With the development of genomics and bioinformatics, especially the extensive applications of high-throughput sequencing technology, more transcriptional units with little or no protein-coding potential have been discovered. Such RNA molecules are called non-protein-coding RNAs (npcRNAs or ncRNAs). Among them, long npcRNAs or ncRNAs (lnpcRNAs or lncRNAs) represent diverse classes of transcripts longer than 200 nucleotides. In recent years, the lncRNAs have been considered as important regulators in many essential biological processes. In plants, although a large number of lncRNA transcripts have been predicted and identified in few species, our current knowledge of their biological functions is still limited. Here, we have summarized recent studies on their identification, characteristics, classification, bioinformatics, resources, and current exploration of their biological functions in plants. As a class of RNAs that have no or little protein-coding potential, the mechanism underlying the functions of non-protein coding RNAs (ncRNAs or npcRNAs) is a fascinating area of research e.g., rRNAs and tRNAs) and processing of pre-mRNA The canonical ncRNAs such as ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) were discovered earlier owing to their important functions in protein synthesis in all living organisms. Small RNAs (sRNAs), for instance, small nucleolar and small nuclear RNAs (snoRNAs and snRNAs) are found in specific cellular locations, which can function through modification of other RNAs  were adjacent to or located as an extension of the annotated genes. Besides poly(A)+ lncRNAs, lncRNAs without poly(A) tails (poly(A)− lncRNAs) were also identified in humans − lncRNAs was revealed in seedlings of A. thaliana under different stress conditions using RNA-seq + lncRNAs, poly(A)− lncRNAs are shorter, have lower expression, and are more specific in response to stresses.About 6480 lncRNAs were identified from 200 cis-NATs were shown to be expressed, among which 503 cis-NATs were expressed under specific conditions Combining both computational and experimental analyses, Xin et al. Arabidopsis, rice, and maize, Qi et al. identified 584 lncRNAs that were responsive to simulated drought stress in foxtail millet by using a deep transcriptomic sequencing approach trans to suppress the expression of target transcripts)-producing loci (PHAS) genes in soybean PHAS genes, although some of these miRNAs can be further regulated by eTMs Brassica rapaBesides lncRNAs identified in the model plant Arabidopsis among model plants. Information on chromatin states should be obtained in other model plants and crops to assist the identification of TARs in the future Other than direct transcriptomics analysis, chromatin signature-based approach was also used to define TARs. K4–K36 domain is usually used to define TARs, since active promoter that is marked by H3K4me3 usually combines with TARs that are marked by H3K36me3. In humans and mice, many lncRNAs were identified by the presence of K4–K36 domains in the intergenic regions As an emerging class of riboregulators, lncRNAs either act directly or are processed to shorter ncRNAs for functioning Arabidopsis. Most siRNAs derived from these five lncRNAs are mapped to both strands of the lncRNA region, suggesting that these lncRNAs are siRNA precursors Arabidopsis, Ma et al. Analysis of the full-length cDNA databases led to the identification of numerous 24-nt siRNAs that were matched with five lncRNAs including npc34, npc351, npc375, npc520, and npc523 in Target mimicry was first found in plants, rising as a novel mechanism for regulating miRNA functions Induced by Phosphate Starvation 1 (IPS1) was first identified in A. thaliana, which functioned as an eTM of miR399 IPS1 binds to miR399 and destroys the miR399-mediated cleavage of its target genes. Thus, IPS1 interferes with the binding of ath-miR399 to its authentic targets as a decoy Arabidopsis and rice were systematically identified in intergenic or non-coding gene regions by Wu and colleagues As an endogenous lncRNA, Target mimicry effects can be induced by both endogenous and engineered artificial miRNA TMs FLOWERING LOCUS C (FLC) gene Flowering time is one of the most important adaptive traits to ensure the transition of reproductive growth and development that occurs under favorable conditions during a plant’s life cycle FLC is a key regulator of flowering time in A. thalianaFLC gene is located at a complex locus. Recent studies have shown that at least two types of lncRNAs are present in this locus. A group of long antisense RNAs, called COLD INDUCED LONG ANTISENSE INTRAGENIC RNAs (COOLAIR) are transcribed in antisense orientation in relation to FLCCOLD ASSISTED INTRONIC NONCODING RNA (COLDAIR), is transcribed from the intron of FLC gene in the sense orientation FLC via epigenetic regulation. Considering that there have been several excellent review articles on this topic long day (LD)-specific male-fertility-associated RNA (LDMAR), is necessary for rice fertility under LD conditions. The transcription level of LDMAR is reduced specifically under LD conditions, which results in programmed cell death (PCD) during rice anther development and causes male sterility Psi–LDMAR, is produced in the promoter region of LDMAR. Overexpression of Psi–LDMAR induced RdDM in the promoter region of LDMAR and resulted in reduced expression of LDMAR. Zhou et al. P/TMS12-1 (another name for LDMAR) encodes a unique ncRNA, which produces a 21-nt sRNA, osa-smR5864w. A C-to-G point mutation present in osa-smR5864w may lead to loss-of-function of the sRNA, eventually resulting in the production of light- and temperature-sensitive male sterile rice Rice is an important crop as well as an important model organism. Breeding of hybrid rice is one of the evolutionary applications of heterosis in agriculture and male sterility lines are essential for this process. However, little is known about the regulatory genes and molecular mechanisms underlying plant male sterility. Ding et al. and Zhou et al. Light is regarded as an important ecological factor to regulate almost all processes of growth and development in plants A. thalianaA. thaliana, with many light-responsive lncNATs related to histone modifications. It would be very interesting to explore the functions of lncRNAs in phototropic responses. Deng et al. HIDDEN TREASURE 1 (HID1), which is involved in the sophisticated photomorphogenic process. By screening their T-DNA insertion mutant collection, they identified a mutant named hid1 later on, which exhibits a hypo-photomorphogenic phenotype under continuous red light (cR). The mutant results from the loss-of-function of the lncRNA gene HID1. Through detailed analyses, the authors discovered that HID1 may function by regulating the expression of the transcription factor, PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), one of the key repressors in photomorphogenesis that modulates light response HID1 could negatively regulate the expression of PIF3 gene. HID1 is located in the nucleus and can associate with chromatin and may bind directly to the promoter region of PIF3 to repress its expression HID1 homologs are found in many plant species OsHID1, the rice homolog, can rescue the phenotype of hid1 mutant in A. thalianaHID1 is the first known lncRNA involved in photomorphogenesis, shedding light on the association of ncRNAs and light response in plants As aforementioned, Wang et al. had identified genome-wide lncNATs in model plant PHOSPHATE2 (PHO2), which encodes a ubiquitin-conjugating E2 enzyme (A). PHO2 can interact with PHOSPHATE1 (PHO1), a membrane protein involved in phosphate loading to the xylem and a key regulator for phosphate homeostasis conserved in plants IPS1 exemplifies the direct involvement of lncRNAs in phosphate homeostasis IPS1 is induced under phosphate deficiency and acts as a target mimic for miR399 (B) cis-natural antisense RNA, PHO1;2cis-NAT, can act as a translational enhancer for the expression of its sense gene, PHOSPHATE1;2  (C) PHO1 in ArabidopsisPhosphate is an essential mineral nutrient for plant growth and development 2 enzyme A. PHO2 cr miR399 B [61]. J C [89], tEarly nodulin 40 (ENOD40) ENOD40 participates in the regulation of symbiosis between bacteria or fungi and leguminous plants ENOD40 expression is rapidly induced by rhizobia in the nodule primordium ENOD40 can play roles in transporting metabolites necessary for cell growth in non-symbiotic plants ENOD40 encodes two short peptides but there lacks long open reading frame ENOD40 exerts its biological activity directly by translation of these two short peptides in barrel medic (Medicago truncatula) ENOD40 mRNA are found in various leguminous and non-leguminous species. Notably, one structural domain conserved in Enod40 is similar to the expansion segments in some structural RNAs ENOD40 mRNA are much more conserved than the encoded short peptides, which suggest that the RNA structure determines the principal functions of ENOD40, whereas more diverse functions, revealed in a minority of plant families, are exerted by short peptides. This hypothesis has been proved in M. truncatulaM. truncatula RNA-binding protein 1 (MtRBP1) directly interacts with ENOD40 in mature nodules, where ENOD40 is expressed at high levels ENOD40 during nodulation in M. truncatula. MtRBP1 protein could be found localized in the cytoplasm only when ENOD40 was co-expressed in these cells. Induction of MtRBP1 relocalization was similarly achieved by ENOD40 transcripts with the initial ATG mutated, indicating that ENOD40-encoded peptides are not involved in this activity. Hence, for the re-localization activity of MtRBP1, RNA structures, and not the encoded short peptides of ENOD40, are required.ArabidopsisAtNSR is mainly expressed in primary and lateral root meristems and regulates development of lateral roots. Transgenic plants overexpressing the ASCO-lncRNA exhibit an altered ability to form lateral roots, which is similar to the phenotypes of the double Atnsr mutants. AtNSRs interact with the ASCO-lncRNA in vivo and affect the splicing patterns of NSR-regulating mRNA targets. It seems that lncRNA can recruit the alternative splicing regulators to modulate the related processes Alternative splicing is an important regulatory layer in gene expression. Multiple variants of proteins or transcripts can be generated from a single gene via alternative splicing, thus increasing the complexity of proteome and transcriptome. Bardou et al. reported the involvement of lncRNA in alternative splicing in Arabidopsis full-length cDNA databases by using bioinformatics approach AUXIN REGULATED PROMOTER LOOP (APOLO) in a recent study PINOID (PID) and is transcribed by two RNA polymerases, RNA Pol II and Pol V APOLO transcription could control the chromatin loop dynamics to regulate the promoter activity of the neighbor PID gene APOLO and PID. Exogenous auxin treatment can activate the demethylation of the APOLO−PID genomic region and the chromatin loop encompassing the promoter region of PID. When the loop is opened, RNA Pol II transcribes the two genes and the accumulation of both PID and APOLO RNAs is increased. Then, the APOLO transcripts produced by RNA Pol II gradually recruit the polycomb repressive complex 1 (PRC1) to close the loop. Then the APOLO transcripts produced by RNA Pol V are recruited by ARGONAUTE4 (AGO4) and trigger DNA methylation. Finally, the APOLO lncRNAs-mediated chromatin loop is reformed and PID expression is down-regulated Dynamic chromatin topology can affect the pattern of gene expression Arabidopsis gene structure and function annotation Arabidopsis, and aims to contain comprehensive information including genomic, transcriptomic, and epigenomic information related to plant lncRNAs. PNRD aims to provide lncRNAs of 150 plant species and now contains 5571 lncRNAs of A. thaliana, Oryza sativa, Zea mays, and Populus trichocarpa only.Mammalian lncRNAs, especially human and mouse lncRNAs, were recorded elaborately in public databases The rapid development of high-throughput RNA-seq and related bioinformatics methods provides revolutionary ways for discovering novel lncRNAs Arabidopsis, rice, maize, wheat, foxtail millet, and soybean lncRNAs reported in plant species are limited to only a few model angiosperm plants such as Functional studies on plant lncRNAs are a challenge. As described in this review, the current functional studies in plants are confined to a few cases The authors have declared that no competing interests exist.


Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators.trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5’-untranslated region (5’UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5’UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5’UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5’UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5’UTRs of complex structure.We demonstrate that oligonucleotide-based trans-acting factors designed to target specific 5’UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene and may have future application in gene therapy strategies to enhance expression of proteins including tumor suppressors.This report represents the first method for gene-specific translation enhancement using selective  Initiat (uORFs)  and inte factors  such as  factors , natural factors  as well  factors  and anti factors .cis-acting element and the inhibitory uORF2 overlapping correct ATF4 ORF in an out of frame manner. Non-stressed, normal conditions allow cells for fast translation of the short uORF1 and ribosome reinitiation at the uORF2, that results in synthesis of truncated proteins. In contrast, stress conditions increase the time required for more accurate scanning that allows the ribosomes to bypass the inhibitory uORF2 and reinitiate at the downstream ATF4-coding region [trans-acting factors [Translation of most human mRNAs occurs via a 5’-cap-dependent mechanism . Certain factors , which r factors , 13. Mor factors . Therefo factors .THRB (GeneID 7068), which encodes β isoforms of human thyroid hormone receptors  [CDKN2A (GeneID 1029) [Many genes have several alternative 5’UTR splice variants that can differentially regulate translation of downstream coding sequences . One exand TRβ4)  and contnd TRβ4) , 20. MulID 1029) . Both geID 1029) , 22 and ID 1029) , 23. TheID 1029) , 23.trans-acting factors that bind to 3’UTR sequences, can also up-regulate protein synthesis [Recent studies have revealed that some naturally occurring microRNAs, considered traditionally as inhibitory ynthesis . Moreoveynthesis . Furtherynthesis . Even thynthesis , showingynthesis , these fRecently, a new type of RNA interference has been shown to result from promoter-specific activation of gene transcription  that is triggered by a novel class of interfering RNAs termed small activating RNAs (saRNAs), which target discrete promoter sequences . The saRcis-acting elements within TRβ1 5’UTRs that were recognized by dGs. Then, the translation-enhancing effects were successfully confirmed by the use of dGs design to target p16INK4a 5’UTR encoded by the CDKN2A gene. dGoligos thus offer the potential for a novel and specific gene-therapy approach to re-express or over-express individual proteins such as tumor suppressors.Here we describe a novel method for 5’UTR-specific enhancement of translation. Protein overexpression is triggered by synthetic, translation-enhancing oligonucleotides, termed dGoligos (dGs), which were originally designed to alter Gibbs energy-dependent secondary structure formation of specific sequences of TRβ1 5’UTRs. Although ΔG is a well-known measure of the stability of higher-order structures of nucleic acid molecules, we used this parameter in a new way, defined in a bespoke dGenhancer calculator. This tool allowed us to determine in vitro transcription-translation (RTS 100 Wheat Germ CECF). The basic luciferase levels served as starting points for further studies on translation-enhancing elements of the 5’UTRs. Initial results demonstrated that variants A-G differently regulate the reporter protein translation efficiency (in vitro translation system based on rabbit reticulocyte lysates [TRβ1 5’UTR splice variants A-G subcloned upstream of the luciferase reporter gene in pKS plasmids  were tesficiency . The meaficiency . We founficiency . In contficiency . These r lysates .cis-acting elements on the levels of protein expression is limited [2 = 0.9746 (p<0.005) showed that protein levels are strictly dependent on Gibbs energies of the TRβ1 5’UTRs ..potentiadatabase . We foundatabase  compareddatabase  without in silico generated 5’UTR sequence variants that differ in a single nucleotide substitution (SNP) altering overall ΔG of the sequences. These artificial variants were created by substitution (base by base) in each nucleotide position of the 5’UTRs  and translation -inhibiting element e3 uORFs,  were come 5’UTRs . Comparie 5’UTRs , locatedgos dGs, , designetrans-acting factors. We synthesized a set of DNA oligonucleotides (dGoligos) directed to interfere with previously identified TRβ1 5’UTR cis-acting elements. High translational regulatory potential was defined as the potential of the translation-regulating elements to enhance protein synthesis from low to high rates. This regulation is thought to be controlled by distant cis- or trans-acting factors specifically binding to the regulatory elements  and the most (F) folded variant of TRβ1 5’UTR cloned upstream of luciferase reporter. Effects of each dGoligo on protein synthesis were assessed in a translation-enhancing assay. Levels of luciferase mRNA and protein (luciferase activity) expressed from plasmid without or with dGoligo supplementation were measured by Real-Time PCR and luminometry. Maximum luciferase activity was observed after 6h .dG1 and dG2 were synthesized as a pair of complementary, sense- and antisense- DNA oligonucleotides. dG1 shares sequence with the most stable secondary structure of the translation-enhancing element e1 (cis-acting elements)   that canlements) .dG3 (sense) and complementary dG4 (antisense) were designed to target sequence at ex2a/ex3 junction of TRβ1 5’UTR  is not affected by other distant sequences of the 5’UTRs and has a key inhibitory role in translational control of TRβ1. These findings support the hypothesis that dGenhancer may be used to identify ΔG-dependent, translation-regulating elements in 5’UTRs that could be targeted by dGs to alter their Gibbs folding energy and regulate the translation efficiency. Finally, the data suggest a role for the multiple alternatively spliced TRβ1 5’UTRs. Strongly folded variants (including variant F) may serve as a reservoir of less-active mRNAs that could be recruited to increase translation efficiency at times of cellular stress, for example, by the use of specific trans-acting factors such as ncRNAs. Interestingly, bioinformatic analysis of microRNA target sites within TRβ1 untranslated regions revealed that hsa-miR-211 could potentially target both TRβ1 3’UTR and 5’UTR , could actually interact with distant cis-acting elements, significantly changing translation efficiency as it was shown in our study  had no or opposite effects , thus cour study .s has an impact on their function we synthesized mismatched control dG5 and control dG6 sharing the same sequence with dG1 and dG2, respectively, but containing a 2–3 nucleotide long insertion in the middle of both oligonucleotides  and termed dG9 and dG10 (s need full complementarity to affect gene expression [To check whether the sequence structure of the dGleotides . Upon bielements . Since n effects . Therefofects [s  and werefects [s  that mayand dG10 . The usepression .scrambled control (dGsc) with a random sequence , a transcript of CDKN2A tumor suppressor. In this in vitro study, dG-mediated regulation of protein synthesis was tested using PCR-amplified linear expression construct containing T7 promoter, p16INK4a 5’UTR and the coding sequence of luciferase allowing for fast and reliable measurements of protein levels  and appropriate dGs , while the action of dG2 and dG4 resulted in 1.3–1.4-fold decrease in the reporter protein levels . The similar effects were observed when using microRNA-like dG9 and dG10 (1.09- and 4.8-fold respectively). These results showed a difference between the in vitro and in vivo studies, wherein the TRβ1 5’UTR targeting microRNA-like dGs exerted the strongest enhancing effects on translation in Caki-2 cells. The observed difference compelled us to introduce an additional 2'-O-methyl RNA modified scrambled control (dGsc) with an irrelevant (random) sequence that was shown to produce no change in luciferase activity, thus, confirming the specificity of the in vivo dG action. All the results of the luciferase activity after dG supplementation were normalized to control pGL3-A plasmid (mock transfected group). Data from three independent experiments were performed in triplicate. The Shapiro—Wilk test was used to determine normality of data distribution. Normally distributed data were analyzed using ANOVA followed by Dunnett’s multiple comparison test.To test our iate dGs . Althougan cells . Therefoan cells . Fig 6b  protein . By contTranslation-enhancing properties of selected dGs were confirmed in Caki-2 cells, where transfection with dGs resulted in increased levels of endogenous TRβ1 protein and its downstream target—type 1 iodothyronine deiodinase DIO1 , 23 Fig. In thiscis-acting element.These studies demonstrate that specific enhancement of gene expression can be achieved at the level of protein translation. We found this phenomenon to be triggered in a specific manner by an exogenous synthetic small enhancing oligonucleotide—dGoligo (dG) targeting a specific 5'UTR THRB gene expression and this may influence tumor progression [in vitro translation efficiency of each 5’UTR and its Gibbs energy (trans–acting factors termed dGoligos (cis-acting domains could result in either enhanced or reduced protein translation [s) or binding to distant cis-acting sequences folding with these domains (sense-like dGs) may enhance or repress protein synthesis  and differential expression of multiple mRNA variants is accompanied by varying levels of TRβ1 protein expression . Althouggression , 44. Thugression  raised ts energy , resultidGoligos  to selecdGoligos . Since aynthesis . To findynthesis . Howevers were added, 1.77-fold and 6.58-fold increases in translation efficiency from weakly folded 5’UTR variant A and strongly folded variant F respectively were observed , and antisense dG4, directly binding to an inhibitory region e3, . When boobserved . At the observed  of variaobserved . These rtastasis . Here, wtastasis . The auttastasis . This repression , 27 but pression , 31.In vitro results revealed that both, sense and antisense dGs can trigger translation-enhancing effects that appear to be mostly dependent on a specific function of a 5'UTR cis-acting element. The action of sense dG1 was thought to increase protein synthesis by releasing translation-enhancing element e1  had no or opposite effects . MoreoveINK4a 5'UTR (CDKN2A) [enhancer calculator to design dGs specifically targeting the ΔG-dependent, translation-regulating elements within this 5'UTR  were obtained by combining dG1p16 and dG6p16   and usedis 5'UTR . The CDKproteins . An analonstruct  revealedonstruct . As was onstruct , the send dG6p16  that is s can regulate protein translation in a cellular context. We used Caki-2 cells transfected with pGL3-derived plasmid [in vitro with RNase H deficient wheat germ extracts, transfection of Caki-2 with DNA based dGs did not alter luciferase activity  are widely used to suppress gene expression by inducing RNase H-mediated mRNA degradation of the target mRNA . The DNAficiency . Translaficiency , 13 . These synthetic In vitro studies were performed with pBluescript-KS(+)-derived plasmid vectors containing different TRβ1 5’UTR variants  or irrelevant leader sequence lacking any TRβ1 UTR (pKS-control) [in vivo analyses, we used pGL3-derived plasmid, carrying variant A of TRβ1 5’UTR (pGL3-A) [control) . 