Recent mapping of 5-hydroxymethylcytosine (5hmC) provides a genome-wide view of the distribution of this important chromatin mark. However, the role of 5hmC in specific regulatory regions is not clear, especially at enhancers.We found a group of distal transcription factor binding sites highly enriched for 5-hdroxymethylcytosine (5hmC), but lacking any known activating histone marks and being depleted for nascent transcripts, suggesting a repressive role for 5hmC in mouse embryonic stem cells (mESCs). 5-formylcytosine (5fC), which is known to mark poised enhancers where H3K4me1 is enriched, is also observed at these sites. Furthermore, the 5hmC levels were inversely correlated with RNA polymerase II (PolII) occupancy in mESCs as well as in fully differentiated adipocytes. Interestingly, activating H3K4me1/2 histone marks were enriched at these sites when the associated genes become activated following lineage specification. These putative enhancers were shown to be functional in embryonic stem cells when unmethylated. Together, these data suggest that 5hmC suppresses the activity of this group of enhancers, which we termed “silenced enhancers”.Our findings indicate that 5hmC has a repressive role at specific proximal and distal regulatory regions in mESCs, and suggest that 5hmC is a new epigenetic mark for silenced enhancers.The online version of this article (doi:10.1186/1471-2164-15-670) contains supplementary material, which is available to authorized users. The Here, we report on a new repressive role for 5hmC at specific regulatory regions in mESCs. We show that 5hmC negatively correlates with nascent transcripts, especially at TFBSs. Interestingly, we discovered that a group of distal TFBSs displays a new epigenetic signature; these sites are exclusively enriched for 5hmC, depleted for activating histone modification marks (H3K4me1 and H3K27ac), and significantly reduced for nascent transcripts or enhancer RNAs (eRNAs). The expression of the genes close to these TFBSs was significantly lower than that of genes close to other classes of TFBSs. In addition, we found that a fraction of these TFBSs becomes enriched for activating histone marks (H3K4me1/2) in neural progenitor cells (NPCs) or endomesoderm cells. RNA polymerase II (PolII) chromatin interaction analysis with paired-end tagging (ChIA-PET) showed tA recent survey had found 5hmC enriched at TFBSs in hESC, mouse nTo further investigate the role of 5hmC in gene regulation in conjunction with other epigenetic marks, we performed an integrative analysis using 5hmC, 5mC, Tet11, H3K4me1To study the epigenetic landscapes surrounding distal TFBSs, we applied the K-means algorithm K = 10) and found clusters marked by various epigenetic modifications  compared with other clusters Figure B. Both 5Next, we interrogated the state of the 5hmC mark in other cell types. In hESCs, we also identified a cluster enriched for 5hmC but deplAdditional fileNext, we analyzed the correlation between 5hmC levels and transcriptional activity of the genes closest to the TFBSs for each cluster. To calculate gene transcription levels, we calculated the reads per kilobase per million mapped reads (RPKM) from GROseq (see Methods). The genes mapping to the TFBSs in cluster 2 had strikingly reduced transcription levels compared to the genes in all other clusters p-value <1.3e-20), even compared to clusters 8 and 10, where the repressive H3K27me3 mark was relatively enriched  associated with PolII in ESCs and NPCs. By usinSorcs2 (TFBS1). This site is also enriched for ChIA-PET[in vitro study suggests that 5hmC-enriched distal TFBSs are bona fide enhancers, which are silent in mESCs when marked with 5hmC.Next, we directly determined if lack of 5hmC activates enhancer activity of the distal TFBSs in mESCs. We selected five highly 5-hydroxymethylated distal TFBSs regions from cluster 2, including the site in ChIA-PET reads. WWe also investigated if 5hmC at distal regulatory regions has a repressive role using the Tet1 shRNA suppression experiments in mESCs. We founThe field of DNA methylation has expanded recently, with the identification of multiple cytosine variants; 5hmC, 5fC, and 5-carboxylcytosine (5-caC), 39. Amoin vitro transcription studies revealed a repressive role of 5hmC at promoter regions[The contribution of the 5hmC modification to gene regulation is actively debated. Recent studies found that 5hmC gain is accompanied by H3K27me3 loss at promoters and in the gene body during neurogenesis, suggesting an activating role of 5hmC. On the  regions. HoweverWe observed that enrichment of 5hmC corresponds with the depletion of eRNAs at distal TFBSs. Considering that eRNAs correlate with gene transcription, 21, we To ascertain if the proposed “silent enhancers” identified above can indeed function as enhancers we employed luciferase reporter assays. We demonstrated that the novel distal elements, characterized by TF binding, high levels of 5hmC, and absence of the H3K4me1 “enhancer” mark, can indeed function as enhancers in mESCs if they are devoid of the 5hmC modification. This experiment is consistent with the notion that 5hmC could inhibit enhancer activity at a subset of distal TFBSs in mESCs.Our findings are different from the work of Sérandour and colleagues, who hadIn hESCs, we also identified a group of distal DHSs with strong 5hmC but weak H3K4me1 and H3K27ac  and repressive marks (H3K27me3) are enriched, but H3K27ac is depleted, 24. 5fC and reprWe found that 5hmC was enriched at distal PPARγ binding sites in fully differentiated adipocytes. These findings suggest 5hmC as a new marker for poised enhancers even in absence of H3K4me1 and H3K27me3. Additionally, we also found enriched 5hmC in NPC at the subset of the active TFBSs (except for cluster 2) in mESCs . For cluTo study the functional roles of 5hmC in various regulatory regions, we employed binding site data of 13 TFs  in mESC.To investigate 5hmC and nascent RNA levels across genes, we divided the genes into promoter (from -1Kbp to 500 bp around the annotated start site), 3′ end (from −500 bp to 500 bp around the annotated termination site), and gene body regions (500 bp from the annotated start site to −500 bp from the annotated termination site). For transcription levels, we calculated RPKM using GROseq reads from 500 bp of the annotated start site to the annotated termination site in order not to include transcriptional pausing at promoters, 48.Genomic DNA was prepared from R1 mouse embryonic stem cells. About 6Figure S2. Comparison of the characteristics of each cluster. Figure S3. Comparison of the 5hmC patterns for each cluster. Figure S4. The 5hmC profile of cluster 2 using TAB-Seq. Figure S5. The 5hmC clusters in hESCs. Figure S6. The 5hmC clusters in mature adipocytes [Figure S7.2 The average profiles of TFs at cluster 2. Figure S8. The gene expression change for the target genes for each cluster. Figure S9. The gene expression changes of the target genes after Tet1 knockdown for each cluster. Figure S10. The 5hmC in mESC and NPC at the TFBSs in mESCs. Figure S11. 5hmC at CTCF binding sites in cluster 2. Table S1. Datasets. Table S2. The frequency of transcription factor occupancy in cluster 2. (DOCX 2 MB)Additional file 1: Figure S1: 5hmC profile at promoters and enhancers. ipocytes . Figure


HSI establishes a domain of noncoding transcription that is ‘looped’ to the hGH promoter as an essential step in initiating hGH gene expression. Thus, defining how HSI generates its domain of noncoding transcription is central to understanding its long-range function. Here, we demonstrate that activation of noncoding transcription reflects an HSI-autonomous activity fully independent of interactions with linked gene promoters and occurring in spatial and temporal synchrony with initiation of GH expression in the embryonic pituitary. HSI establishes its noncoding transcription start sites (TSS) over a defined distance from its core determinants and in a manner independent of local primary sequences. The interval between HSI and it TSS co-maps with a domain of disordered and/or highly mobile nucleosomes specific to the pituitary locus. Thus, a localized chromatin reconfiguration by HSI and consequent establishment of an adjacent domain of noncoding transcription constitute initiating events in long-range enhancer function within the hGH locus.The  The elooping’ ,9, and dlooping’ . In somein trans –12. Impohuman growth hormone (hGH) gene is under the control of a long-range enhancer. This enhancer, HSI, constitutes the major pituitary-specific transcriptional determinant within the multicomponent hGH locus control region (LCR)   Figure  13). HS. HShumanr Figure . It is fene loci . In vivo) Figure  17). Pi. PihumanGH locus  and is epromoter . Deletiopression . Thus, HhGH locus revealed that a B-cell specific gene, hCD79b, is situated between HSI and the hGH-N promoter .The initial mapping of the r Figure  20). hC. hChGH lreceptor . Igβ is receptor . It was ransgene . While icription , subsequatotrope ,23. ThushCD79b transcription in B cells and in the pituitary are under distinct regulatory controls. In B cells, hCD79b transcription is dependent on the interaction of B-cell specific transcription factors with promoter and promoter-proximal determinants  reveals that they are positioned 1.6 ± 0.2 kb 3′ to the HSI core determinants. This intervening distance co-maps to a domain of disordered and/or highly mobile nucleosomes specific to the pituitary. These data support a model in which HSI reconfigures local chromatin structure to support a robust set of noncoding TSSs. These