNA (1 μg) isolated from HCT116 cells treated with 5 μM 5-aza-2′-deoxycytidine for 6 days or untreated control was sonicated into 150-bp fragments, bisulfite treated, library prepared, and sequenced on the Illumina NextSeq 500 platform with 150-bp paired-end reads for whole genome bisulfite sequencing analysis. Adaptor and low quality sequences (Phred score <20) were trimmed and reads were mapped to hg19 using Bismark with Bowtie2 . CpG met


Transposable elements (TEs) comprise nearly half of the human genome and play an essential role in the maintenance of genomic stability, chromosomal architecture, and transcriptional regulation. TEs are repetitive sequences consisting of RNA transposons, DNA transposons, and endogenous retroviruses that can invade the human genome with a substantial contribution in human evolution and genomic diversity. TEs are therefore firmly regulated from early embryonic development and during the entire course of human life by epigenetic mechanisms, in particular DNA methylation and histone modifications. The deregulation of TEs has been reported in some developmental diseases, as well as for different types of human cancers. To date, the role of TEs, the mechanisms underlying TE reactivation, and the interplay with DNA methylation in human cancers remain largely unexplained. We reviewed the loss of epigenetic regulation and subsequent genomic instability, chromosomal aberrations, transcriptional deregulation, oncogenic activation, and aberrations of non-coding RNAs as the potential mechanisms underlying TE deregulation in human cancers. LINE1 sequences represent up to 17% of the human genome [LINE1 is considered the most active mobile element in a human, around 500,000 copies no longer have the ability to mediate retrotransposition due to significant LINE1 truncations [LINE1 would be able to encode proteins that are very efficient to “copy and paste” into a new genetic location and hamper the associated gene expression and/or drive oncogenic process through the transcription of chimeric proteins [Over two-thirds of the human genome is composed of repetitive sequences . Transpon genome ,4. AlthoActive TEs are considered highly mutagenic and are associated with the multiple steps of cancer development and progression ,6. TEs hBRCA2 [APC [RB1 [LINE1 promoter is able to activate an alternative transcript that encodes a truncated and constitutively active MET protein in bladder cancer [LINE sequences cause an alternative transcription of a new isoform in an ALK (anaplastic lymphoma kinase) gene [ALK isoform is specifically expressed in around 11% of melanomas that further show a specific response to the ALK inhibitor [FABP7); that are known to promote lymphomagenesis [ERBB4 in ALK-negative anaplastic large cell lymphoma (ALCL) [ERBB4 transcripts are found in almost a quarter of ALK-negative ALCL patients [A growing body of evidence has documented the essential role of TEs in human carcinogenesis. The insertion of TEs into genes that are responsible for DNA repair including BRCA2 , APC [13APC [RB1  can causAPC [RB1 . Methylar cancer . De novose) gene . The newnhibitor . A compragenesis . The reaa (ALCL) . Two abepatients . DespiteTEs are tightly regulated from early embryonic development and during the entire human life . EpigeneLINE1 has been reported in colorectal cancer [TEs have been suggested to play an essential role in maintaining genome integrity  and therl cancer , hepatocl cancer , and brel cancer . Figure LINE1 is reported in both solid tumors and leukemia, and is frequently correlated with unfavorable clinical outcomes [LINE1 and Alu in circulating blood has potential value for cancer diagnoses [LINE1 demethylation, as well as subsequent activation, and is followed by oncogene activation [The hypomethylation of outcomes ,35. In aiagnoses . Exposurtivation . The humtivation . In additivation . Ovariantivation .SUV39 cause TE upregulation [SINEs, VNTRs, Alus), suggesting a potential role of BORIS in the regulation of active TEs in cancer cells [Heterochromatin formation has also been associated with the regulation of TE silencing in cooperation with DNA methylation and small RNAs . Histonegulation . In addigulation ,41. Specgulation ,31. Chrogulation . Severalgulation ,44. A reer cells . DDM1, Ler cells . Especiaer cells . Howeverer cells ,46. Uponer cells ,48. Howeer cells ,50.Alu sequences are commonly found to create new exons in mature transcripts upon insertion [Alu insertions in the exons caused either a frame-shift or premature stop codon [Alu elements [Alu element [TP53 gene cause an elevated activity of RNA polymerase III [Alu sequences accompanied by transcriptional repression can trigger the deregulation of multiple genes [TEs are able to modulate gene transcription by insertion into transcriptional regulatory regions . TE insensertion ,53. Soreop codon . In addiop codon , causingop codon , SP1 [56op codon , P53 [58op codon , have bi element . Mutatiorase III . Levels rase III . It seemle genes .cis-regulatory elements of gene expression [TE insertions within the genome are considered to be non-random events, as the insertions might affect transcriptional regulation ,63. It hpression . The effAlu element into the NF-1 gene has been shown to cause deletion and reading frame-shift, leading to the development of neurofibromatosis [TEs also interact with non-coding RNAs, including microRNAs and lncRNAs, to modulate transcription, as discussed below. TEs use other mechanisms to influence gene expression, including the spreading of repressive chromatin signals to adjacent regions in order to silence the nearby genes . In addiomatosis .Alu and short sequence repeats that are inversely inserted during DNA replication tend to form a secondary hairpin structure that is susceptible to double-strand breaks (DSBs) [Alu elements in the human genome is relatively rare. In some parts, there is a tendency for the direct orientation of newly adjacent Alu insertion to prevent secondary hairpin formation [Alu insertion, the fact that unregulated Alu insertions frequently occur in cancer has increased the probability of de novo inverted insertions [The vast majority of TE insertions target particular genomic loci that are specifically recognized by endonuclease domains causing alterations of adjacent gene expression and significant genomic deletions . Under cs (DSBs) ,91. Howeormation . Despitesertions .The second mechanism of TE-mediated genomic instability is the induction of unstable microsatellite seeding after TE insertions . MutatioTE-mediated genomic instability is also associated with alterations in the overall transcription in cancer. As previously mentioned, TE mobility can induce aberrant and recombinant gene transcription . The intBRCA2 has been affected by multiple TE insertions in cancers [APC gene is also disrupted by multiple TE insertions (Alu and LINE1) and is suggested to play a role in colorectal carcinogenesis [LINE1 transposition causes DNA breakage and, even after DNA repair, this event still produces the deletion of a few base pairs [LINE1 activation and is associated with the initiation of carcinogenesis [The induction of genomic instability by TE insertion will induce host DNA repair signaling. The recruitment of the DNA repair machinery to the TE insertion loci is accompanied by cell cycle arrest affecting the relative efficiency of TE mobility. However, TE insertion also targets genes that are involved in the DNA repair pathway. The breast cancer susceptibility gene  cancers ,98. In aogenesis ,99. Voinogenesis . LINE1 eogenesis . In addise pairs ,102. In ogenesis . TE-assoAlu sequences are frequently found in several breakage points of chromosomal translocations and are closely associated with cancer [LINE1 and Alu insertions [LINE1 insertions often target endogenous elements, suggesting a preference for specific incorporation sites [TE insertions have also been associated with chromosomal rearrangement. h cancer . TE insesertions ,122. TE-on sites ,121. Chron sites ,123.Alu repeats within or near the duplication intersections [Alu repeats can initiate segmental duplications [Alu and other TE species induce segmental duplications remain unclear. Homologous repair is able to generate duplications by arranging replicons tandemly. However, the mechanism underlying large duplication events with a distance of more than 1 Mb remains elusive. The relatively frequent Alu element insertions at the junction of the segmental duplication, accompanied by the involvement of evolutionarily young and identical elements, suggest that homology sequences guide these events [TE-associated chromosomal rearrangements are also mediated by segmental duplications that are relatively common in the human genome . Segmentsections . In humaications ,125. Howe events ,125. TE-LINE1, Alu, and SVA are among the most common TEs that frequently induce insertional mutagenesis [Random integrations of TEs into specific sites of the human genome increase the chance for insertional mutagenesis followed by the activation of signaling pathways leading to carcinogenesis ,15. LINEagenesis . The actagenesis ,134. In agenesis ,127.Alu is abundantly found in tumors with TP53 mutations [Alu insertions tends to impose an inverted orientation, leading to the loss of p53 functions [Alu sequences are also involved in mismatch repair (MMR) by disrupting MLH1 and MLH2 genes [Alu insertion and the presence of Alu in the MLH1 