5'UTRs control) . For in (pGL3-A) , which w(pGL3-A) . pGL3-co(pGL3-A) .Manual method allowed us to identify higher-order structures within 5’UTR cis-acting sequences (IRESs or uORFs stretches). Folding predictions from RNAstructure version 5.2, together with sequence analysis using NCBI tools were combined to select putative cis-acting elements containing the most stable secondary structures (the most negative ΔG). As a second method, dGenhancer—an excel-based calculator was used to automatically identify putative ΔG-dependent translation-regulating elements within 5'UTR sequences (in silico introduced single nucleotide substitutions (SNPs) of the 5’UTR sequences. These artificial SNPs differently affected overall sequence ΔGs (Gibbs energies) that were drawn by the dGenhancer to show regions where substitution can alter ΔGs the most, indicating putative cis-acting elements with the highest translational regulatory potential. The software that implements the calculations can be accessed here: http://www.serwer1448847.home.pl/biotechnology/dGenhancer.xlsxTwo methods were used. equences . The algcis-acting elements of TRβ1 5’UTRs  using standard DNA or 2'-O-methyl-modified phosphoramidites .Sense-, antisense- or microRNA-based DNA oligonucleotides were designed  to targe1 5’UTRs . For in Real-Time PCR  and two pairs of PCR primers  with the Synergy2 luminometer  in conditions recommended by the manufacturers.500ng of each plasmid were simultaneously transcribed and translated in 0.2mL-PCR tubes using RTS 100 Wheat Germ CECF Kit . The translation assay was conducted in 20μL of Reaction Solution, supplemented with 20uL of Feeding Solution after initial 3h-incubation. All reactions were maintained at 37°C for 6h with shaking at 600 rpm, using the RTS ProteoMaster Instrument . After reaction, DNA levels of appropriate pKS plasmids (plasmid copy number per each reaction) were measured by semi-quantitative Real-Time PCR and served as internal controls of transcription and translation efficiency . mRNA le primers . The rea dGoligo  or in th2 atmosphere. For all the experiments, Caki-2 cells were seeded into 75cm2 bottles, 6- or 12-well culture plates at density 13x103 cells/cm2, 24h before transfection. Three independent in vivo experiments were performed in triplicate.The human clear cell renal carcinoma cell line (Caki-2) was used . Caki-2 cells were grown in McCoy’s 5A medium with L-glutamine  with 10% fetal bovine serum  and 1x penicillin-streptomycin solution . The cells were maintained at 37°C in 5% CODual-luciferase assay. The protein measurements were performed using dual-luciferase assay  in the Synergy2 luminometer , according to the manufacturer’s instructions.24 hours after seeding, cells were transfected with 100 ng of control pRL-TK  and 1ug of pGL3-A plasmid , using 1in vitro assay.Total RNA for real-time PCR was purified from the second part of the collected cells as it was described for in vitro study. The transcript levels of Firefly luciferase were compared with Renilla using specific primers ((-ΔΔCt).Reverse transcription and Real-time PCR of luciferase pGL3-A and pRL-TK control was performed according to the protocol used for  primers . Relativs were tested as complementary sense and antisense sequences  of 5’UTR secondary structures was performed using RNAstructure version 5.2 [Total Gibbs energy prediction Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Gene expression control is a fundamental determinant of cellular life with transcription being the most important step. The spatial nuclear arrangement of the transcription process driven by RNA polymerases II and III is nonrandomly organized in foci, which is believed to add another regulatory layer on gene expression control. RNA polymerase I transcription takes place within a specialized organelle, the nucleolus. Transcription of ribosomal RNA directly responds to metabolic requirements, which in turn is reflected in the architecture of nucleoli. It differs from that of the other polymerases with respect to the gene template organization, transcription rate, and epigenetic expression control, whereas other features are shared like the formation of DNA loops bringing genes and components of the transcription machinery in close proximity. In recent years, significant advances have been made in the understanding of the structural prerequisites of nuclear transcription, of the arrangement in the nuclear volume, and of the dynamics of these entities. Here, we compare ribosomal RNA and mRNA transcription side by side and review the current understanding focusing on structural aspects of transcription foci, of their constituents, and of the dynamical behavior of these components with respect to foci formation, disassembly, and cell cycle. Proper regulation of gene expression is of utmost importance to ensure maintenance of cell integrity, coordinated cellular differentiation, and adequate responses to external and internal cues.Nuclear transcription is exerted by three RNA polymerases, RNA polymerase I (Pol I) exclusively dedicated to transcription of ribosomal RNA (exception: 5S rRNA), RNA polymerase II (Pol II) transcribes mRNA and noncoding RNAs, and RNA polymerase III (Pol III) produces tRNAs and 5S rRNA.With the introduction of labeled nucleosides that become incorporated into nascent RNA, it was realized that nuclear transcription does not occur randomly distributed throughout the nuclear volume but rather is found in discrete regions within the nuclear space  is present in a unique structure, rDNA is the only target of Pol I, and transcription is spatially confined to a conspicuous nuclear organelle, the nucleolus , a procIn the nucleolus, the regulation of rRNA transcription is tightly coupled to metabolic demands of the cell. The minimal essential Pol I transcription machinery consists of Pol I, SL-1, and UBF [reviewed in  have been shown to be important epigenetic modifiers of gene transcription. It could be demonstrated that they are crucial for NoRC targeting and thus for rDNA silencing  being mobile and the other (25 %) being transiently immobile  to near-continuous (strongly expressed genes during differentiation) to continuous transcription (rDNA) can be observed. Morphologically, alterations occur at very different timescales, seconds in Pol II transcription factories and minutes for nucleoli when, e.g., treated with transcriptional inhibitors or enhancer drugs.The number of foci of ongoing Pol II transcription inside nuclei is much lower than the number of genes transcribed. Indeed, it has been shown that TFs contain more than one transcribed gene. This requires chromatin to be organized such that genes can come into contact. Loops established by promoter–enhancer interactions (PEIs) are pivotal, although other interactions such as promoter–promoter, enhancer–enhancer, or insulator–insulator looping occur.Differing models have been proposed on the mechanisms by which PEIs form in nuclei. It has been shown that a fraction of Pol II is retained in nuclei after extraction procedures implying that they are tethered to an underlying structural support (nucleoskeleton) (Jackson and Cook hGH-N (human pituitary growth hormone), it was shown that the DNase I-hypersensitive site I (HSI) of the LCR acts as important factor to sustain association of the gene with transcription factories over many years of development . This mark is transmitted through mitosis into G1 of daughter nuclei and is thought to ensure robust expression of the same set of genes active in the mother cell. The bookmarking mechanism includes specific histone modifications, histone variant replacement or maintenance of the general transcription factor TIFIID binding to promoters [for review bookmarking, see  NORs throughout mitosis [reviewed in (Hernandez-Verdun Interestingly, the number of nucleoli varies even within one population of a cell line, although there exist less active NORs than the ten NORs in humans (Carpenter et al. Genetic engineering approaches have been conducted to gain insight into the requirements for nucleolus formation in human cells. The ectopic insertion of an UBF-binding sequence in a cell line created a “pseudo-NOR,” i.e., secondary constriction at the integration site upon UBF loading (Mais et al. The example of the co-localization of neo-NORs and endogenous NORs in one nucleolus is somewhat reminiscent of co-localization of several genes in one Pol II transcription factory. Similar to transcription factories also in the case of nucleoli, a structural fixation of active Pol I molecules in the FC was demonstrated (Dickinson et al. RNA polymerase I transcription has often been put forward as paradigmatic for nuclear transcription. Concerning the constituents, Pol I and Pol II transcription are largely different. However, on a functional level, analogies can be found which may rely on general principles of biological organization. Chromatin loop formation is one hallmark that acts as prerequisite of focal transcription in both transcription factories and nucleoli. Another common property is the high mobility of some of the constituents.The underlying organizing principles of focal transcription and of nuclear organization in general are controversially discussed and range from fixation to an underlying support such as nuclear matrix or nucleoskeleton to self-organization and compartmentalization based on stochastic biochemical and biophysical principles. The methodological armory for studying biophysical forces is restricted, and novel approaches are needed to reveal if one of these possibilities or combinations thereof occur.In addition to basic consideration on nuclear organization, focal nuclear transcription might have consequences relevant for medicine. In this respect, it was postulated that the proximity of genes present in an open and transcriptionally active state at transcription factories might have potentially detrimental consequences for genome stability and chromosomal translocations ultimately leading to cancer Osborne , and it


Escherichia coli and its orthologs globally regulate metabolism, motility, biofilm formation, stress resistance, virulence of pathogens and quorum sensing by activating the transcription of genes for regulatory sRNAs, e.g. CsrB and CsrC in E. coli. These sRNAs act by sequestering the RNA binding protein CsrA (RsmA) away from lower affinity mRNA targets. In this study, we used ChIP-exo to identify, at single nucleotide resolution, genomic sites for UvrY (SirA) binding in E. coli and Salmonella enterica. The csrB and csrC genes were the strongest targets of crosslinking, which required UvrY phosphorylation by the BarA sensor kinase. Crosslinking occurred at two sites, an inverted repeat sequence far upstream of the promoter and a site near the -35 sequence. DNAse I footprinting revealed specific binding of UvrY in vitro only to the upstream site, indicative of additional binding requirements and/or indirect binding to the downstream site. Additional genes, including cspA, encoding the cold-shock RNA-binding protein CspA, showed weaker crosslinking and modest or negligible regulation by UvrY. We conclude that the global effects of UvrY/SirA on gene expression are primarily mediated by activating csrB and csrC transcription. We also used in vivo crosslinking and other experimental approaches to reveal new features of csrB/csrC regulation by the DeaD and SrmB RNA helicases, IHF, ppGpp and DksA. Finally, the phylogenetic distribution of BarA-UvrY was analyzed and found to be uniquely characteristic of γ-Proteobacteria and strongly anti-correlated with fliW, which encodes a protein that binds to CsrA and antagonizes its activity in Bacillus subtilis. We propose that BarA-UvrY and orthologous TCS transcribe sRNA antagonists of CsrA throughout the γ-Proteobacteria, but rarely or never perform this function in other species.The two-component signal transduction system BarA-UvrY of  E. coli and other γ-proteobacteria that is extensively interconnected with transcriptional regulatory circuits  KH2PO4, 0.6% [wt/vol] yeast extract containing 0.5% [wt/vol] glucose). Growth was determined by monitoring OD600 and/or by assaying total cellular protein.LB medium  was used for culture of bacteria unless stated otherwise. The antibiotics ampicillin (100 μg mL−1), tetracycline (15 μg mL−1), kanamycin (50 μg mL−1), and chloramphenicol (25 μg mL−1) were included in growth media as needed. Bacterial cultures were stored at -80°C in medium containing ~15% glycerol. To revive cultures, LB medium (2 mL) was inoculated from the frozen stock cultures and incubated with shaking (250 rpm) at 37°C overnight. The overnight cultures served as inoculum (1:1000) for LB medium or Kornberg medium (1.1% [wt/vol] KE. coli) and carboxy-terminally 3XFLAG®-tagged constructs  were