HSI activities are proposed to constitute initiating events in its long-range enhancement of hGH-N gene expression.In the present study, we explore the mechanisms by which HSI activates its adjacent domain of noncoding transcription. Our studies reveal that this HSI activity is autonomous of interactions of HSI with linked promoters. The activation of this HSI-dependent noncoding transcriptional activity is further shown to be developmentally appropriate, tracking both spatially and temporally with the initiation of hCD79b fragment and a 1.6-kb mζ-globin fragment were hybridized using corresponding α-dCTP-labeled probes. Each band signal was scanned and quantified using Typhoon FLA9500 Phosphorimager  and the normalized ratio was used to establish the transgene copy number. The transgenic lines hGH BAC, λΔCD/hGH BAC, CDΔ0.7/hGH BAC, CDΔ1.6/hGH BAC and −8.0CD79b have been described previously (Transgenic lines were generated by microinjecting purified transgene DNAs into fertilized mouse oocytes (C57BL/6XSJL) by Transgenic and Chimeric Mouse Facility (TCMF) of the University of Pennsylvania. Founders were identified by PCR of tail DNA and transgene copy numbers were determined by Southern blot analyses of purified genomic DNA digested with NcoI; a 3.5-kb eviously ,25,26.hCD79b, Hs00236881_m1; λ; mGAPDH, Mm99999915_g1). SDS2.4 software was used for analyses.Total RNA was extracted from mouse pituitaries with RNA-Bee (Tel-Test), treated with RQ DNase (Promega) at 37°C for 1 h, and purified using an RNeasy Minikit (Qiagen). In all cases, 1 μg of purified RNA was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and RNA levels were assessed by real-time PCR using TaqMan Universal PCR Master mix (Applied Biosystems) and a 7900HT platform with corresponding probes  and cloned into the pGEM-T easy vector (Promega). DIG-labeled anti-sense RNA probes were generated by SP6 or T7 in vitro transcription, treated with DNase I (New England Biolabs), precipitated with ethyl alcohol, dissolved in TE buffer ), and stored at −80°C until use. Embryos from defined transgenic lines were collected at specified days post coitum (dpc) and fixed overnight at 4°C in 4% paraformaldehyde in phosphate buffered saline (PBS). The fixed embryos were immersed in 30% sucrose in PBS at 4°C, embedded in OCT (Sakura), and quick-frozen on dry ice. Twenty micrometers slices were generated by cryostat and in situ hybridizations were performed as described by Nissim et al. . The amplified cDNAs were cloned into the pGEM-T Easy Vector (Promega) and individually sequenced .Total RNA isolated from transgenic mouse pituitaries was reverse-transcribed by Superscript III reverse transcriptase (Invitrogen) at 55°C for 1 h using a sequence-specific antisense primer (Supplementary Table SI). Free residual dNTPs were removed by G-50 Spin column , a poly G tail was added to the 3′ end of the transcribed cDNA using terminal dinucleotidyl transferase (Promega), and the tailed cDNA was then amplified between an adaptor-polyChGH BAC transgenic mice  were crossed with GRF transgenic mice . The oligonucleotides used for library preparation are listed on Supplementary Table SI. The concentration and size distribution of each library was validated using an Agilent Bioanalyzer (Agilent Technologies). Single-end sequencing (50 bp) was performed on each library using a HiSeq2000 by Next Generation Sequencing Core (NGSC) at the University of Pennsylvania.A chimeric genome assembly was constructed by concatenating the standard mm9 assembly with an additional human sequence contig (hg19) spanning the transgene locus (chr17:61932183–62053367). Reads were aligned to this chimeric genome using bwa-0.7.8  (PMID 19451168). Alignments were merged and coordinate sorted using samtools-0.1.18 (PMID 19505943). Nucleosome coverage and nucleosome positioning stringency were estimated using NuMap-0.9  . CoveraghCD79b gene . A single remaining line was expressed at substantially lower levels, but still clearly above background. An inclusive compilation of these data from all 11 lines  was replaced by an identically sized (3.6 kb) segment of bacteriophage λ DNA  removing the B-cell specific hCD79b promoter and a more extensive 1.6 kb deletion extending further 3′ through a secondary promoter located within intron 1 (CDΔ1.6/hGH BAC) (−8.0CD79bΔ1.6) transgene  by an identically sized segment of λ DNA) . These data support the conclusion that HSI is able to activate noncoding TSS at a mean distance of 1.6 ± 0.2 kb 3′ from HSI and in a variety of primary sequence environments. How this particular spacing between HSI and its noncoding TSS is established was next assessed.TSSs located 3′ to HSI were initially mapped in the pituitaries of mice carrying the intact 123 kb e Figure , top. ThhGH BAC) . 5′ RACEhCD79b promoter-proximal elements (see 'Introduction' section). To explore the basis for the activation of TSS in the pituitary, we directly compared nucleosome architecture in the pituitaries and B cells of hGH BAC mice. Mononucleosome preparations generated from somatotrope-enriched pituitaries and splenic B cells were digested with micrococcal nuclease for 10, 20 or 30 min . These timed digestions were designed to release mononucleosomes from ‘open’ as well as ‘compacted’ regions of the genome and to minimize any loss of DNA associated with loosely bound nucleosomes. Mononucleosome populations were gel purified, pooled and sequenced to a minimum depth of 10-fold genome coverage (Table hGH BAC transgene was mapped for each cell type (Figure The activation of noncoding TSSs by HSI in the pituitary appears to reflect a non-canonical mechanism dependent on HSI actions. The TSS in the pituitary are solely dependent on HSI function while those in the B cells are solely dependent on e Figure  and corre Figure  and C.hGH-N promoter and that bridging HSI and hCD79b (Figure hGH-N promoter (Figure hGH-N promoter itself aligns with a 200-bp nucleosome free region that is flanked by positioned nucleosomes in both pituitary and B cells although this nucleosome-free region is more pronounced in the pituitary, the site of hGH expression (Figure hCD79b promoter co-maps in both cell types with a nucleosome-free region flanked by strongly positioned nucleosomes (Figure The detailed analysis of nucleosome occupancy focused on the regions encompassing the r Figure  containsn Figure . The hCDs Figure .hGH-N and hCD79b promoters in pituitary and B cells, the chromatin structures at HSI and in the region between HSI and the hCD79b promoter in these two tissues are clearly divergent (Figure hCD79b promoter and HSI in the two tissues were next compared (Figure In contrast to these conserved nucleosomal organizations surrounding the t Figure . The HSIt Figure , blue. Tt Figure , red, cot Figure . The 600d Figure . This red Figure , blue, dd Figure .Recent advances in genome-wide technologies have generated significant insights into linkages between chromatin structures and gene regulation. A finding of particular interest is the association of enhancer elements with localized regions of noncoding transcription . While whGH gene is under the control of a defined enhancer determinant, HSI, located 14.5 kb 5′ to the target hGH-N promoter (GH gene in the embryonic pituitary (Figure hGH-N gene expression.The promoter ,18,34. Tpromoter ,16 and dpromoter ,18. HSI promoter . HSI enhpromoter . In the y Figure . These dhCD79b gene and by replacing the hCD79b gene with a segment of λ-DNA (Figure How HSI activates its domain of noncoding transcription was addressed by mapping the position of the corresponding TSSs. 5′ RACE studies revealed that the TSSs that define the 5′ terminus of the noncoding transcriptional domain are established at a mean distance of 1.6 ± 0.2 kb from the HSI core elements. This distance was maintained despite the introduction of large deletions in the A Figure . These dhGH BAC in the pituitary revealed that in addition to the activation of a cluster of TSSs at the conserved spacing of 1.6 ± 0.2 kb, two additional sets of TSSs are generated further 3′ (Figure hGH-N promoter in the pituitary locus (hCD79b promoter (hGH locus in pituitary and B-cell.The 5′ RACE analysis of the intact ry locus . This copromoter . While tin vivo remain incompletely understood. Nucleosomes can be organized in phased arrays or they can lack specific positioning, taking an unstructured or ‘fuzzy’ configuration (hGH locus in pituitary in B cells may be particularly informative.Structural parameters that dictate nucleosome positioning and occupancy in the mammalian genome guration . Determiguration . Tightlyguration ,37. A laguration . This unguration . In thishGH BAC transgenic mice reveals strong similarities as well as distinct differences. The chromatin architecture encompassing the hGH-N promoter is marked in both tissues by a nucleosome-free regions bracketed by strong peaks of nucleosome occupancy (Figure hCD79b promoter (Figure hCD79b promoter comprises a 600-bp region that is occupied by nucleosomes but lacks an organized nucleosomal configuration. While the exact structure of this ‘fuzzy’ domain flanked by a set of strong nucleosome occupancy peaks remains to be further explored (Figure hGH-N promoter would place the target promoter within a microenvironment that strongly enhances transcriptional activity.The comparison of mononucleosome-protected DNA fragments generated from pituitary and B cells of y Figure . A nucler Figure . This tyr Figure . In contr Figure . HSI in r Figure  and its r Figure . The secd Figure , its lacSupplementary Data are available at NAR Online.