and MLH2 proteins are associated with hereditary non-polyposis colorectal cancer [Alu elements within the BRCA1 gene are associated with the important deregulation of genomic integrity in breast cancer [Alu repeats have also contributed significantly to chromosomal translocations, including BCR/ABL rearrangement in chronic myelogeneous leukemia [Alu has also caused myoblastosis (MYB) duplication to T-cell acute lymphoblastic leukemia [Alu recombination [Alu rearrangements have been reported in the tumor suppressor gene von Hipple-Lindau (VHL) [Alu sequences [Non-allelic homologous recombination causing deletions or duplications in the presence of utations . The clounctions . Alu seqH2 genes . In addil cancer . Recombit cancer ,98. Alu leukemia . The recleukemia . Chromosbination . Especiabination . Additioau (VHL) . Compareequences .LINEs, and SINEs [hsa-mir-548 that derives from inverted-repeat transposable elements [The interconnection between TEs and non-coding RNAs (ncRNAs) has been recently delineated, especially in the biogenesis of small ncRNAs, including microRNAs that are associated with TEs. MicroRNAs are small non-coding RNAs that regulate post-transcriptional gene expression and modulate several important oncogenic pathways establishing a dynamic network of cell homeostasis . A nd SINEs . The firelements . The derelements ,145.Alu insertions are able to create a targeting site for mRNA decay by forming stable RNA/mRNA interactions through intra- or intermolecular base pairing with 3′UTRs and complementary binding with RNA targets [Alu sequences that flank exon and the alternative formation of inverted Alu pairs are suggested to greatly contribute to RNA circularization [Alu and LINE-embedded sequences also have regulatory roles for the transcription of lnc-RNAs and the stability of mRNA products [SINEB2 and Alu elements that can interact with targeted mRNAs to inhibit the translation. [SINE/Alu elements that might interact with 3′UTR mRNAs, suggesting an important role of TE-embedded regions in the post-transcriptional regulation of gene expression [It has also been indicated that  targets . Hundred targets . Numerourization ,149. Theproducts . Since lproducts ,150,151.slation. . They alpression .trans enrolment of Ago complexes to subsequently regulate genome instability through transcriptional repression and repetitive DNA element recombination [Alu sequences occupy a large portion of microRNA genes, as well as 3′UTR mRNA targets [Reciprocally, small non-coding RNAs including microRNAs have also been reported to regulate genomic stability through direct or indirect transcriptional and post-transcriptional TE repression . As cytobination . In addibination . An initbination . Another targets . In geneAlu-derived piRNAs are able to bind some nuclear proteins, suggesting their potential roles in DNA repair, chromatin reprogramming, and cell proliferation [LncRNAs have been associated with myriads of important signaling pathways, most of which require an interaction with proteins, as well as transcription factors. TE-associated lncRNAs are reported to bind to some important transcription factors for cell proliferation, including p53, sp1, and NF-Y . In respferation . TherefoLINE1 sequences [LINE1 sequence [The PIWI-piRNA axis has also been implicated in the silencing of transposable elements and contributes to the development of human cancers . Transcrequences . One of equences ,163. In equences . In the equences . The piRsequence . Correspsequence .The above compiled results and observations suggest an important role of TEs in transcriptional control, genomic instability, chromosomal rearrangements, non-coding RNA regulation, and oncogenic activation. Although a direct association between TE insertion and cancer initiation still has to be clarified, the deregulation of TEs is closely linked to cancers of various etiologies. Bioinformatic screens have revealed mutagenenic TE insertions in different types of cancers. TE insertions also function as a reservoir of endogenous gene regulatory factors that have been co-opted by the human genome to control gene expression, as well as cellular phenotypes. After co-option or exaptation, insertions of TEs can provide novel transcription factor binding sites in the promoters, enhancers, or insulators, leading to cancer specific activation. TEs flanking ncRNA genes are also involved in ncRNA biogenesis. In addition, repetitive sequences within TEs are important loci for the complementary binding of ncRNAs acting as a reservoir for ncRNA inactivation. Therefore, the deregulation of TEs affects genomic stability, transcription, and non-coding RNA regulation, leading to cancer development and progression (please see