introduced by the Red recombinase method, as described  of the following composition: 7.5% separating gel  with a 4% stacking gel . UvrY-P was resolved by electrophoresis at constant 150 V at 4°C for 70 min using modified MOPS running buffer . Gels were subsequently stained with Coomassie blue and the signals were imaged using a ChemiDoc XRS+ system (Bio-Rad) and quantified using Quantity One image analysis software (Bio-Rad).For E. coli and Salmonella strains carrying biologically functional UvrY-FLAG  5’-end-primers  per reaction, for 1 min at 37°C. The DNase I was inactivated by heating at 75°C degrees for 10 min. Reactions were then mixed with formamide loading buffer , denatured at 95°C for 5 min, and separated by electrophoresis on a 6% denaturing polyacrylamide gel. The gel was vacuum dried, radioactive signals captured by phosphor imaging  and analyzed with Quantity One software.Double stranded DNA for footprinting of -primers . The 5’-csrB-lacZ and csrC-lacZ carrying plasmids, pLFXcsrB-lacZ and pLFXcsrC-lacZ, were constructed by first amplifying the 502 nt  and 304 nt  genomic regions using the primer pairs csrB lacZ Fwd / csrB lacZ Rev and csrC lacZ Fwd / csrC lacZ Rev . The UvrY-P (2.3 μM), ppGpp (250 μM) and DksA (2 μM) were included, as indicated, in the reactions. IHF (1μM) was added to all of the reactions that contained pLFXcsrB-lacZ plasmid. DksA protein was a generous gift of Prof. Richard Gourse, University of Wisconsin, Madison. IHF protein was a generous gift from Prof. Anca Segall, San Diego State University. Incorporation of 35S-methionine into protein products was determined after SDS PAGE separation by using phosphorimaging. Signal intensity was determined using Quantity One software.eviously , except http://www.ncbi.nlm.nih.gov/genome/), using the NCBI Prokaryotic Genome Annotation Pipeline v2.0 to assign orthology  GTP (2.5 μCi). This suggests that unknown factors may be required for csrB/C transcription (data not shown). We next used coupled transcription-translation in S-30 extracts to determine whether the addition of ppGpp and DksA would directly regulate expression of csrB/C in the presence of other cellular factors present in the S-30 extracts but not the defined transcription reactions. As previously reported [csrB-lacZ (csrC-lacZ (csrB-lacZ reaction caused a modest increase in expression (csrB-lacZ expression (1.5-fold). DksA alone also had weak effects in the presence of UvrY-P (1.4-fold), while the addition of both ppGpp and DksA led to a 1.7-fold increase in csrB-lacZ expression. Addition of ppGpp alone, in the absence of UvrY-P, resulted in a slight increase in csrC-lacZ expression  database [E. coli genome using the first six bases of the 18 bp (TGTAAGNNNNNNCTTACA) UvrY binding sequence, followed by manually checking the presence of the rest of the IR DNA sequence in the regulatory region of each discovered putative target. In this way, we identified 19 putative target genes containing the 18-bp UvrY-P IR sequence either perfectly or near perfectly conserved in their regulatory regions  . Hence, database . We scan regions . Next, w regions . When we regions , supportE. coli, the pathogen Salmonella, as well as a variety of other γ-proteobacterial pathogens [csrA homologs, but have not been shown to express CsrA-inhibitory sRNAs. Recently, Mukherjee et al. have shown that the FliW protein of B. subtilis binds to CsrA and antagonizes its activity, thus preventing it from binding to the flagellin mRNA, hag [E. coli and Salmonella and fliW is absent in species known to produce CsrA-inhibitory sRNAs [-205) and genus level   . Of the urvum sp . InteresAccurate and complete identification of orthologs across a broad taxonomic range is a challenging problem, with potential for both false-positives and false-negatives . AlthougE. coli within the similarity and coverage thresholds (see methods) such as Pseudomonas , Xanthomonas , Pseudoxanthomonas (NC_014924), Shewanella , and other members of the γ-Proteobacteria class : 15675047, 13622200, 21904470), peptide methionine sulfoxide reductases , and putative CsrA homologs that lack the N-terminal and C-terminal conserved regions of CsrA . Most of the main false negatives that we identified are BarA and UvrY from genomes that match the representative homologs from ia class .These false positives and false negatives caused a small increase of 2% in the negative correlation, reinforcing the present conclusions  . These rE. coli and Salmonella. We discovered that the csrB/C genes are by far the strongest direct targets of UvrY in these species. UvrY binds specifically to an 18 nt palindromic sequence in the promoter regions of csrB/C and exhibited an almost absolute requirement for phosphorylation by BarA for this binding in vivo under growth conditions examined. UvrY-P requires IHF for optimal binding to and activation of csrB but not csrC. CsrA activates csrB/C transcription by activating uvrY expression by an undefined mechanism, which may require the noncoding mRNA leader, but does not involve the other known posttranscriptional regulator of uvrY, the DeaD-box RNA helicase DeaD [csrB/C transcription in E. coli [csrB DNA in vivo without affecting the expression of other factors known to activate csrB expression. This suggests that SrmB may regulate unknown factor(s) involved in csrB transcription. The stringent response factors ppGpp and DksA activate CsrB/C expression in vivo [csrB expression in vitro in S-30 extracts. Whether this involves direct binding to RNA polymerase remains to be determined.Using ChIP-exo , we probase DeaD . The RNA E. coli , promote in vivo  and wereE. coli and 301 genes in Salmonella, respectively , 14028S strain with sirA-FLAG fusion integrated at the native sirA locus and sirA and csrB deletion strains (B). CsrB levels in MG1655, uvrY deletion and UvrY-His6 (expressed from pET24-a expression vector) E. coli strains (C). For Western blotting, RpoB loading controls shown (A). For Northern blotting, the 16S/23S rRNA loading controls are shown. Cultures were grown in LB to mid-exponential growth phase (OD600 of 0.6).Western blot showing expression of SirA-FLAG (A) and Northern blots showing CsrB levels in 14028S (wild type (TIFF)Click here for additional data file.S2 FigcsrB, lacY and 16S rDNA (rrsH) were used to amplify the promoters of csrB, lacY and/or 16S rDNA genes from DNA that was crosslinked and immunoprecipitated from E. coli (panel A) or Salmonella (panel B). In these analysis, csrB was used as a positive control and lacY and 16S rDNA (rrsH) were used as negative controls for E. coli and Salmonella, respectively.Polymerase chain reaction was used to confirm the specificity of ChIP assay. Primers  annealin(TIFF)Click here for additional data file.S3 FigbarA, ∆dksA, ∆relA, ∆ihfA, ∆ihfB, csrA::kan and ∆uvrY strain. Cultures were grown in LB to mid-exponential growth phase (OD600 of 0.6). The relative levels and % of phosphorylation of UvrY in the WT, barA, dksA, relA, ihfA, ihfB and csrA are: 1.0, 1.2, 1.0, 1.0, 1.2, 1.2, 0.23 (UvrY levels) and 7%, 1%, 6.8%, 6.9%, 9%, 9% and 8% (% of UvrY phosphorylation), respectively.Phos-tag SDS-PAGE with Western blotting was used for detection of the phosphorylated (P-UvrY-FLAG) and non-phosphorylated (UvrY-FLAG) protein levels expressed in a WT (MG1655 expressing UvrY-FLAG) and isogenic ∆(TIFF)Click here for additional data file.S4 Fig32P-end labeled DNA probe (reverse strand) that included both the upstream and downstream putative UvrY binding sites was used Click here for additional data file.S5 FigihfA and ∆ihfB strains. Cultures were grown in LB to mid-exponential growth phase (OD600 of 0.6). RpoB loading control is also shown.Western blotting of UvrY-FLAG levels in strains MG1655 (no FLAG fusion), WT (MG1655 expressing UvrY-FLAG), and isogenic ∆(TIFF)Click here for additional data file.S6 Fig600 of 2.0). The 16S/23S rRNA loading controls are also shown. Western blot showing effect of uvrY deletion on FhuF-FLAG protein (B). Cultures were grown in LB to mid-exponential growth phase (OD600 of 0.6), at which point dipyridyl was added to culture . Samples were collected before and 10 min after the addition of dipyridyl. RpoB loading control is also shown. Electrophoretic gel mobility shift assay showing UvrY binding to cspA, spf, fhuF and csrB DNA (C). Phosphorylated (UvrY-P) UvrY-His6 binding to spf, cspA, fhuF, csrB and rrlE DNA was tested by EMSA as shown. The spf, cspA, fhuF and csrB DNA probes used in this experiments encompass the ChIP-exo derived putative UvrY binding sites discovered in the promoter region of each gene  were also used as non-specific and specific competitors, respectively. The DNA-protein complexes were resolved in a non-denaturing 7% polyacrylamide gel. Shifted protein-DNA complex is indicated in black arrows. Effect of UvrY on the expression of putative sRNA targets (D). Northern blots showing effect of UvrY on the expression of several sRNA genes. Cultures were grown in Kornberg, supplemented with 0.5% glucose, to mid-exponential (OD600 of 0.6), transition to stationary (OD600 of 1.2) and stationary growth phases (OD600 of 3.0). The 5S rRNA loading control is also shown.Northern blot showing effect of UvrY and Csr factors on SpoT42 sRNA (A). Cultures of MG1655 and the isogenic mutants indicated were grown in Kornberg medium containing 0.5% glucose, to early stationary growth phase (ODshown in . The DNA(TIF)Click here for additional data file.S7 Fig(TIFF)Click here for additional data file.S8 FiguvrY-FLAG fusion integrated at the uvrY locus) and isogenic csrA mutant. Cells were grown in LB to mid-exponential growth phase (OD600 of 0.6) at which point tetracycline and chloramphenicol were added and cultures were sampled thereafter at the times shown. Western blotting of IhfA-FLAG and IhfB-FLAG proteins (B) examined in MG1655 or an isogenic csrA::kan mutant expressing ihfA-FLAG or ihfB-FLAG fusions from the native genomic loci (WT). Cultures were grown in LB to mid-exponential growth phase (~OD600 of 0.6). RpoB loading controls for these analyses are also shown.Western blot UvrY-FLAG protein stability (A) in MG1655 (no FLAG fusion), WT (MG1655 with a (TIFF)Click here for additional data file.S9 Fig35S-labeled methionine into protein products was detected by SDS PAGE with phosphorimaging. Signal intensity was determined using Quantity One software. The fold-effects of regulatory factors were determined with respect to the control reaction lacking these factors, after normalization against β-lactamase as an internal control. Absolute deviation was determined from two independent experiments.Reactions contained pLFXcsrC-lacZ (4 μg), UvrY-P (2.3 μM), ppGpp (250 μM) and/or DksA (2 μM) as indicated. Incorporation of (TIFF)Click here for additional data file.S10 Figin silico analysis using the Ab Initio Motif Identification Environment (AIMIE) database (46). This was done by scanning the E. coli genome using the first six bases of the 18bp-long (TGTAAGNNNNNNCTTACA) UvrY consensus binding sequence, followed by manually checking the presence of the rest of the IR DNA sequence in the regulatory region of each discovered putative target. The predicted UvrY binding sequence in the promoter region of each putative target in comparison to the UvrY consensus sequence is shown. Distance from the center of the predicted UvrY binding sequence to the known transcription start site (TSS) of each putative target is shown. Nucleotides marked in red are the mismatch between the UvrY consensus sequence and the predicted UvrY binding sequence.19 Putative UvrY targets were derived by (TIFF)Click here for additional data file.S11 Figin silico analysis Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Tablehttp://www.ncbi.nlm.nih.gov/genome/). The results were compared to other orthology databases and filtered by sequence similarity and alignment coverage in order to minimize the number of false positives/negatives (see Methods). At both species and genus levels, the results exhibited a strong anti-correlation between BarA-UvrY and FliW.We identified CsrA, BarA, UvrY and FliW orthologs from all fully-sequenced bacterial genomes in the NCBI genomes database ((XLSX)Click here for additional data file.