Most of the recent advances in our understanding of the bidirectional [Ni-Fe] hydrogenase (Hox) came from investigations performed in the widely-used model cyanobacterium Synechocystis PCC6803 where Hox is the sole enzyme capable of combining electrons with protons to produce H2 under specific conditions. Recent findings suggested that the Hox enzyme can receive electrons from not only NAD(P)H as usually shown, but also, or even preferentially, from ferredoxin. Furthermore, plasmid-encoded functions and glutathionylation (the formation of a mixed-disulfide between the cysteines residues of a protein and the cysteine residue of glutathione) are proposed as possible new players in the function and regulation of hydrogen production.In order to use cyanobacteria for the biological production of hydrogen, it is important to thoroughly study the function and the regulation of the hydrogen-production machine in order to better understand its role in the global cell metabolism and identify bottlenecks limiting H Even hydrogen (H2), which has a higher energy content than oil (142 MJ/kg for H2vs. 44.2 MJ/kg for oil) and burns cleanly, producing only water as its by-product, is not yet a clean biofuel because it is mostly produced from burning oil  hydrogenase, which does not use ATP  bidirectional hydrogenase , where it is the sole enzyme capable of combining electrons with protons to produce H2 under specific conditions  hydrogenase sub-complex HoxHY the NAD(P)H-transported electrons produced by photosynthesis and/or sugar catabolism . Recentlavodoxin , similaravodoxin . During avodoxin , and subavodoxin ,14. The avodoxin ; it has oduction  and it ioduction . Hence, 2 production. In order to use cyanobacteria for the biological production of hydrogen, it is important to thoroughly study the regulation of the hydrogen-production machine in order to better understand its role in the global cell metabolism  and idenhypABCDEF genes that are scattered in the Synechocystis chromosome  and –10 (TAacAa) located at correct distances from each other (18 bp) and from the transcription start site (7 bp). As such canonical promoters were long ago shown to be strongly active in Synechocystis [In contrast to the romosome , the hox Sll1225 ,20. ThisoxE gene . The proy active  though ihoxEFUYH operon is regulated by various environmental conditions, such as hydrogen, light, nitrate, nickel, oxygen and sulfur availabilities [2 production can be increased in media with optimized concentrations of these elements [hox genes are increased (five to sixfold) under microaerobic conditions [hoxEF in darkness [The bilities , in agreelements . The tranditions ,28, withdarkness .hox genes elicited by nitrogen starvation is consistent with the increased H2 production triggered by inhibition of nitrate assimilation, suggesting that hydrogenase serves as an alternative sink for photosynthetic electrons no longer used for nitrate reduction [The upregulation of eduction .hox genes are also regulated by inhibitors of the photosynthetic electron transport chain in a way suggesting that the redox state of the plastoquinone pool does not play a prominent role in hox regulation [2 production [hoxEFUYH operon is controlled by three transcription factors  strain and ΔlexA mutants where we measured the expression of the cat (chloramphenicol acetyl transferase) gene driven by the hox promoter. Similar CAT activities were obtained in the WT and ΔlexA strains growing in standard photoautotrophic conditions, indicating that the activity of the hox promoter is not affected by the absence of LexA (data not shown). This finding challenges the notion that LexA is a direct transcription regulator of the hox operon.The occurrence of two LexA binding sites in the  bending , a well- bending . To testome copy . Then, whox expression was isolated on the basis of its specific interaction with the hox promoter region. This regulator, AbrB1, resembles the AbrB regulator of B. subtilis, which regulates about 100 genes involved in various processes , though the cyanobacterial AbrB1 protein has its putative DNA-binding domain in its C-terminal region, instead of in the usual N-terminal region as occurs in non-cyanobacterial AbrB proteins [Synechocystis AbrB1 protein was found to be indispensable to cell life in both the wild-type strain [abrB1 gene is expressed from an atypical promoter harboring an extended –10 element (5'-tgtTATAtT-3') [secA gene [By using DNA affinity assays, another transcription factor positively acting on proteins ,33. The e strain  and the e strain . The abrTAtT-3') , which mecA gene .Synechocystis, and appeared to be dispensable to the growth of both the glucose-tolerant mutant [hox operon [hoxW, hyp, lexA and abrB1 were not regulated by AbrB2, even though it appeared to regulate a large number of genes , as shown by transcriptome analysis with pan-genomic microarrays [Synechocystis, though it is one of the more intensively studied cyanobacterium. Many these unknown genes belong to the endogenous plasmids, suggesting that they play a role in hydrogen production. Because it resembles to AbrB1, the AbrB2 has also been studied in t mutant  and the t mutant . In the t mutant ,35,36. Ix operon . The othroarrays . The higcysAPUW (SO42−), fecBCD (Fe2+), in agreement with the Hox enzyme using a Ni-Fe and several Fe-S redox clusters to produce hydrogen [abrB2-deleted mutant, the increased expression of the anti-oxidant genes cydAB (cytochrome bd-quinol oxidase that can reduce O2) and norB (nitric oxide reductase) are consistent with the increased production of hydrogen [Synechocystis to both metal and oxidative stresses triggered by nickel and the thiol oxidizing agent diamide [The AbrB2 master regulator was also found to regulate several transport genes, including hydrogen . In thishydrogen , which ihydrogen ,15,17,18 diamide . abrB2 promoter region was also analyzed, using transcriptional fusion to the promoter-less cat reporter gene of the plasmid pSB2A that replicates stably in Synechocystis at one copy per chromosome copy [abrB2 gene was found to expressed from an atypical promoter, which harbors an extended –10 element (5'-TGTATAAT-3'), likely compensating the absence of a genuine –35 box , similarly to the genes secA [gap2 [abrB1 [5)-AAC, was also proposed for AbrB2 binding [The ome copy . The abrnes secA , gap2 [3cA [gap2  and abrB2 [abrB1 . In addi binding .Synechocystis, Germer and co-workers used the light-inducible promoter of the photosynthetic gene psbAII to increase the expression of the endogenous hoxEFUYH operon and the heterologous NoshypABCDEF operon from Nostoc PCC7120 [i.e., from 2.9 nmol H2·min−1·mg·chlorophyll−1 in wild-type cells up to 9.4 nmol H2·min−1·mg·chlorophyll−1 in mutant cells) for three main possible reasons. First, the light-inducible psbAII promoter used to increase the expression of the hoxEFUYH and the NoshypABCDEF genes is more active under high light that increases the photosynthetic production of O2, which inhibits hydrogenase activity. Second, the Nostoc HypABCDEF proteins might be not fully active on the Synechocystis PCC6803 HoxEFUYH proteins. Third, it is also possible that the Nostoc HypABCDEF proteins might somehow interfere with the function of the endogenous Synechocystis HypABCDEF proteins.To attempt increasing hydrogenase activity in  PCC7120 . The gaihoxEFUYH operon and the hypABCDEF genes, we have used the strong λRp promoter [hoxEFUYH operon by the λRp promoter, and we cloned the hypABCDEF genes under the control of the same λRp promoter, in a plasmid that replicates at the same 10–20 copies per cell as the chromosome [hoxEFUYH operon and the hypABCDEF genes (about 100-fold), and exhibited a 20-fold higher levels of active hydrogenase than the WT strain. These findings show that an increased production of active hydrogenase is not detrimental to cell life, and other factors than hoxEFUYH and hypABCDEF transcription limit hydrogen production (see below).To increase the expression of the promoter ,24. Pracpromoter  natural Though, the complex hydrogenase enzyme is neither ubiquitous in all cyanobacteria nor essential to the standard photoautotrophic growth of model strains , the ana2O2, through the accidental autoxidation of redox enzymes [2O2 tolerance of the WT strain to that of mutants which either lack the hoxEFUYH operon, or overexpress it, alone or in combination with the hypABCDEF genes [hoxEFUYH operon and the hypABCDEF genes contribute to the protection against H2O2, positively (hoxEFUYH operon) or negatively (hypABCDEF). Future experiments will be required to test whether the higher H2O2 tolerance directed by the overexpression of the hoxEFUYH operon is due to the increased abundance of (i) the HoxHY hydrogenase enzyme per se; (ii) the HoxEFU diaphorase enzyme; and/or (iii) the Sll1222, Ssl2420 and Sll1225 proteins of as yet unknown function.All aerobic organisms invariably produce reactive oxygen species, such as H enzymes , which o enzymes . As the  enzymes ,17, we hEF genes . The datArthrospira (Spirulina) maxima [Cyanothece ATCC 51142 [Synechocystis growing under an otherwise normal light fluence, probably because these reduced metabolites somehow decrease the electrons-consuming CO2-assimilation, thereby allowing spared electrons to recombine with O2 and generate ROS [hoxH-deletion mutant [2 to generate toxic reactive oxygen species [In addition, we have tested the influence on hydrogenase deletion or overproduction mutants of the reduced-carbon metabolites glucose and glycerol, a cheap surplus of industries  that sti) maxima  and CyanCC 51142 . Both glrate ROS . The levrate ROS , in agren mutant . Collect species .hox operon repressor AbrB2 exists under two uncharacterized posttranslational modification forms [hox operon play a role in the tolerance to oxidative stresses [hox operon-promoter DNA; its repression of a wealth of other genes; and its stability at the non-standard temperature of 39 °C [hoxEFUYH regulator, AbrB1, which interacts with AbrB2 and also regulates the hoxEFUYH operon [in vitro like AbrB2. Thus, it will be interesting in the future to analyze the influence of the glutathionylation of AbrB1 and AbrB2 on their interaction and their regulation of hydrogen production. To our knowledge, AbrB2 and AbrB1 are the first cyanobacterial regulators reported to undergo glutathionylation  of two cysteinyl residues to form disulfide bonds (-S-S-) between proteins, or between a protein and a molecule of the, crucial , anti-oxon forms ,48, and stresses ,38, promof 39 °C ) and staH operon ,33,36, cnylation ), emphasnylation .Stimulated by the findings that both AbrB1 and AbrB2, and the mercuric reductase enzyme , can be Synechocystis PCC6803, important questions remain.Though significant progress has been made recently in our understanding of the function and regulation of the complex hydrogen Ni-Fe hydrogenase of the model cyanobacterium in vitro data suggested that the Hox enzyme can receive electrons from not only NAD(P)H as usually shown, but also, or even preferentially, from ferredoxin. This proposal will certainly stimulate the investigation of the selectivity/redundancy of the nine ferredoxins of Synechocystis, which are conserved in cyanobacteria.Recent hoxEFUYH operon operates in the defense against redox stresses triggered by H2O2 or reduced carbon or nitrogen metabolites. These findings strengthen the proposal that hydrogenase operates as an electron valve to prevent the supernumerary electrons to recombine with O2 to generate toxic reactive oxygen species. Hence, the hydrogenase complex can be viewed as an important enzyme in cyanobacteria like Synechocystis, which can grow in biofilm, a thick network of auto-aggregated cells where they are inevitably exposed to H2O2 and reduced metabolites released by their neighbors (living or dying and lysing). This view is strengthened by the absence of hydrogenase enzyme in most planktonic cyanobacteria living in open oceans. It will be interesting in the future to investigate the influence of the Synechocystis hydrogenase enzyme in the growth and response to environmental stresses of cells incubated not only in agitated liquid cultures but also in static conditions or on solid media to favor the formation of biofilm.Using gene deletion and over-expression, it has been shown recently that the 2 tolerance of the hydrogenase enzyme to increase hydrogen production. The recently achieved simultaneous overproduction of the HoxEFUYH and HypABCDEF proteins within the same cells led to a 20-fold increase in active hydrogenase. These sophisticated mutants with a higher hydrogenase content and a healthy growth will be very useful cell factories for the purification of large hydrogenase quantities for structural analyses, the data of which should enable the design of a meaningful mutational strategy to increase the low natural OhoxEFUYH and hypABCDEF genes than that of active hydrogenase, indicating that limiting post-transcriptional factors should be dealt with. Thus, it will be important to pay particular attention to glutathionylation (the formation of a mixed-disulfide between the cysteines residues of a protein and the cysteine residue of glutathione) because it was recently reported that the AbrB1 and AbrB2 hydrogen regulator and the HoxH (diaphorase) and HoxF (hydrogenase) protein-subunits can be glutathionylated. These findings will certainly stimulate the analysis of the redox crosstalk between hydrogen production and the oxidative stress-responsive glutathionylation-and-deglutathionylation process. All hydrogenase-overproducing mutants displayed higher levels of expression of the


The GRCh37.p13 primary assembly of the human genome contains 20805 protein coding mRNA, and 37147 non-protein coding genes and pseudogenes that as a result of RNA processing and editing generate 196501 gene transcripts. Given the size and diversity of the human transcriptome, it is timely to revisit what is known of VDR function in the regulation and targeting of transcription. Early transcriptomic studies using microarray approaches focused on the protein coding mRNA that were regulated by the VDR, usually following treatment with ligand. These studies quickly established the approximate size, and surprising diversity of the VDR transcriptome, revealing it to be highly heterogenous and cell type and time dependent. With the discovery of microRNA, investigators also considered VDR regulation of these non-protein coding RNA. Again, cell and time dependency has emerged. Attempts to integrate mRNA and miRNA regulation patterns are beginning to reveal patterns of co-regulation and interaction that allow for greater control of mRNA expression, and the capacity to govern more complex cellular events. As the awareness of the diversity of non-coding RNA increases, it is increasingly likely it will be revealed that VDR actions are mediated through these molecules also. Key knowledge gaps remain over the VDR transcriptome. The causes for the cell and type dependent transcriptional heterogenetiy remain enigmatic. ChIP-Seq approaches have confirmed that VDR binding choices differ very significantly by cell type, but as yet the underlying causes distilling VDR binding choices are unclear. Similarly, it is clear that many of the VDR binding sites are non-canonical in nature but again the mechanisms underlying these interactions are unclear. Finally, although alternative splicing is clearly a very significant process in cellular transcriptional control, the lack of RNA-Seq data centered on VDR function are currently limiting the global assessment of the VDR transcriptome. VDR focused research that complements publically available data (e.g., ENCODE Birney et al.,  An appreciation of the diversity of transcription across the human genome has undergone a rapid expansion in recent years, in large part thanks to the efforts of integrative genomic approaches such as those of ENCODE consortium Birney, . From thIn this context it is timely to review the functions of the vitamin D receptor (VDR/NR1I1)  is found in the promoter regions of actively transcribed genes. This mark is mutually exclusive with H3K9me, which instead is associated with transcriptionally silent promoter regions. Acetylation of H3K9 is found along with methylation of H3K4 at active promoter regions. Similarly, H3K27 can be either acetylated or methylated, with acetylation associated with active gene transcription and methylation associated with gene silencing.2D3) or synthetic analogs, can lead to a highly dynamic exchange of co-factors by releasing co-repressors and inducing a receptor conformation that attracts binding of co-activator proteins s Figure . In the s Figure . The seqs Figure , 2009.Whilst these scenarios are relatively well characterized for the positive regulation of gene expression, it is probably not a complete understanding as the distribution of VDR binding in the genome 2D3 and cell background, with longer treatment time points tending to be associated with more binding sites. However, as yet there are not uniform standards for the analyses of NGS data, and therefore it is likely that different analytical approaches are influencing the number and significance of the VDR enriched peaks identified.To identify VDR binding sites through the genome several groups have now applied ChIP-Seq approaches in different human cell types including immortalized lymphoblastoids  spaced by 3 nucleotides (DR-3), which was identified by traditional biochemical approaches in candidate gene studies, appears to be the minority genomic element that directly binds the receptor. Fewer that 30% of genomic VDR binding sites contain a DR-3, although this number is increased following ligand treatment when there is increased enrichment for VDR binding to DR-3 elements  the concept has emerged that receptor binding is guided by the actions of more dominant so-called pioneer factors including the Forkhead (FKH) family members ) 0/G1 of the cell cycle, coupled with up-regulation of a number of cell cycle inhibitors. Candidate promoter characterization studies have demonstrated a series of VDR binding sites in the promoter/enhancer region of CDKN1A  is mechanistically enigmatic, and included translational regulation and enhanced mRNA translation, and attenuating degrading mechanisms  and p27(kip1) principally mediate G1 cell cycle arrest, but 1α,25(OH)2D3 has been shown to mediate a G2/M cell cycle arrest in a number of cancer cell lines via direct induction of GADD45A 2D3 in breast cancer cell models 2D3. Furthermore, these studies examined the epigenetic basis for the transcriptionally inert state of these targets in resistant models  to several days  clones enabled relatively large pieces of DNA, upto 300 kb to be inserted for sequencing . They elegantly demonstrated a role for VDR to down-regulate miR181a, which when left unchecked degrades p27(kip1), initiates cell cycle arrest and commits cells toward differentiation. These studies illuminated the earlier ones that suggested that p27(kip1) protein levels appeared to be regulated by a range of post-transcriptional mechanisms, such as enhanced mRNA translation, and attenuating degradative mechanisms  in a feed-forward loop and determine the final extent of the cell cycle arrest. 1α,25(OH)2D3 regulates the DNA helicase MCM7  through the balance of MCM7 and CDKN1A  miR-98  contribute negative regulatory aspects to normal gene regulation, for example as part of feed-forward loop motifs  vitamin D3 levels are associated with increased risk of either cancer initiation and/or progression  vitamin D3, are strongly correlated with the generation of the active hormone 1α,25(OH)This raises the very exciting possibility that generating integrated models of VDR binding, the impact of genetic variation, the tissue specific differences in the transcriptome and identifying miRNA contained within transcriptional circuits offers the opportunity of exploiting their serum expression levels of 25(OH) vitamin D3, genetic variation and miRNA expression will be able to be exploited to predict accurately the capacity of VDR function.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hox gene Ultrabithorax and show that its protein-coding region also contains alternative splicing regulatory information. Taken together our experiments indicate that UCEs emerge as a result of combinatorial gene regulatory roles and highlight common features in mammalian and insect UCEs implying that similar processes might underlie ultraconservation in diverse animal taxa.Ultraconserved elements (UCEs) are discrete genomic elements conserved across large evolutionary distances. Although UCEs have been linked to multiple facets of mammalian gene regulation their extreme evolutionary conservation remains largely unexplained. Here, we apply a computational approach to investigate this question in Drosophila, exploring the molecular functions of more than 1,500 UCEs shared across the genomes of 12 Drosophila species. Our data indicate that Drosophila UCEs are hubs for gene regulatory functions and suggest that UCE sequence invariance originates from their combinatorial roles in gene control. We also note that the gene regulatory roles of intronic and intergenic UCEs (iUCEs) are distinct from those found in exonic UCEs (eUCEs). In iUCEs, transcription factor (TF) and epigenetic factor binding data strongly support iUCE roles in transcriptional and epigenetic regulation. In contrast, analyses of eUCEs indicate that they are two orders of magnitude more likely than the expected to simultaneously include protein-coding sequence, TF-binding sites, splice sites, and RNA editing sites but have reduced roles in transcriptional or epigenetic regulation. Furthermore, we use a Drosophila cell culture system and transgenic Drosophila embryos to validate the notion of UCE combinatorial regulatory roles using an eUCE within the  Evolutionary conservation of genomic sequences is highly heterogeneous: poorly conserved regions are commonly intermingled with sequences that show perfect conservation across large evolutionary distances. This latter category includes the remarkable class of ultraconserved elements (UCEs), originally defined as sequences of at least 200 nt that are identical across the human, mouse, and rat genomes . In spitMammalian UCEs have been linked to a diverse set of regulatory functions including transcriptional enhancers  and noncDrosophilamelanogaster and related species harbor an independent set of UCEs to those found in mammals —and observed that mutation of one ultraconserved exon of Ubx affects alternative splicing in Drosophila cells in culture and during embryonic development.Here, we investigate the functional roles of 1,516 UCEs shared across 12 Drosophila genomes. In contrast to previous findings we find that intronic and intergenic Drosophila UCEs (iUCEs) are indeed overrepresented within annotated regulatory elements and show significant enrichments and depletions for binding of specific TFs. Our analysis of individual DNA-binding factors also shows that UCEs are enriched for Polycomb-group (PcG) protein binding, suggesting a role for UCEs in chromatin regulation during development. We further show that exonic UCEs (eUCEs) are strongly enriched for multifunctional sequences and are about 100-fold more likely to combine protein-coding capacity with the presence of RNA editing sites, splicing regulators and TF binding sites compared with randomly chosen exonic elements of identical size. To explore whether such predicted multi-functionality was relevant to gene expression we studied one of the Drosophila genes bearing the highest number of UCEs—the This study therefore contributes to the understanding of the mechanisms that lead to the existence of UCEs and suggests that constraints derived from their multiple enrolment in diverse gene regulatory processes can explain the high level of evolutionary conservation observed in UCEs.D. melanogaster genome and checked for their presence in the other 11 genomes using the short read mapper Bowtie .In this study we define UCEs as DNA elements of at least 50 nt that are identical across the genomes of 12 Drosophila species. Our design is such that the evolutionary distance between the most divergent fruitfly species in our dataset exceeds that of humans and reptiles . Althougr Bowtie . All uniD. melanogaster chromosome arm 3R  in the 15-way multiple alignment available from the dm3 release of the UCSC Genome Browser (https://genome.ucsc.edu/) . We found that 345 out of the 466 UCEs (74.0%) were