As a model of phenotypic plasticity, we study abdominal pigmentation of Drosophila melanogaster females, which is temperature sensitive. Abdominal pigmentation is indeed darker in females grown at 18°C than at 29°C. This phenomenon is thought to be adaptive as the dark pigmentation produced at lower temperature increases body temperature. We show here that temperature modulates the expression of tan (t), a pigmentation gene involved in melanin production. t is expressed 7 times more at 18°C than at 29°C in female abdominal epidermis. Genetic experiments show that modulation of t expression by temperature is essential for female abdominal pigmentation plasticity. Temperature modulates the activity of an enhancer of t without modifying compaction of its chromatin or level of the active histone mark H3K27ac. By contrast, the active mark H3K4me3 on the t promoter is strongly modulated by temperature. The H3K4 methyl-transferase involved in this process is likely Trithorax, as we show that it regulates t expression and the H3K4me3 level on the t promoter and also participates in female pigmentation and its plasticity. Interestingly, t was previously shown to be involved in inter-individual variation of female abdominal pigmentation in Drosophila melanogaster, and in abdominal pigmentation divergence between Drosophila species. Sensitivity of t expression to environmental conditions might therefore give more substrate for selection, explaining why this gene has frequently been involved in evolution of pigmentation.Phenotypic plasticity is the ability of a given genotype to produce different phenotypes in response to distinct environmental conditions. Phenotypic plasticity can be adaptive. Furthermore, it is thought to facilitate evolution. Although phenotypic plasticity is a widespread phenomenon, its molecular mechanisms are only beginning to be unravelled. Environmental conditions can affect gene expression through modification of chromatin structure, mainly  Drosophila melanogaster. Here we show that temperature affects female abdominal pigmentation by modulating the expression of tan (t), a gene involved in melanin production, in female abdominal epidermis. This effect is mediated at least partly by a particular regulatory sequence of t, the t_MSE enhancer. However we detected no modulation of chromatin structure of t_MSE by temperature. By contrast, the level of the active chromatin mark H3K4me3 on the t promoter is strongly increased at lower temperature. We show that the H3K4 methyl-transferase Trithorax is involved in female abdominal pigmentation and its plasticity and regulates t expression and H3K4me3 level on the t promoter. Several studies have linked t to pigmentation evolution within and between Drosophila species. Our results suggest that sensitivity of t expression to temperature might facilitate its role in pigmentation evolution.Environmental conditions can strongly modulate the phenotype produced by a particular genotype. This process, called phenotypic plasticity, has major implications in medicine and agricultural sciences, and is thought to facilitate evolution. Phenotypic plasticity is observed in many animals and plants but its mechanisms are only partially understood. As a model of phenotypic plasticity, we study the effect of temperature on female abdominal pigmentation in the fruit fly  Drosophila melanogaster, female abdominal pigmentation is a plastic trait as it is darker in females grown at 18°C than at 29°C  of control flies and of t loss-of-function mutant flies   , S2 Fig.entation , this ret expression could be mediated by its cis-regulatory sequences. An enhancer essential for driving t expression in the epidermis of abdominal segments A5 and A6 in males, t_MSE, was previously mapped upstream of t, between the genes CG15370 and Gr8a [t_MSE-nEGFP reporter transgene [ebony-nEGFP transgene in which nEGFP is under control of the regulatory sequences of ebony [nEGFP and not stability of the nEGFP protein was responsible for the effect observed with the t_MSE-nEGFP transgene. Interestingly, the fold change observed with the t_MSE-nEGFP transgene between 18°C and 29°C was lower than that of t expression t expressransgene  in youngof ebony , a pigmepression . This cot_MSE activity by temperature prompted us to analyse the chromatin structure of this enhancer in epidermes of female abdominal segments A5, A6 and A7 at 18°C and 29°C -qPCR experiments, a methodology allowing detection of open chromatin [VG01 enhancer of vestigial (vg), which recapitulates vg expression in wing and haltere imaginal discs, was specifically open in these tissues, but not in leg imaginal discs where vg is not expressed [vg is not expressed in the abdominal epidermis either   enrichment by chromatin immunoprecipitation experiments (ChIP-qPCR), which showed a higher nucleosome concentration on the VG01 enhancer than on t_MSE, but no difference between 18°C and 29°C for both enhancers  and 29°C (p<0.05). However, we detected no significant H3K27ac enrichment on t_MSE at 18°C compared to 29°C  of active genes, which includes the promoter, is known to be depleted in nucleosomes [t TSS  tended to be less compact at 18°C than at 29°C , a gene nearby t (CG12119 and t (NC). Highly transcribed genes are enriched in H3K4me3, with a maximum of enrichment 50–750 bp downstream of the TSS [t TSS  or for NC  . Howevernhancers . We thennhancers . t_MSE w to 29°C . This releosomes . FAIRE-qnearby t  that wasnearby t , or for 19 and t , NC. Hig the TSS . We founr for NC .t promoter in the posterior abdominal epidermis of females.In conclusion, our results show that temperature modulates chromatin compaction and H3K4me3 enrichment on the t, we addressed the role of genes involved in H3K4 methylation in pigmentation and its plasticity. In D. melanogaster, H3K4 mono-, di- and tri- methylations are catalysed by three complexes of the COMPASS family called Trithorax (Trx), Trithorax-related (Trr) and Set1. These complexes are characterised by their histone methyl-transferase subunit encoded by the genes trx, trr and Set1, respectively [As temperature modulates deposition of the H3K4me3 active mark on ectively . The hisectively . Whereasectively , Set1 isectively . Indepenectively –47.trx, trr or Set1 using UAS-RNAi transgenes and the late pupal driver y-Gal4 to analyse their implication in abdominal pigmentation . Nevertheless, the interaction between genotype and temperature was highly significant  that participates in leaf margin dissection is modulated by temperature and has been repeatedly involved in leaf shape evolution through cis-regulatory sequence variation or gene loss [Several studies have also linked  species ,19,26. R species . Furtherin t_MSE . Intereselection . The recvolution . In otheene loss . Therefow1118 inbred line as wild-type. The UAS-t line was a gift from Dr. Nicolas Gompel, whereas t_MSE-nEGFP was from Dr. Sean Carroll's lab. The ebony-nEGFP line (ebony-(ABC+intron)-nEGFP) was from Dr. Mark Rebeiz. The UAS-RNAi-t (GD18124) and UAS-RNAi-Set1 (GD40683) lines were from the VDRC Stock Center. The pnr-Gal4 (BL3039), y-Gal4 (BL44267), P(XP)td07784 (BL19282), e1 (BL1658), trxj14A6 (BL12137), as well as the VALIUM UAS-RNAi lines  against trx (BL33703), trr (BL29563 and BL36916), Set1 (BL33704) and GFP (BL41556) were from the Bloomington Stock Center. The homozygous lethal trxj14A6 allele that corresponds to an insertion of a w+ P transposon was used in this study. This allowed us to introgress this allele in the w1118 background (ten generations), so that the mutation is in the same genetic background as the control. Complementation test with a well characterized trx loss-of function allele . Gal80 inactivation was performed by shifting the progeny at late pupal stage from 18°C to 29°C. We tested that all lines allowing trx, trr or Set1 down-regulation induced lethality with the ubiquitous daughtherless-Gal4  driver. Efficiencies of BL29563 (UAS-RNAi-trr) and GD40683 (UAS-RNAi-Set1) were previously published [UAS-RNAi-trx line (BL33703), quantification of trx expression level in da-Gal4>UAS-RNAi-trx embryos showed a 1.5 fold down regulation as compared to control embryos, thus proving its efficiency.We used a e (trxE2 ) indicat-Gal4 BL467, P(XP)ublished . For theAdult females between 3 and 5 days old were stored for 10 days in ethanol 75% before dissection. Abdominal cuticles were cut just beyond the dorsal midline, which was therefore entirely included in each preparation. After dissection, cuticles were dehydrated 5 minutes in ethanol 100% and mounted in Euparal (Roth). For nEGFP observations, abdomens were dissected in PBS, fixed 20 minutes in 3.7% paraformaldehyde in PBS, washed twice 10 minutes in PBS and mounted in Mowiol.t (611 bp) and e (639 bp) were amplified by PCR (primer sequences are listed in pBlueScript vector (Invitrogen). Sense and antisense DIG-labelled RNA probes were synthesized using the appropriate RNA polymerase. In situ hybridizations were performed according to the Carroll's lab protocol (http://carroll.molbio.wisc.edu). Specificity of the antisense probe was assessed by comparison with signal from the sense probe. For t, we also performed in situ hybridization with the t antisense probe on UAS-RNAi-t/pnrGal4 females and observed a strong decrease of the signal in the pnr domain  separates the two hemi-segments. Cuticle pigmentation in hemi-tergites A5, A6 or A7 was measured as mean grey value using ImageJ. This value was subtracted from 255 to get a final pigmentation value comprised between 0 (white) and 255 (black).Adult cuticles and abdominal t_MSE-nEGFP and ebony-nEGFP females were imaged using a Macro-Apotome (Zeiss). nEGFP intensity was measured in hemi-tergites A5, A6 or A7 using ImageJ in Maximum Intensity projections of 40 picture stacks.Abdominal epidermes of rp49 and Act5C; rp49 and eIF2,) was used for normalization [RNA was extracted from pools of dissected female posterior abdominal epidermes  with the RNAeasy Mini kit (Qiagen) . We could not use developmental time to stage pharates as it is temperature sensitive. We therefore used morphological markers  to collect pharates grown at 18°C or 29°C at a similar developmental stage. This stage corresponds to the stage P12(i) described by Bainbridge and Bownes . For eaclization . Primersper qPCR reaction. qPCR experiments were carried out in a CFX96 system (Biorad) using SsoFast EvaGreen Supermix (Biorad). Primers used are listed in Chromatin immunoprecipitation (ChIP) experiments were performed as previously described  with minper qPCR reaction. qPCR experiments were carried out in a CFX96 system (Biorad) using SsoFast EvaGreen Supermix (Biorad). Primers used are listed in 75 posterior abdominal epidermes  of females between 0 and 2h after hatching were used for each FAIRE experiment. Fixation and lysis protocols were similar to those used for ChIP except that fixation was performed for 5 minutes at room temperature in PBS-1% paraformaldehyde with gentle shaking. Chromatin sonication was performed in 300μl in a Bioruptor sonifier (Diagenode) with 8 cycles of 30'' ON, 30'' OFF, High power, allowing to obtain chromatin fragments between 300–400 bp. 100μl of chromatin preparation was kept as the input . The rest (200μl) was submitted to phenol-chloroform extraction and the aqueous phase containing the decompacted chromatin (FAIRE chromatin) was kept. Input and FAIRE DNA were purified with the Ipure kit (Diagenode) in 150μl of water and 4μl were used t (trx (http://statprogramsplus.com/OpenStatMain.htm). Normality of the residual distributions was checked with a Shapiro-Wilk test .To analyse the effect of temperature on A5, A6 and A7 pigmentation, we performed a one-way ANOVA (or Welch’s ANOVA when variances were heterogeneous) with temperature as factor. To analyse the effect of t  or trx (t (trx  on pigmehttp://anastats.fr) and then used the appropriate option of t-test.For t-tests, we checked first homogeneity of variance using a Levene Test  or ebony  mutant background at 25°C. In a wild-type background, tan down-regulation strongly reduced pigmentation in the 6th abdominal segment (*), whereas tan over-expression increased melanin production in all segments. In contrast, the modulation of tan expression had no effect on pigmentation in an ebony mutant background.Down-regulation or up-regulation of (TIF)Click here for additional data file.S2 Figtd07784 or w1118 females (n = 10 per condition).Reaction norms of pigmentation in A5, A6 and A7 abdominal segments of (TIF)Click here for additional data file.S3 Figin situ hybridization of ebony expression pattern in the abdominal epidermis of freshly hatched w1118 females. Left and middle: ebony antisense probe at 18°C and 29°C. Right: Sense ebony control probe at 29°C. Note the similar expression patterns of ebony at 18°C and 29°C in A5, A6 and A7 segments.  Expression of ebony at 18°C and 29°C monitored with the ebony-nEGFP transgene in the abdominal epidermis of freshly hatched females. (B) nEGFP fluorescence in abdominal epidermes. At 29°C, the fluorescence on the left part of the tissue is from the pleura and the bright region in the bottom marked by an asterisk is a part of the genitalia. (C) Quantification of nEGFP fluorescence in A5, A6 and A7 hemi-tergites at 18° and 29°C (n = 10 per temperature). In A6 and A7, nEGFP intensity is higher at 29°C that at 18°C .(A) Analysis by (TIF)Click here for additional data file.S4 Figvg (A) and CG12119 (B) is not significantly modulated by temperature. Note that vg is expressed at a very low level. In A and B, n = 3; error bars: standard deviations.RT-qPCR experiments showing that expression of (TIF)Click here for additional data file.S5 FigVG01, t_MSE, t-TSS-down, t-ex2, CG12119-TSS-down and NC. (B) H3K27ac IP signal normalized to input signal for VG01 and t-MSE. (C) H3K4me3 IP signal normalized to panH3 IP for t-TSS-down, t-ex2, CG12119-TSS-down and NC. In A, B, C, n = 3, error bars: standard deviations.(A) PanH3 IP signal normalized to input signal for (TIF)Click here for additional data file.S6 Figy-Gal4>UAS-RNAi-trx and y-Gal4>UAS-RNAi-GFP females grown at 18°C . .Quantification of pigmentation gene expression in posterior abdominal epidermes  from young (TIF)Click here for additional data file.S7 Figtrxj14A6 heterozygous and w1118 females (n = 30 per condition).Reaction norms of pigmentation in A5, A6 and A7 abdominal segments of (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Interestingly, chromosome-wide distributions of Asf1 and one of the condensin subunits, Brn1 show a nearly identical pattern. Moreover, Brn1 shows reduced occupancy at various condensin-binding sites in asf1Δ cells. These results along with high association of Asf1 with heterochromatic centromeres and telomeres ascribe novel roles to Asf1 in condensin loading and chromatin dynamics.Genome-wide participation and importance of the histone chaperone Asf1 (Anti-Silencing Function 1) in diverse DNA transactions like replication, repair, heterochromatic silencing and transcription are well documented. Yet its genome-wide targets have not been reported. Using ChIP-seq method, we found that yeast Asf1 associates with 590 unique targets including centromeres, telomeres and condensin-binding sites. It is found selectively on highly transcribed regions, which include replication fork pause sites. Asf1 preferentially associates with the genes transcribed by RNA polymerase (pol) III where its presence affects RNA production and replication-independent histone exchange. On pol II-transcribed genes, a negative correlation is found between Asf1 and nucleosome occupancy. It is not enriched on most of the reported sites of histone exchange or on the genes, which are misregulated in the  They often involve localized chromatin assembly/disassembly via eviction/deposition of the histones by specific histone chaperones, which bind dimers of canonical or variant histones H2A/H2B or H3/H4 Organization of the eukaryotic genome into chromatin enables its compaction inside the cell nucleus and concomitant regulation of DNA-related processes ASF1 are sensitive to DNA damaging agents, as it plays important role in checkpoint signaling and genomic stability Yeast cells lacking As evident from the above account, significant advances have been made in understanding the functional diversity of Asf1. However, despite genome-wide functional studies, its genome-wide association map in the budding yeast is not reported yet. Our high-resolution mapping of Asf1 along the 16 chromosomes of the budding yeast revealed that Asf1 specifically associates with highly active pol II and pol III-transcribed loci, which are also known sites of replication fork arrest 600 of 0.7. Chromatin Immunoprecipitation (ChIP)-grade anti-HA was from Millipore; anti-Myc, anti-GFP and anti-HA antibodies were from Santa Cruz Biotechnology.List of the yeast strains and sequences of the primers used in this study are given under the ASF1-18myc from its genomic locus 3 gene were used for histone exchange assay As detailed in the Supplementary Information, yeast cells carrying a plasmid that contains Since Asf1 is a general H3/H4 chaperone, we used peak calling criteria similar to nucleosome peak calling (peak size 150 bp and spacing of peaks by 300 bp), with a FDR of 0.1%. Analysis of the ChIP-sequencing data revealed only 590 chromosomal locations  associatSNR6, SNR52, RPR1 and SCR1) transcribed by pol III  tRNA genes  and othe pol III  as well  pol III  belong t pol III . Pol IIImcm mutants, Asf1 has a minor role in chromosome maintenance The presence of Asf1 on the telomeres agrees with its previously established role in telomeric silencing ARG1, a gene reported as repressed under growth on enriched media Our Asf1 enrichment data have under-representation of ORFs . We divided ∼4000 pol-II transcribed ORFs into five categories, based on their transcriptional activity Yeast pol II-transcribed genes generally have a nucleosome-free region (NFR) at the 5′ end of genes, flanked by two positioned nucleosomes, −1 and +1 asf1Δ cells (color coded pink and green), only 89 genes were common, while only 26 genes were found misregulated  between the two. Validation of the ChIP-Seq data for Asf1 occupancy at different pol III-transcribed genes by ChIP-qPCR shows high occupancy of Asf1  is highest of all targets , 3A. Usi of Asf1  on tRNA  of Asf1 . On non- of Asf1 .SNR52 and some tRNAs in asf1Δ cells have been suggested as related to transcription activity SNR6 and SNR52) pol III-transcribed genes, in asf1Δ cells as compared to the wild-type cells, although RPR1 shows only marginal increase  Asf1 targets  decrease in asf1Δ cells . A comparison of the peak distributions and a close up view of both the arms of chromosome V show that Asf1 and Brn1 peak distributions are nearly identical  in the same data Asf1 is required for H3K56 acetylation by Rtt109 asf1Δ cells. Since Asf1 is a highly conserved H3/H4 chaperone, its targets in higher eukaryotes may be similar. The outcomes of this study also propose that identifying genome-wide targets of other histone chaperones can resolve known redundancy and specific or novel functions of different histone chaperones.Further probing of the functional and physiological implications of its high association with centromeres and genome-wide co-localization with condensin may reveal novel functions of Asf1. An earlier study Figure S1ChIP efficiency check. (A) Quality check for ChIP DNA preparations. After crosslinking, chromatin was fragmented using Bioruptor sonicator to a mean size of 150–300 bp. Chromatin fragments were separated and an aliquot was analyzed by agarose gel electrophoresis. Input DNA preparations for both replicates are shown (lane 1 and 2); lane M depicts 100 bp-ladder, used as size marker. ChIP and Mock experiments were performed using the same input DNA . Schematic diagrams in the right panels show amplicon positions used for ChIP-qPCR experiments on SCR1 (B), RPR1 (C) and SNR6 (D) loci. Two genes transcribed by RNA polymerase III, viz. SCR1 and RPR1, were selected due to their relatively longer length than other pol III-transcribed genes.(PDF)Click here for additional data file.Figure S2Asf1 association profile at some of the pol III-transcribed genes. Screen shots from UCSC genome browser showing Asf1 association at (A) SNR52, (B) SNR6, (C) SCR1 and (D) RPR1 genes. Y-axis represents normalized Asf1 tag counts; mock tag count shows background. Distribution of Asf1  is shown at different pol III-transcribed genes.(PDF)Click here for additional data file.Figure S3Asf1 preferentially occupies the genes highly transcribed by pol II. Heat maps depicting Asf1 ChIP-Seq signal at different pol II ORFs. Color code is shown at the bottom. Bent arrow represents TSS. Asf1 occupancy 500 bp upstream and downstream of the TSS for all pol II ORFs was calculated. Heat maps of 500 lowest expressed  and 500 highly expressed  ORFs are shown. Vertical arrow shows increasing order of transcription activity of ORFs in each category.(PDF)Click here for additional data file.Figure S4Asf1 deposits nucleosomes on pol III-transcribed genes. (A) Data from the previous study asf1Δ cells, as compared to RNA levels in the wild-type cells, set to 1. SCR1 was used as an internal control. The averages of five independent experiments with error bars are shown. (C) and (D) show results of nucleosome occupancy measured on various pol III-transcribed genes in wild-type and asf1Δ cells. Mononucleosomal DNA isolated from chromatin digested with micrococcal nuclease in situ was used for qPCR. Averages from three independent estimations with error bars are shown. Amplicons A–F for SCR1 regions (panel D) are described in the (PDF)Click here for additional data file.Table S1List of the yeast strains used.(PDF)Click here for additional data file.Table S2List of the Primers used.(PDF)Click here for additional data file.Table S3Different categories of the genome-wide chromosomal features targeted by Asf1.(PDF)Click here for additional data file.Table S4Functional distribution of Asf1-occupied 242 pol II genes.(PDF)Click here for additional data file.Table S5Transcription activity at 242 ASf1-occupied pol II ORFs.(XLSX)Click here for additional data file.Methods S1This file contains details of the methods followed and the supplementary references cited in the Supporting Information files.(PDF)Click here for additional data file.