intact in the alignment: these elements aligned across the 12 Drosophila species without mismatches or gaps . A total of six UCEs (1.3%) could not be verified to be present in a syntenic location in all genomes, typically because they occurred in multiple copies, due to duplications and/or assembly errors. Thus, our alignment-free method greatly increases the sensitivity of UCE detection in these 12 Drosophila genomes, at the cost of a small false positive rate in the order of 1%. As a case in point, a recent study of the same 12 species identified 98 UCEs of at least 80 nt based on the multiple alignment used above . The largest UCE clusters overlapped with key developmental factors, including the pair-rule gene odd-skipped (odd), the Hox genes Antennapedia (Antp) and Ultrabithorax (Ubx) (see below) and the Hox co-factor homothorax (hth) . Thus, our novel approach applied to distantly related Drosophila species points toward a general and robust association between ultraconservation and the genetic control of animal development, in line with earlier findings regarding Drosophila and mammalian UCEs   which reian UCEs .Table 1The detection of multiple links between Drosophila UCEs and developmental genes and processes (see above) together with the fact that in mammals many UCEs serve as developmental enhancers  promptedP = 0.015, χ2 test). Motivated by this finding, which suggests a functional parallel between mammalian and Drosophila UCEs, we decided to undertake a more detailed investigation of the contributions of UCEs to transcriptional regulation in Drosophila.In contrast to the findings of an earlier study  we foundFor this we intersected our UCE annotations with data from the modENCODE consortium  providinWe further reasoned that, if the enrichment of TFs within UCEs is biologically relevant in the context of Drosophila development, we might observe differential TF enrichment at various developmental time points. For one of the enriched TFs, Caudal (Cad) , data weHox genes (The protein Polycomblike (Pcl) was one of the most consistently enriched TFs in our analysis A. Becausox genes . To examox genes . All fouox genes B. Pc andDue to their involvement in protein encoding eUCEs are likely to be subjected to distinct functional requirements from those found in iUCEs. Apart from constraints on coding sequences, previous work highlighted RNA editing and splicing as two individual processes associated with eUCEs in flies and mammals , but theP = 1.9 × 10−8, Mann–Whitney test) suggesting that multifunctionality is a core property of UCEs. As an example, our analysis showed that eUCEs are ∼100-fold more likely than reference elements to be tetrafunctional, i.e., they are located in a protein-coding region, overlap with a splice site, contain an RNA editing site and are bound by at least one TF .To test whether this was the case we calculated the degree of multifunctionality observed for UCEs and reference elements by assessing each UCE in terms of protein-coding potential, presence of RNA editing sites , overlapP = 6.8 × 10−12, χ2-test) suggesting an important role for ultraconservation in the generation of alternative transcripts. This observation prompted us to consider specific eUCEs to experimentally test the hypothesis that ultraconservation might be related to multifunctional roles played by UCEs (see below).Notably, our analysis of eUCEs also revealed that almost all (96%) eUCEs overlap with alternatively spliced exons , as well as of an additional small exon, microexon II (mII), generates functionally distinct Ubx isoforms  in the Drosophila gene Ubx A. Given  mammals  we decidisoforms . The dem species  brought Musca domestica, the tsetse fly Glossina morsitans, and the Mediterranean fruit fly Ceratitis capitata . We then estimated the rate of nonsynonymous substitutions relative to the rate of synonymous substitutions (dN/dS) with codeml  identical across all 12 investigated Drosophila species. Focusing on the third position of each codon, we found that 29 out of 57 sites within the homeodomain had synonymous substitutions. Using this as our reference we were thus able to exclude that the lack of synonymous mutations at the 15 synonymous sites within the mI exon was due to chance  supporting our hypothesis that coding constraints contribute to, but cannot fully explain, ultraconservation within the mI exon.To evaluate this hypothesis, we first investigated to what extent the mI protein-coding sequence showed signs of purifying selection. Using BLAST , we wereh codeml . The dN/Ubx mI (mI-UCE) to test the possibility that it functions as a protein-coding element as well as a docking region for splicing factors. We reasoned that if the latter were true, an experiment where coding sequence capacity is maintained while the nucleotide sequence is modified should potentially expose such splicing-related functions. To test this model, we decided to carry out a series of experiments where the original sequence of the mI-UCE was replaced by one in which the coding potential was unaffected but the nucleotide composition distorted  given that the splicing patterns of Ubx are complex within this tissue  promoter and endogenous gene), and during the formation of adult appendages—that is regulation of wingless, araucan, and spalt genes in wing and haltere imaginal discs  have different abilities to bind to DNA targets in vitro , (2) disal discs , (3) peral discs  and durial discs , and (4)al discs .in vivo support a model by which the mI-UCE performs functions that affect the process of alternative splicing; these data support our hypothesis that UCEs may have retained their sequences over long evolutionary periods due to their intrinsic multifunctionality in regards to gene expression control.All in all, the data of our experimental analysis in cultured cells and Despite a decade of research since the discovery of UCEs the molecular basis of ultraconservation remains unknown. So far, most UCE studies have been carried out in mammals, yet Drosophila UCEs are currently receiving renewed attention , not leaPrevious work suggested that Drosophila UCEs are not involved in transcriptional regulation . In contIn contrast, eUCEs show less pronounced patterns of TF binding enrichment and depletion, suggesting that they do not primarily function as transcriptional regulators. Indeed, eUCEs have been associated with other regulatory processes, such as alternative splicing and RNA editing . In thisFurthermore, our analysis shows that multiple functional layers are frequently superimposed on a single eUCE sequence. For example, we show that eUCEs are nearly 100-fold more likely than expected to simultaneously contain protein-coding sequence, TF binding sites, splice sites, and RNA editing sites. This finding adds to the growing literature on regulatory sequences embedded within coding regions  and suggHox gene Ubx. Although the protein-coding sequence is under strong purifying selection, our comparison between the mI sequence and that of the well-conserved homeodomain showed that protein-coding constraints alone were insufficient to explain the ultraconservation of this exon. As the mI exon is alternatively spliced, we used a previously developed splicing minigene system , D. erecta (droEre2), D. grimshawi (droGri2), D. melanogaster (dm3), D. mojavensis (droMoj3), D. persimilis (droPer1), D. pseudoobscura (dp4), D. sechellia (droSec1), D. simulans (droSim1), D. virilis (droVir3), D. willistoni (droWil1), and D. yakuba (droYak2) were downloaded from the UCSC Genome Browser , which was previously used by D. melanogaster. Instances where we did not find our annotated UCE in a syntenic position, and where this could not be explained by alignment or assembly errors, were due to multiple occurrences of the UCE and highly similar sequences in one or more of the nonmelanogaster genomes. In these cases, the annotated UCE in D. melanogaster had been aligned to a UCE-like sequence, which typically contained only a single mismatch .Unlike previous approaches, our method does not rely on whole-genome alignments and does not incorporate information on synteny. To assess whether our dataset included UCEs that did not occur at syntenic positions across the 12 genomes, we carefully inspected the 466 UCEs located on the supplementary table 4, Supplementary Material online). To explore the association between UCEs and protein-coding genes, we chose to focus on the 15 largest clusters (each comprising at least 10 UCEs), as the limited quality of some of the analyzed genomes prevented us from performing a global clustering and synteny analysis for all species. For these clusters, we checked the overlap with protein-coding genes in the D. melanogaster, D. pseudoobscura, and D. virilis genomes (We grouped UCEs if they occurred within less than the median distance (18 kb) of each other. This resulted in 288 UCE clusters, comprising 1,043 UCEs ( genomes .D. melanogaster genome into fragments with the same length distribution as the UCE set. All reference elements were required to map uniquely to the genome (bowtie -v 0 -m 1). The reference elements were grouped into functional classes  in the same manner as the UCEs. We used BEDTools  using a χ2-test. Correction for 117 multiple tests was performed using the Benjamini–Hochberg method.We analyzed ChIP-chip and ChIP-seq data from the modENCODE consortium . TFs werD. melanogaster coordinates for constitutive and nonconstitutive exons from Ensembl release 79  and recovered mI in the common housefly M.domestica (C.capitata (http://www.hgsc.bcm.edu), but not in more distantly related species, such as the mosquitoes Anopheles gambiae and Aedes aegypti, the flour beetle Tribolium castaneum or the honey bee Apis mellifera. We used the same method to identify the mI exon in the genome of the tsetse fly G.morsitans  fly lines. Embryos were collected at 25C in the dark on apple juice plates supplemented with yeast paste following standard procedures.Virgins of transgenic UAS-lines were crossed to males of 69B-Gal4 (Bloomington 1774) or Ubx wild-type minigene, was originally developed in the laboratory of Javier Lopez .Ubx.mini.HA.short. Subsequently, the minigene was released by sequential SpeI/blunting through T4 Polymerase/KpnI digestions and transferred to the transformation vector pUASP.K10.attB   using the ZH-attB-51C landing site (To create UAS-Ubx minigene expression constructs bearing wild-type (wt) or mutated versions of the mI microexon (mutA) the following procedures were employed. For the creation of pUAS.Ubx.mini.HA.short.attB (HA.short) the zerland)  that hading site .Ubx-mini transgene expression was detected by antibody-stains using enzymatic, alkaline phosphatase detection using standard protocols. In brief, we used anti-hemagglutinin (HA)  followed by anti-mouse-biotin  and streptavidin-alkaline phosphatase conjugates ; enzymatic detection was performed using NBT/BCIP (Roche) substrate.At the protein level Drosophila S2 cells were transfected using Effectene transfection reagent  according to the manufacturer’s protocol. Typically, 1.5 million cells were transfected with 200 ng DNA  and incubated for ∼65 h before collection of RNA.Total RNA was isolated (Sigma RNA Minikit) followed by DNase treatment. cDNA was generated with oligo-dT primer using either 1 or 3 μg of total RNA with M-MuLV reverse transcriptase from NEB . 1 μl of cDNA was used as a template for PCR to amplify isoforms. PCR primers Ubx_E1F: TGGAATGCCAATTGCACCATC3′/Ubx3′R 5′CGCGTCTTCGCAGACCATTT3′ were used to detect the different isoforms [PCR cycles: 31× ]. More PCR cycles amplified other isoforms but gave inconsistent results indicating that the reaction was not in linear range and were not considered further. Minigene expression level was monitored by primers expF: 5′AGTGGAAGGAGCGCAGATTA3′/expR: 5′TCGAGCGAATCCTCTTGAAT3′ [PCR cycles 25× ] amplifying a product of 103 nt. Products were separated on an 8% nondenaturing polyacrylamide gel and quantified with MultiGauge (Fujifilm). Background corrected intensity values were normalized to the general gene level and the average and standard error from the three replicas was calculated presented in log2 scale.Supplementary tables S1–S5 are available at Molecular Biology and Evolution online .


Bisphenol A (BPA) is one of the commonest Endocrine Disruptor Compounds worldwide. It interferes with vertebrate reproduction, possibly by inducing deregulation of epigenetic mechanisms. To determine its effects on female reproductive physiology and investigate whether changes in the expression levels of genes related to reproduction are caused by histone modifications, BPA concentrations consistent with environmental exposure were administered to zebrafish for three weeks. Effects on oocyte growth and maturation, autophagy and apoptosis processes, histone modifications, and DNA methylation were assessed by Real-Time PCR (qPCR), histology, and chromatin immunoprecipitation combined with qPCR analysis (ChIP-qPCR). The results showed that 5 μg/L BPA down-regulated oocyte maturation-promoting signals, likely through changes in the chromatin structure mediated by histone modifications, and promoted apoptosis in mature follicles. These data indicate that the negative effects of BPA on the female reproductive system may be due to its upstream ability to deregulate epigenetic mechanism. Endocrine Disruptor Compounds (EDCs) are chemicals that can interfere with vertebrate reproductionBisphenol A (BPA) is commonly used in the manufacturing of polycarbonate plastics and epoxy resins, making it one of the most widespread EDCs worldwide. According to recent estimates, 100 ton of BPA per year may end up in the environmentBPA alters gene expression by binding to nuclear estrogen receptors (ERs) ERα and ERβ. It is defined as a selective ER modulator because its effects are pro-estrogenic in some tissues and anti-estrogenic in others5As an environmental pollutant, BPA is probably able to affect epigenetic mechanisms through methylation of CpG sitesesr1 and esr2a, steroidogenic acute regulatory protein (star), and a member of the cytochrome P450 family 11 (cyp11a1)8910pgrmc1 and pgrmc2 for oocyte growth and maturation; and bone morphogenetic protein 15 (bmp15) and growth differentiation factor 9 (gdf9) as local signalsThe first goal of the present study was to determine the effects of BPA concentrations consistent with environmental exposure  on reproductive physiology in female vertebrates. To do this, the expression of several genes critical for reproduction was monitored in the ovary: ERs The second goal of the study was to explore whether the changes in the expression levels of these genes were caused by histone modifications, as measured by lysine 4 and 27 trimethylation in the amino terminal of histone 3 (respectively H3K4me3 and H3K27me3). H3K4me3 is involved in the activation of transcriptionDanio rerio, has a fully sequenced genome and shares with humans 70% gene orthology and body plan similarity17The experimental model selected for the study, th to the 21st day of BPA treatment. The results showed blocked ovulation (0 ± 0 eggs) 8 days into treatment in fish receiving the lowest BPA concentration (5 μg/L), whereas the intermediate (10 μg/L) and the highest (20 μg/L) concentration did not induce significant changes (respectively 314 ± 8 and 330 ± 9 eggs) compared with control fish (333 ± 12 eggs).Fertility was measured as mean number ± standard deviation (SD) of fertilized eggs per female per day from the 8The lowest of the 3 concentrations tested, 5 μg/L, induced the most severe effects. Molecular analysis demonstrated that BPA can inhibit reproduction, particularly by affecting oocyte maturation.esr1 and esr2a    . It also pgrmc1) , whereaspression .cyp11a1 gene  and beclin1 (caspase3 (tp53)   was up-rOocyte viability was assessed by histology, to document the effects of 5 μg/L BPA on the ovary.Findings showed that total follicle number did not change in any experimental group, and that pre-vitellogenic follicles grew normally and developed into vitellogenic and mature ones. However, nearly all exposed mature follicles exhibited morphological evidence of atresia, including membrane disintegration, follicle cell proliferation, zona radiata breakdown, and yolk resorption .star, fshr and lhcgr, which are related to steroidogenesis, oocyte growth, and oocyte maturation.To establish whether the gene expression alterations caused by exposure to 5 μg/L BPA could be caused by epigenetic changes, H3K4me3 and H3K27me3 enrichment was assessed in the TSS region, in its two flanking regions, and in other gene body regions by evaluating star_4 region  induced DNA hypomethylation in the epigenome of the offspring, demonstrating a role for it in epigenetic changesThis study examined whether BPA concentrations similar to those found in the environment affect female reproduction by altering endocrine signaling and/or local factors at the level of the ovary, and whether the well-established harmful effects of BPA are exerted via deregulation of epigenetic mechanisms. These working hypotheses are based on a studyThe relationship between histone modifications and BPA has been explored only by Doherty and co-workers21star and cyp11a1 were selected as biomarkers of steroidogenesis. The former is involved in cholesterol transfer through the mitochondrial membranecyp11a1 has proved to be a valuable biomarker of the effects of EDCs on fishTo shed some light on the issue, this study adopted a multidisciplinary approach including gene expression analysis and monitoring of histone modifications. The expression of some key genes involved in different phases of oogenesis was analyzed to elucidate the effect of BPA on the process. In particular, esr1, esr2a, and esr2b) were investigated for their important role in follicle development, given the ability of EDCs to interact with genes presenting the estrogen response element (ERE) regionfshr was included because it is part of the hormonal cascade, which comprises the synthesis of 17β-estradiol in the gonads and of vitellogenin in the liver, that ultimately leads to oocyte growthThe three ER isoforms found in zebrafish ovary , and pgrmc1 and pgrmc2, which induce the first meiotic division, or germinal vesicle breakdown.The signals responsible for oocyte maturation were also examined for BPA-induced effects. The most important are bmp15 and gdf9 also play a large role, since they inhibit the early maturation of oocytes2526Even though oogenesis is mainly controlled by pituitary gonadotropins and their receptors, local factors such as Finally, the demonstration by our groupcaspase3 and tp53, two markers of apoptosis, in the group exposed to 5 μg/L BPA, and a lack of effect on the autophagy process, suggesting inhibition of reproduction. The other findings suggest that the lowest BPA concentration was the most harmful, since it significantly down-regulated esr1, esr2a, lhcgr, and pgrmc1, but not esr2b, star, cyp11a1, and fshr expression.The results showed higher mRNA levels of Therefore, since the signals related to steroidogenesis and oocyte growth were not affected, it may be speculated that BPA damages oocyte maturation even at a concentration as low as 5 μg/L. These data agree both with the histology findings, which documented a greater incidence of atresia in mature oocytes from fish treated with 5 μg/L BPA compared with controls, and with the block of reproduction seen only in fish exposed to this concentration.star and fshr genes decreased in the exposed groups. Their unaltered gene expression pattern may be explained by a balancing out of reduced levels of both promoter and repressor, resulting in unchanged gene transcription.The results of ChIP-qPCR in zebrafish exposed to 5 μg/L BPA are also in line with the findings of molecular analyses, since enrichment of H3K4me3 and H3K27me3 histone modifications in the TSS region of the lhcgr, which was down-regulated by the lowest BPA concentration, showed a different modulation of the epigenetic pattern, with a decrease and an increase, respectively, in H3K4me3 and H3K27me3 marks in its TSS region. Histone 3 lysine 4 trimethylation has been reported to promote gene transcription and specifically marks the TSS region, whereas the increment of histone 3 lysine 27 trimethylation in the promoter region often leads to repression of gene expression29lhcrg downregulation in fish treated with the low BPA concentration is due to an increase of H3K27me3 in the TSS region, demonstrating the adverse effect of BPA on the epigenome.In contrast, dnmt1 to 8). It has been suggested that each has a specific function - dnmt1 would maintain methylation while dnmt3-8, especially dnmt3 and dnmt8, would be involved in de novo methylation and share similarities with mammalian DNMT3DNMTs have recently been suggested to mediate the interplay between histone modifications and DNA methylation15Moreover, a relationship has been suggested between human disease and the deregulation of epigenetic mechanisms3334dnmt1 and dnmt3 in fish exposed to 5 μg/L BPA could be related to the histone modifications, further supporting the notion of BPA-induced epigenetic deregulation.The above considerations suggest that upregulation of D. rerio oocyte growth and maturation, and down-regulating the signals involved in the last phase of oogenesis.In conclusion, the lowest BPA concentration consistent with environmental exposure proved to be the most harmful, affecting These findings agree with previous data highlighting that EDCs do not follow the principle “the dose makes the poison”, but rather exhibit a U-shaped and inverted U-shaped, non-monotonic, dose response curve, where the strongest responses may be elicited by the lowest and highest doses, or by intermediate concentrations, respectively3738Even though the present data are preliminary, and further work is needed, particularly to gain additional information on the methylation pattern of the genes involved in reproduction, these findings strongly suggest that the adverse effects of BPA on reproduction are due to its upstream ability to deregulate epigenetic mechanisms. Given the high synteny between the zebrafish and human genome, this evidence may be a starting point to explore the role of EDCs in the decline of human fertility17D. rerio, AB wild-type strain) were placed in eight 10-L aquaria (6 fish/tank) with oxygenated water under controlled conditions (28.0 ± 0.5 °C) and maintained on a 14/10 h light/dark cycle. They were fed 4 times a day, twice commercial food  and twice Artemia salina. There were a control and 3 exposed groups, which received 5, 10, or 20 μg/L BPA  for 3 weeks. All tanks were maintained in duplicate.A total of 48 adult female zebrafish  buffered to pH 7.4. Five half ovaries from each experimental group, in duplicate, were removed and fixed in Bouin’s solution; the remaining ovaries were used for Real Time semi-quantitative Polymerase Chain Reaction (qPCR)  and Chromatin ImmunoPrecipitation (ChIP) analysis (7 whole ovaries). All procedures complied with Italian animal experimentation laws and were approved by the Ethics Committee of Padua University (Prot. 112/2015-PR).th day of treatment, the 3 groups of BPA-exposed females and control females were crossed with untreated males, and fertility was determined during the following 15 days. Fertilized eggs were counted and the fertility rate was calculated as the mean ± standard deviation (SD) of fertilized egg number/female/day from the 8th to the 21th day of treatment.Starting on the 8Total RNA was