Compared to gamma-retroviral and lentiviral vectors, the SIN-ASLV vector maintains a preference for open chromatin regions, but shows no bias for transcriptional regulatory elements or transcription units, as defined by genomic annotations and epigenetic markers (H3K4me1 and H3K4me3 histone modifications). Importantly, SIN-ASLV integrations do not cluster in hot spots and target potentially dangerous genomic loci, such as the EVI2A/B, RUNX1 and LMO2 proto-oncogenes at a virtually random frequency. These characteristics predict a safer profile for ASLV-derived vectors for clinical applications.Gene transfer vectors derived from gamma-retroviruses or lentiviruses are currently used for the gene therapy of genetic or acquired diseases. Retroviral vectors display a non-random integration pattern in the human genome, targeting either regulatory regions (gamma-retroviruses) or the transcribed portion of expressed genes (lentiviruses), and have the potential to deregulate gene expression at the transcriptional or post-transcriptional level. A recently developed alternative vector system derives from the avian sarcoma-leukosis alpha-retrovirus (ASLV) and shows favorable safety features compared to both gamma-retroviral and lentiviral vectors in preclinical models. We performed a high-throughput analysis of the integration pattern of self-inactivating (SIN) alpha-retroviral vectors in human CD34 He genome ,14 and te genome ,16. These genome ).Small-scale surveys of integration sites of vectors derived from alpha-retroviruses, such as the avian sarcoma-leukosis virus (ASLV), in different cell types indicated a more random pattern compared to other retroviruses, with a slight preference for transcription units, but no apparent preference for promoters and transcription start sites (TSSs) ,18,19,20+ hematopoietic stem/progenitor cells (HSPCs), which was carried out to evaluate their comparative genotoxic potential in a clinically relevant target cell. We show that the SIN-ASLV integration profile is close to random, with no preferential targeting of TSSs or transcribed genes compared to SIN-MLV and SIN-HIV. The SIN-ASLV vector does not target CpG islands, conserved non-coding regions (CNCs) or elements enriched in transcription factor binding sites (TFBS), is less frequently associated with epigenetically defined promoter and enhancer regions compared to SIN-MLV and is randomly associated with repetitive elements in the genome. Similarly, we observed no preference for transcribed regions compared to SIN-HIV. Heterochromatic regions are excluded by the integration pattern of all three vectors. Interestingly, the ASLV vector showed no apparent clustering in the genome and has no association with the typical integration hot spots observed for MLV- and HIV-based vectors. These results highlight a safer integration profile of alpha-retroviral vectors in human cells, supporting their development as a clinical gene transfer tool.We and others previously reported that MLV, SIN-MLV and SIN-HIV integrations are highly clustered in the human genome, with cell-specific patterns that correlate with the transcriptional program and the epigenetic landscape of each cell type ,24,25,26+ HSPCs were purified form umbilical cord blood, pre-stimulated for 48 h in serum-free Iscove’s modified Dulbecco medium supplemented with 20% Fetal Calf Serum (FCS), 20 ng/mL human thrombopoietin, 100 ng/mL Flt-3 ligand, 20 ng/mL interleukin-6 and 100 ng/mL stem cell factor, as previously described [Human CD34escribed . HSPCs w6 CD34+/GFP+ cells enriched by fluorescence-activated cell sorting, after a brief period in culture to dilute unintegrated vectors. 3'-LTR vector-genome junctions were amplified by LM-PCR adapted to the GS-FLX Genome Sequencer (Roche/454 Life Sciences) pyrosequencing platform, as previously described [Genomic DNA was extracted from a pool of 3.5 × 10escribed . Raw seqescribed  and mappescribed . All UCSescribed . Raw seqescribed  were annp-value < 0.01.For the association of the integrations with epigenetically defined chromatin states, we used publicly available ChIP-Seq data (NIH Roadmap Epigenomics Mapping Consortium database) that we re-annotated in the UCSC hg19 release of the human genome. We analyzed the distribution of integration sites around histone modifications  using the seqMINER platform . Previou+ cells with a previously described SIN-ASLV vector carrying a GFP expression cassette under the control of the intron-less, 240-bp version of the elongation factor-1α (EFS) promoter [+ cells in comparable experimental conditions, and a set of in-silico generated normalized random sites  [To generate a high-definition alpha-retroviral integration profile in human HSPCs, we transduced umbilical cord blood-derived CD34promoter . Cells wpromoter . Raw seqpromoter  and mapp ,26 were p < 0.01). Similarly, SIN-ASLV showed only a slight tendency to integrate into genes (49.48% vs. 40.58% of random sites), significantly lower than that observed for SIN-HIV vectors . As a consequence, the frequency of SIN-ASLV integration outside transcription units was only slightly lower than random (43.55% vs. 56.26%) and significantly higher than those observed for the other two vectors   compared to SIN-HIV and random sites , which was significantly lower than that observed for the SIN-MLV vector  . A plot  < 0.01) .This analysis indicates that SIN-ASLV vector integrations have an almost random distribution in the human genome, with only a modest preference for genes and promoter regions compared to SIN-HIV and SIN-MLV vectors, suggesting entirely different modalities of target site selection.vs. 17.68%; TFBSs: 55.70% vs. 69.95%; CNCs: 5.49% vs. 8.42%, p < 0.01 in all cases)  ,25,26. Sl cases) , suggestvs. 50%), while both SIN-MLV and SIN-HIV integrations were significantly under-represented in repetitive regions  (vs. 15%), probably as a consequence of the fact that SINEs are often located in transcribed regions and contain PolII promoters [vs. 0.35%), while both SIN-MLV and SIN-HIV integrations were significantly under-represented in these regions   . By lookromoters ,31. On t < 0.01) .Overall, these data indicate a remarkably random pattern of integration for the ASLV-derived vector, which shows none of the characteristic preferences of gamma-retroviruses and lentiviruses for genes and genetic elements associated with gene function and regulation.+ HSPCs, MLV integrations are strongly associated with histone modifications marking transcriptionally active PolII promoters and enhancers, while HIV integrations correlate with epigenetic markers of active PolII elongation within transcription units [+ HSPCs, we analyzed the association of SIN-MLV, SIN-ASLV and SIN-HIV integrations with specific histone modifications defining active or poised PolII promoters, enhancers, transcribed regions and heterochromatin .Many studies have reported a strong correlation between MLV and HIV integration sites and distinct epigenetic markers in different cell types (reviewed in ). In humon units ,32. We ti.e., enhancer (H3K4me1+) and promoters (H3K4me3+), at a much lower frequency compared to SIN-MLV , a tendency comparable to that observed for SIN-HIV (10% in enhancers and 4% in promoters) and slightly higher than that of the random sample . Moreover, SIN-ASLV integrations were poorly associated with a marker of transcribed gene bodies (H3K36me3) compared to SIN-HIV . All three vectors were under-represented in heterochromatic regions marked by H3K27me3 compared to random sites, with the SIN-ASLV vector showing the highest association . Only 21% of all SIN-ASLV integrations are clustered, compared to 56% and 51% of SIN-MLV and SIN-HIV integrations, respectively (p < 0.01)  . MoreoveAlthough the SIN-ASLV integration profile shows none of the features typical of MLV- and HIV-derived vectors, it is not completely random and shows a general preference for euchromatic regions. It is now know that both MLV and HIV pre-integration complexes (PICs) are targeted to chromatin by a tethering mechanism involving the interaction of the viral integrase with host cell factors: the LEDGF/p75 chromatin component interacts with the HIV integrase and directs its integration into transcribed gene bodies ,39, whilOvercoming the genotoxic consequences of retroviral vector integration in the host cell genome is one of the major issues for the application of retroviral-based gene transfer in clinical trials. The strong preference to target TSSs, active regulatory elements or transcribed genes, together with the high frequency of clustering around hot spots, is a characteristics shared by all retroviral vectors currently used in clinical gene therapy. These characteristics are at the basis of the potential of retroviral insertion to deregulate gene expression at the transcriptional or post-transcriptional level, which has been observed to cause clonal expansion and contribute to neoplastic transformation in a number of cases (reviewed by ). Many eex vivo gene therapy applications.Based on a genome-wide analysis of >8000 integration sites in human HSPCs, we show that a SIN-ASLV vector has a quasi-random integration pattern that privileges active chromatin regions, but is not associated with active regulatory elements, like MLV, or with transcribed genes, like HIV. More importantly, the SIN-ASLV vector showed no integration hot spots and no preferences for subsets of genes with a defined ontology or genes that were previously identified as being activated by retroviral insertion into tumors. Previous evaluations of ASLV-derived vectors in pre-clinical models proved its ability to sustain long-term transgene expression in murine and human hematopoietic progenitors and to correct the pathology in a mouse model of X-linked Chronic Granulomatous Disease (X-CGD), with no evidence of post-transcriptional interference ,22. Comb