isolated from the ovary with RNAzol solution (Sigma Aldrich) according to the manufacturer’s instructions. Its final concentration was determined using Nanophotometer TM P-Class , whereas integrity was established by Gel Red staining of 28S and 18S ribosomal RNA fragments on 1% agarose gel.Total RNA was treated with DNAse to remove genomic DNA . Then 1 μg total RNA was used for cDNAs synthesis with iScript cDNA Synthesis Kit , and kept at −20 °C until use.star, dnmt6, esr2a, gdf9, ambra1a, beclin1, tp53, caspase3, and cyp11a1, 59 °C for dnmt1, dnmt8, dnmt3, dnmt5, fshr, pgrmc1, pgrmc2, bmp15, and 58 °C for dnmt4, dnmt7, esr1, esr2b, and lhcgr, and 20 sec elongation at 72 °C. Fluorescence was monitored at the end of each cycle. Dissociation curves for primer specificity and absence of primer-dimer formation check were performed and consistently showed a single peak.Relative quantification of gene expression was performed with the SYBR Green method in an iQ5 Multicolor Real-Time PCR Detection system (Bio Rad). All samples were analyzed in duplicate. Reactions contained 1 μl cDNA diluted 1/10, 5 μl 2X SYBR Green PCR Master Mix (Bio Rad) containing SYBR Green as a fluorescent intercalating agent, 0.1 μM of forward and reverse primers ED , and 3.818S rRNAarp 43Genes Five ovaries per fish group were fixed in Bouin’s solution and prepared for histological examination using standard biological procedures. Gonads were embedded in paraffin and sectioned (7 μm) with a microtome. Each ovary was fully sectioned, processed for hematoxylin-eosin staining, and observed at 200× final magnification under a light microscope . Atretic follicles were identified based on specific morphological markers of follicular atresia  as described by Üçüncü and Çakıcı (2009)Images  were captured using a high-resolution digital camera  and atretic follicle number was counted and expressed as a percentage of the number of atretic/total follicles.Student’s t-test and ANOVA followed by Duncan’s test for multi-group comparisons were performed, as appropriate, to assess the significance of differences. Data were expressed as percentages and reported as mean values ± SD.Chromatin was prepared from frozen ovaries following the instructions of the ChIP-IT High Sensitive Kit . Chromatin was fragmented by sonication with Active Motif’s EpiShearSonicator  to produce fragments ranging from 200 to 600 bp. Approximately 200 μl of chromatin was used for each immunoprecipitation reaction. Then, 50 μl was removed from each sample and used as input control. ChIP was performed using antibodies specific for H3K4me3 (Abcam) and H27K4me3 (diagenode).in silico PCRs on the UCSC Genome Bioinformatics website. Primer sequences are reported in ED ChIP-qPCR was performed with real time PCR using the SYBR Green method. The reaction consisted in 5 μl SYBR Green Reaction Mix, 1 μl 0.1 μM primer pairs, 3 μl sterile water, and 1 μl DNA sample (ChIP or Input), for a total volume of 10 μl. Gene mapping and information on regions of interest were obtained using the UCSC Genome Browser. Primers were designed to target the TSS, the two flanking regions, and the gene body region using Pick Primers from the NCBI website, and were tested by qPCR analyses were performed in a 7900 HT Fast Real-Time PCR System (Applied Biosystems) using technical triplicates. The thermal protocol was as follows: initial 10-min denaturation at 95 °C, followed by 40 cycles of 95 °C for 15 sec; 60 °C for 30 sec; and 72 °C for 30 sec. ChIP-qPCR signals were calculated as percentage of input and SD was measured and represented as error bars. Data were obtained from 7 biological replicates.p < 0.05. All procedures were performed with GraphPad Prism 6. Numerator and denominator degrees of freedom are respectively 3 and 17. F and P values were also calculated for each gene analyzed and are reported in the legends.Statistical analysis of gene expression was performed with one-way ANOVA followed by Tukey’s multiple comparison test. Significance was set at How to cite this article: Santangeli, S. et al. Bpa-Induced Deregulation Of Epigenetic Patterns: Effects On Female Zebrafish Reproduction. Sci. Rep.6, 21982; doi: 10.1038/srep21982 (2016).


We show that silencing is achieved by co-transcriptional silencing, associated with repressive histone marks at the endogenous gene. This is accompanied by secondary siRNA accumulation, strictly limited to the open reading frame of the remote locus. Our data shows that in this mechanism, heterochromatic marks depend on a variety of RNAi components. These include RDR3 and PTIWI14 as well as a second set of components, which are also involved in post-transcriptional silencing: RDR2, PTIWI13, DCR1 and CID2. Our data indicates differential processing of nascent un-spliced and long, spliced transcripts thus suggesting a hitherto-unrecognized functional interaction between post-transcriptional and co-transcriptional RNAi. Both sets of RNAi components are required for efficient trans-acting RNAi at the chromatin level and our data indicates similar mechanisms contributing to genome wide regulation of gene expression by epigenetic mechanisms.Across kingdoms, RNA interference (RNAi) has been shown to control gene expression at the transcriptional- or the post-transcriptional level. Here, we describe a mechanism which involves both aspects: truncated transgenes, which fail to produce intact mRNA, induce siRNA accumulation and silencing of homologous loci  RNA interference has been described to be an important epigenetic regulator in almost all eukaryotes. This includes post-transcriptional inactivation by mRNA cleavage or inhibition of translation. Moreover, newest insights describe a highly dynamic cross-talk between transcription, siRNA accumulation, and histone modifications: small RNAs guide heterochromatin formation by recruitment of the histone modifying machinery –3.Three core components are described for RNAi in plants, animals, fungi and protists –7: RDR, S. pombe led to our current view of transcriptional silencing: siRNAs guide enzyme complexes to nascent transcripts to establish repressive chromatin marks. Thus, RNAi-induced heterochromatin formation requires active transcription  if the target gene position is close to heterochromatic loci, (ii) in knockouts of the exoribonuclease ERI1, (iii) or in HP1 (Swi6) over-expression lines at targets showing antisense transcription  and a somatic Macronucleus (Mac) in a single cell. A developmental-specific RNAi pathway has been described in inations . Apart fg siRNAs –22. In P control . Furtherechanism .trans silencing in P. tetraurelia. RNAi against endogenous genes can be triggered by introduction of truncated transgenes  infusion medium bacterized with Klebsiella pneumonia supplemented with 0.8 μg/ml β-sitosterol, unless otherwise stated. RNAi by feeding was induced as described , inducing the chromosomal Plac driven T7 polymerase gene followed by growth in LB medium for 2 h. Induced bacteria were washed and resuspended in WGP medium and applied to Paramecium cultures. All transgenic cultures were fed with induced bacteria for three days before RNA isolation.escribed ,28, by chttp://www.R-project.org/) and Bioconductor . The method for significance testing was Fisher's exact test with P-values <0.05. The analysis is based on previously published microarray data (Microarray data processing was done using R (R Development Core Team (2012), onductor . An annoar model  using thery rate  <0.05 anst topGO  on GO teray data  depositeray data  under ac® Xtra EF Kit , linearized at the BglI restriction site in the vector sequence, phenol extracted and filtered with a 0.22 μm Ultrafree-MC filter . After dissolving at a concentration of 5 mg/ml, ∼3–8 pl DNA was injected in the macronucleus of cells younger than 6 divisions after autogamy. Before, cells were washed in Dryl′s phosphate buffer  supplemented with 0.2% bovine serum albumin and immobilized in an aqueous drop under paraffin oil. The pTI+ and pTI- constructs harbouring a constitutive bidirectional promoter from scaffold 93 position 237470–239396 are described in ref. . Denaturing gel electrophoresis and northern blotting was carried out as described before . Gel slices were smashed and small RNAs were extracted by overnight incubation in 3Vol of 0.3M NaCl. After precipitation with 3 Vol EtOH and 70ng/μl glycogen, the small RNA fraction was cloned using the NEBNext® small RNA library prep Kit , following the manufacturer's instructions (18 h 3′-adapter ligation at 16°C). Sequencing was carried out on a HiSEQ2500 Illumina platform using the default RTA analysis software. Long RNA libraries were prepared as described before in  were gel purified starting from 50 μg total RNA. Small RNAs (17-25nt) were extracted from 17.5% urea acrylamide gels stained in SYBRefore in . Ribo dehttp://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) that uses Cutadapt  and filtered for lengths between 17 and 25nt. Sequencing data of long RNAs were first checked for adapter contamination. Adapter sequences were trimmed using Trim Galore  then the read is said to be un-spliced. From un-spliced and spliced read candidates we computed splice rates.P. tetraurelia MAC genome (version 51) and coding sequence annotation (version 1.91) downloaded from ParameciumDB (http://www.ebi.ac.uk/ena) under study accession no. PRJEB13116.Small RNA reads have been aligned using Bowtie2 . AlignmemeciumDB . Only reP. tetraurelia, this second class of methods was not applicable.As the sequencing runs produced different numbers of reads, we had to normalize read counts for comparison between samples. Sequencing statistics and read count of Mac mapping reads can be found in Supplementary Figure S8. We are not aware about normalization methods specialized for small RNA-seq data sets. However, many methods have been proposed to normalize mRNA-seq data. There are two prevalent classes of mRNA-seq normalization methods, (i) total count scaling (TCS) methods and (ii) methods that analyze the median log fold change, or a similar quantity, among all genes between mRNA-seq experiments . The latK below. Further, we removed small RNAs from highly abundant structural RNAs that are known to produce small RNAs, e.g. ribosomal RNAs, which are mostly RNA degradation products. In addition, other small RNAs that may have been introduced due to feeding cultures are removed, leaving only small RNA reads mapping to the rest of the Paramecium genome, this quantity is denoted T below.Therefore we used a variant of TCS as introduced for mRNA-seq data , which nR for a region of interest that we want to compare between samples. Denote as T the total number of reads mapping to the genome, as described above, and denote as K the number of small RNA reads mapping to the knockdown gene. We compute the normalized read count M is the maximum over all values (T1 – K1),…,(nT – nK) over all n samples. The normalization and comparison functions for small RNA seq data are accessible as an analysis pipeline under https://github.com/SchulzLab/RAPID.In detail, assume read count Paramecium histone H3 in spite of one amino acid mismatch to the immunogen peptide . All ChIPs were carried out in biological triplices, thus three cultures (wild type or feeding) of the same transgenic line with one