Applications of these techniques illuminate the developmental mechanisms underlying reptilian corticogenesis, which provides significant insight into the evolutionary steps of different types of cortex and the origin of the mammalian neocortex.The mammalian neocortex is a remarkable structure that is characterized by tangential surface expansion and six-layered lamination. However, how the mammalian neocortex emerged during evolution remains elusive. Because all modern reptiles have a homolog of the neocortex at the dorsal pallium, developmental analyses of the reptilian cortex are valuable to explore the origin of the neocortex. However, reptilian cortical development and the underlying molecular mechanisms remain unclear, mainly due to technical difficulties with sample collection and embryonic manipulation. Here, we introduce a method of embryonic manipulations for the Madagascar ground gecko and Chinese softshell turtle. We established  The mammalian cerebral cortex is a remarkable brain structure that is responsible for intricate social behaviors and intelligence. The cerebral cortex is characterized by tangential expansion of its surface area, which is particularly enhanced in the primate and human neocortex, and a six-layered laminar structure composed of multiple types of excitatory and inhibitory neurons , which develops in the dorsal part of the telencephalon in all vertebrate species  and the Chinese softshell turtle (Pelodiscus sinensis). Surgical techniques on developing reptilian embryos enable us to utilize various experimental approaches. We established the introduction of exogenous genes into the reptilian cortex by in ovo electroporation. Furthermore, we developed an ex ovo culture system for gecko and turtle embryos, which remarkably increased accessibility to the embryos and improved the efficiency of gene introduction. Successful manipulation techniques of non-mammalian embryos are valuable for studies of the evolutionary developmental biology of the cerebral cortex.Here, we describe a method of embryonic manipulation techniques for two reptilian species: the Madagascar ground gecko (P. pictus) is a ground crawling gecko that commonly lives on Madagascar Island. Gecko, lizard, snakes and Tuatara (Sphenodon) are included in the group of lepidosaurs  is a freshwater-living turtle that is widely distributed in eastern and southeastern Asia ; after these stages, the eyes and jaws increase rapidly in size, which make it difficult to access and electroporate to brains.After oviposition, the laid eggs should be isolated from the mother animal to avoid accidental crushing of the eggs. In our laboratory, the eggs are immediately transferred to a plastic container filled with dried vermiculite Figure . To mainin ovo electroporation, the egg is placed on a depression slide  with moistened papers  with a non-toxic dye (0.01% fast green). Typically, we prepare 2.5–5 μg/μL of plasmid solution for the electroporation. Holding the head of the embryo with a fine forceps, the DNA solution is injected into the left or right side of the lateral ventricle with a fine glass capillary  that is connected to a mouth-pipette  or mini-injector . Subsequently, needle-type electrodes  is inserted into the extra-embryonic space. Because DNA is negatively charged, a positive electrode was positioned at the target region , and a negative electrode was placed at the opposite side of the head .n Figure . FertiliThe position of an embryo within the egg can be monitored by illuminating the egg with a fiber light. To open the turtle eggs, a small hole is made in the shell by drilling the top of the shell with a micro drill  under the dissecting microscope Figures . After oex ovo culture system for the middle stages of reptilian embryos  provides higher expression of transgenes in amniotic brains, particularly in the neural stem/progenitor cells .To perform immunohistochemical analysis, embryos are fixed with standard fixative  for overnight at 4°C and immersed in 20% sucrose for cryoprotection. The samples were embedded in OCT compound , and 14 μm of cryosections are made with a cryostat . Several commercial antibodies are potentially applicable for immunohistochemistry in gecko and turtle embryos Table , 2012, aThe expression of exogenous genes can be monitored soon after electroporation. We collected gecko and turtle embryos at several time points after the electroporation, and examined the expression of fluorescent reporters under a fluorescent microscope. At 2–4 days after electroporation, intense GFP expression was detected in the dorsal part of the gecko and turtle telencephalon Figures . Even atAt 2 days after electroporation, GFP expression was exclusively detected in mitotic neural stem/progenitor cells that were localized at the ventricular zone of the developing gecko cortex. These neural stem/progenitor cells have a radial fiber similar to the mammalian radial glial cells, but the fibers extend in a curved manner at the neuronal layer as in the case of avian cortical radial fibers  multipolar neurons in the reptilian cortex do not exhibit mitotic activity and (2) multipolar-to-bipolar transition of migratory modes is not detected during the reptilian corticogenesis. These data support the idea that both amplification of IPCs  lineage tracing of neural stem/progenitor cells, (2) quantification of reporter activities for signaling molecules, and (3) gain- and loss-of-function analyses of specific genes in the developing reptilian cortex , a related species to the gecko, have been published .Recently, whole genome sequences of Chinese softshell turtle and sew turtle have been performed, which have confirmed that turtles must be positioned phylogenetically in archosaur groups in amniotes . Because reptilian eyes and jaws rapidly increase in size during embryogenesis, positioning the electrodes to target dorsal cortex is technically difficult at later embryonic stages. Application of an ex ovo culture system for gecko and turtle embryos is also limited for 3–4 days, most likely due to the lack of some essential nutrients and/or sufficient oxygen supply. Further improvements of gene delivery tools and/or culture conditions are required to manipulate embryos at any developmental stage.Currently, we have only successfully performed in vivo delivery of CRISPR/Cas vectors induces direct somatic recombination in target tissues, which enables the site-specific mutation of endogenous genes (Xue et al., Electroporation with a transposon-mediated genomic integration system provides permanent lineage tracing in mammalian and non-mammalian vertebrates (Garcia-Moreno et al., 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.


The regulation of specific target genes by transcription factors is central to our understanding of gene network control in developmental and physiological processes yet how target specificity is achieved is still poorly understood. This is well illustrated by the Hox family of transcription factors as their limited in vitro DNA-binding specificity contrasts with their clear in vivo functional specificity.Drosophila Kc167 cells, revealing clear target specificity and a striking influence of chromatin accessibility. In the absence of the TALE class homeodomain cofactors Exd and Hth, Ubx and Abd-A bind at a very similar set of target sites in accessible chromatin, whereas Abd-B binds at an additional specific set of targets. Provision of Hox cofactors Exd and Hth considerably modifies the Ubx genome-wide binding profile enabling Ubx to bind at an additional novel set of targets. Both the Abd-B specific targets and the cofactor-dependent Ubx targets are in chromatin that is relatively DNase1 inaccessible prior to the expression of Hox proteins/Hox cofactors.We generated genome-wide binding profiles for three Hox proteins, Ubx, Abd-A and Abd-B, following transient expression in Our experiments demonstrate a strong role for chromatin accessibility in Hox protein binding and suggest that Hox protein competition with nucleosomes has a major role in Hox protein target specificity in vivo.The online version of this article (doi:10.1186/s13072-015-0049-x) contains supplementary material, which is available to authorized users. Drosophila and their vertebrate Pbx/Meis homologues, increase the specificity of Hox binding in vitro  were grouped according to transcription factor and plotted as one dimensional dot plots together with median using R. The Biostrings R package [2(frequency in peak sequences/background frequency) using 2 kb sequences upstream of TSSs from D. melanogaster genome BDGP release 5 as background (this is the same background as used for PWMEnrich). FIMO [HOMER  and MEME package  was used package  in the sh). FIMO  was usedMinor groove widths were predicted using the DNAshape online tool  using 2 Average GC content was calculated using a Perl script from the Berkeley Drosophila Transcription Network Project (base_composition_across_peaks.pl written by Stewart MacArthur) using 2 kb sequences centred on the peak summits, a window size of 100 bp and a step of 10 bp.−1 DAPI in PTX at room temperature for 2 h. Coverslips were then washed three times in PTX and mounted in Citifluor AF1 Glycerol/PBS solution (Agar Scientific) for immunofluorescence microscopy .Cells transfected with the pMT-Hth2AGFPUbx bicistronic expression vector were induced for 4 h as described above, and fixed using 4 % formaldehyde (Sigma-Aldrich F8775) in PBS for 10 min at 23 °C in an Eppendorf Thermomixer at 700 rpm. Transfected (GFP+) and non-transfected (GFP−) cells were sorted as described above. Sorted cells were seeded onto 1 × 0.8 cm coverslips in the wells of a 12-well plate each containing 1 ml PBS. Cells were allowed to adhere for 30 min. The PBS was then replaced by 1 ml PTX (PBS/0.5 % Triton X-100) and cells were incubated at room temperature for 30 min. Coverslips were then transferred to microfuge tubes containing mouse anti-Exd B11M monoclonal antibody  and incThe ChIP-Seq data are available from GEO under accession number GSE69796. Table S1 in Additional file