ChIP per antibody each and three technical PCR replicates each.Chromatin isolation, sonication, fixation and immunoprecipitation were carried out as described previously using shearing fragments of 300–500 bp . The ant peptide . ImmunogRDR3 knockdown cultures revealed that ∼20% of annotated genes are differentially expressed genes (DEGs) including the surface antigen multigene family (RDR3 and heterochromatin formation (detailed gene lists of diff. regulated GO terms can be found in Supplementary Data File 2).We previously identified RDR3, a highly divergent RDR, involved in transgene-induced silencing of endogenous genes as well as in endogenous regulation of the transcriptionally controlled multigene family of surface antigens . Microare family . Interesin trans.We therefore asked for the silencing trigger, small RNAs, and further RNAi components to be associated with RDR3-mediated silencing. For this approach, the transgene-induced silencing mechanism served as a model system to mimic endogenous silencing ND169 (endo ND169) gene, serving as a reporter gene here. Its gene product is necessary for membrane fusion during trichocyst discharge. The plasmid map of pTI+, which uses a bidirectional promoter for expression of GFP and the native version of the ND169 gene is shown in Figure ND169 transgene induces silencing of the endo ND169 locus. This effect was described for the 3′-truncated version pTI- which lacks a part of the 3′-CDS and 3′-UTR . Thus, mRNA transcription appears to impede siRNA synthesis, which is in agreement with our previous data showing that pTI+ does not induce siRNA accumulation  revealed only a slight difference of the proportion of spliced transcripts for the five sense introns between the native endogenous gene (average of 90%), the poly-A enriched (78.6%) and in the non-polyA enriched (70%) pTI-/- data sets. Thus, un-spliced transcripts accumulate in the presence of the transgene. As the vast majority of the detected derives from the transgene and not from the endogenous gene . Thus, splicing of antisense- but not sense introns in antisense RNAs clearly demonstrates bidirectional transcription at the transgene locus.As RNAi mechanisms usually start with dsRNA triggers, we aimed to analyse antisense RNA (asRNA) for the presence of sense introns or sense intron junctions to distinguish whether this RNA results from bidirectional transcription or RDR activity. However, sequencing of long RNA yielded only few antisense reads. We did not find any long antisense RNAs showing sense exon/exon junctions which would indicate RDR activity on spliced sense RNAs. However, we found two antisense introns in antisense reads. As these reads also contain the transgene junction, they need to derive from the transgene and not from the endogenous locus. Figure ND169 locus. In Figure ND169 mapping siRNAs were predominantly antisense in almost the entire transgene region (NDgene), except for the ultimate 5′- region (ND-5′). The region between the transcription start site and the first intron showed a clear sense bias, suggesting that siRNAs at the 5′-end have distinct accumulation criteria.To analyse siRNAs of the transgene, deep sequencing of small RNA was carried out and reads were mapped to the endogenous ND169, which are not part of the transgene (ND-1 and ND-2), thus demonstrating that siRNA production and transitivity occurs at the remote locus. In the following, we refer to these siRNAs as secondary siRNAs. For the subsequent analyses we had to consider that reads mapping to the transgene represent a mix of primary (1°) and secondary endo ND169 (2°) siRNAs. The coverage of 2° siRNAs was drastically lower compared to the transgene sequence , thus we conclude that the majority of transgene-mapping siRNAs represent 1° siRNAs. The read length distribution showed all siRNAs to be predominantly 23nt .This is likely due to normalization: if more small RNAs map to the Paramecium genome after phosphatase treatment, the relative number maps to the transgene becomes smaller. Vice versa, this indicates that other small RNA species exist in Paramecium. Whereas we show here that the occurrence of 2° siRNAs is a result of a high copy transgene in the pTI-/- line . Pyrophosphatase treatment did not alter the predominant length of sequenced RNAs of 23nt, nor the ratio between 1° and 2° siRNAs . We previously demonstrated that NDgene mapping siRNAs are 5′-monophosphorylated using northern blots . Therefoe Figure , we can A series of RNAi components have been described in previous studies to be involved in transgene-induced silencing by phenotypic analysis. These components include RDR3, the Piwi ohnologs PTIWI08 and PTIWI14, and a set of RNAi components which are also involved in PTGS: RDR2, CID2, DCR1 and PTIWI13 –22. ThisND169 regions by deep sequencing. As the importance of PTIWI08 in transgene-induced silencing could not be clearly distinguished from that of its highly similar ohnolog PTIWI14 . Two samples per RNAi component were chosen from different transgene lines for RNA analysis. Figure RDR3 and PTIWI08/14), as the decrease of transgene siRNAs correlates with the number of dsRNA-feeding associated siRNAs, representing the feeding efficiency and thus the knockdown efficiency of the RNAi component . It seems tempting to speculate here that RNAi against RDR3 and PTIWI08/14 (which our data suggest to be associated in CTGS as discussed below) may involve a self enforcing loop of RNAi stabilization. The reduction of transgene-induced silencing would then not solely represent the function of RDR3 and PTIWI08/14 silencing efficiency.Our data shows that no RNAi component exclusively affected 2° siRNAs (ND-1 and ND-2). In contrast, knockdown of any RNAi component reduced siRNAs mapping to the NDgene region. Their reduction cannot be due to a pure loss of 2° siRNAs, because their maximum coverage in the ND-1 region was only ∼5% of that of the NDgene region: therefore, 2° siRNAs appear to have lower abundance and their loss cannot explain the reduction of siRNAs in the NDgene region. The finding that all components are indeed involved in 1° siRNA accumulation is confirmed by the replicate series shown in Supplementary Figure S6B. As mentioned above, these lines show a weaker reduction of transgene siRNAs compared to the first series Figure . The weaDCR1 silencing compared to ND-2, otherwise the ND-2 region shows the greatest fold change in RDR2 knockdown lines. Therefore, the data indicates different behaviour of 2° siRNAs in the 5′- and the 3′ -region, thus suggesting different accumulation pathways.Consequently, this indicates that all RNAi components are involved in 1° siRNA accumulation and we can exclude that a subset is exclusively necessary for 2° siRNAs. In our data, the interpretation of 2° siRNA fold changes appears difficult, as additional to the knockdown of RNAi components also the abundance of 1° siRNAs greatly affects 2° siRNA synthesis, as mentioned above. Although tempting, we cannot attribute any RNAi component to be additionally involved in 2° siRNA accumulation by their stronger effect on 2° siRNAs. However, the data shows that both regions specific for 2° siRNAs do not react in the same manner/uniformly to the knockdown. In both replicate series, ND-1 shows for instance less sensitivity to RDR2 and CID2 knockdown lines altered the strict antisense ratio of 1° siRNAs . The new ND-3 region is specific for 2° siRNAs and the transgene K28 region can produce 1° siRNAs exclusively  are involved in 1° siRNA accumulation and to get a deeper insight into 2° siRNA synthesis, we constructed another transgene, pTI-/-K28. For this, we eliminated a third region of y Figure .ND169 sequence is reduced in the pTI-/-K28 transgene.The tric- phenotype of the pTI-/- K28 transgene was usually weaker  compared to both other pTI constructs, meaning that only ∼50% of the cells showed decrease of discharged trichocysts Figure . This isImportantly, the coverage plot of siRNAs mapping to the K28 transgene sequence Figure  supportsPTIWI13/8-14 and RDR2/3  on the ND-2 region but not on the ND-1 and ND-3 region let us again hypothesize that RDR2 together with PTIWI13 have a specific role in 3′-transitivity. Although this effect was not obvious in the pTI-/- lines, silencing of both components showed the strongest reduction in the ND-2 region in all samples, together with CID2  and RNAi against DCR1, RDR2 and RDR3. Figure ND169 locus compared to the pTI- ChIPs in Figure. The only apparent difference between the ChIPs of both transgenes is that pTI-/- ChIPs show a much higher pull-down with the H3K27me3 in the promoter region. These data clearly shows that both, histone occupancy and H3K27me3, depend on RDR2, DCR1, and RDR3, thus indicating that these components are involved in heterochromatin formation. We conclude that RDR2 and associated components do not just contribute additional PTGS, but are also essential for heterochromatin formation in trans.To test whether RDR2 mediated siRNAs just represent an additional post-transcriptional silencing of transgene- and endogenous transcripts independent of heterochromatin formation, ChIP-experiments were carried out with un-injected and pTI-/- injected lines undergoing control feeding  have been used in different studies, demonstrating that non-expressible transgenes injected at high copy numbers cause homology dependent silencing  is not located to known heterochromatic loci. However, trans silencing requires injection of the triggering transgene construct at high copy numbers .Our results show that all analysed RNAi components are involved in 1° siRNA accumulation. Therefore, 2° siRNAs may have either entirely different genetic requirements, or the same as primaries. Although we cannot dissect individual components of 2° siRNA accumulation, the similarities of 1° and 2° siRNAs let us hypothesize that the very same mechanism acts on the transgene and at the endogenous S.pombe which possesses only one Dicer, one RDR and one Ago, the genome of Paramecium reveals eight Dicers  with different nucleotidyltransferase subunits and in vitro experiments suggest that uridinylation at the 3′-end of primary transcripts support conversion into dsRNA by RDR  . In theprotein) . Also inaffected . This muaffected , was alsaffected  whose coWith this study, we contribute more knowledge on detailed mechanisms how non-coding genes can produce siRNAs without requiring the formation of hairpin precursors. As indicated by a recent transcriptome analyses, siRNA-mediated heterochromatin formation might not only contribute to the regulation of a large number genes, but also to an epigenetically determined robustness counteracting rapid transcriptome dynamics .It remains to be noted that the siRNAs described here, as well as their endogenous counterparts, may be responsible for transgenerational inheritance of gene expression: transgene-induced silencing as well as PTGS by dsRNA feeding was described to cause deletions of homologous DNA sequences during the development of a new Mac in the sexual progeny . HoweverAlthough we cannot answer this question, we know that at least DCR1 and RDR3 are also involved in accumulation of 23nt siRNAs of surface antigen genes . As this multigene family can pass on its expression pattern to sexual progeny  without