Chromatin consists of ordered nucleosomal arrays that are controlled by highly conserved adenosine triphosphate (ATP)-dependent chromatin remodeling complexes. One such remodeler, chromodomain helicase DNA binding protein 1 (Chd1), is believed to play an integral role in nucleosomal organization, as the loss of Chd1 is known to disrupt chromatin. However, the specificity and basis for the functional and physical localization of Chd1 on chromatin remains largely unknown.SET2) did not affect either Chd1 occupancy or nucleosome organization genome-wide.Using genome-wide approaches, we found that the loss of Chd1 significantly disrupted nucleosome arrays within the gene bodies of highly transcribed genes. We also found that Chd1 is physically recruited to gene bodies, and that its occupancy specifically corresponds to that of the early elongating form of RNA polymerase, RNAPII Ser 5-P. Conversely, RNAPII Ser 5-P occupancy was affected by the loss of Chd1, suggesting that Chd1 is associated with early transcription elongation. Surprisingly, the occupancy of RNAPII Ser 5-P was affected by the loss of Chd1 specifically at intron-containing genes. Nucleosome turnover was also affected at these sites in the absence of Chd1. We also found that deletion of the histone methyltransferase for H3K36  contains supplementary material, which is available to authorized users. In vitro DNA-histone reconstitution assays and in vivo micrococcal nuclease (MNase) digestion experiments have shown that preferred DNA sequences and structural features on nucleosomes determine nucleosomal organization[Nucleosomes represent the basic unit of chromatin and their post-translational modifications and positions play a critical role in both the structure and transcriptional regulation of chromatin. High renization, 5, 6. Inization. High-renization.Overall nucleosome positions across the genome are typically not strongly disrupted by the loss of a single chromatin remodeler yet tend to be significantly disrupted by double or triple deletions–11, suggSchizosaccharomyces pombe, nucleosome arrays at highly transcribed genes become disorganized in a strain deleted for the ortholog of Chd1 (i.e. hrp3Δ)[S. pombe, however, showed that genes with high and low transcription rates were equally disrupted[In . hrp3Δ). A more isrupted. Though chd1Δ showed that the extent of disruption was particularly strong at the +2 and more downstream nucleosomes, implying that Chd1 works in nonpromoter regions[Studies on individual genes have shown that Chd1 localizes on highly transcribed genes and interacts with transcription elongation factors, 15. Con regions, 12, 13. regions. Resolviin vitro, Chd1 binds to nucleosomes methylated and unmethylated at H3K36 with a similar affinity[CHD1 has been shown to cause a shift in the distribution of H3K36me3 upstream in gene bodies[Chd1 has a double chromodomain motif, which in other proteins typically mediates interactions with methylated peptides such as tri-methylated histone H3K4 (i.e. H3K4me3). Howeveraffinity. H3K4me3affinity, which iaffinity. Deletioe bodies, 24, sugchd1Δ strains. Our novel approach revealed that the deletion of CHD1 specifically disrupts nucleosomal organization at highly transcribed genes. Chd1 occupancy at highly transcribed genes is strikingly similar to phosphorylated RNAPII at Ser 5. Interestingly, RNAPII Ser 5-P occupancy was dramatically altered but specifically at intron-containing genes in the absence of Chd1. Finally, we tested the possibility that Chd1 recruitment is mediated by H3K36me3 by examining Chd1 occupancy in a strain lacking the histone methyltransferase SET2, and found that its recruitment to transcribed regions occurs independently of H3K36me3.In this study, we first quantitatively compared nucleosome occupancy profiles between wild-type (WT) and chd1Δ strains of budding yeast[chd1Δ strain with a different resistance marker  and ng yeast. We founng yeast, 12, 13 chd1Δ strains, focusing on the region between the transcription start site (TSS) and the polyadenylation site (PAS)[MET14 has four distinct nucleosomes within the gene body, which could be detected in both the WT and in chd1Δ; in accordance with the small nucleosome shifts observed between the two strains, shapeDiff measured a high correlation for the nucleosomes . This anchd1Δ reported by shapeDiff was significantly lower at highly transcribed genes, indicating that the loss of Chd1 leads to greater nucleosome disorganization at loci with high transcription rates , are strongly repressed by heat shock, and show significant nucleosome depletion[chd1Δ , but RNAPII Ser 5-P peak shapes were clearly affected by the deletion of CHD1 (median correlation =0.38) , we examined the effect of  Figure . When thchd1Δ strains[et al. measured the histone exchange rate by using yeast strains that expressed constitutive Myc-tagged H3 and galactose-inducible Flag-tagged H3. The histone exchange rate was defined as the occupancy levels of Flag-H3 relative to Myc-H3. As reported by Smolle et al., we confirmed that nucleosome exchange is slower at 5′ end but faster at the 3′ end of genes in chd1Δ   was used as a wild type strain and background genotype for chd1Δ and set2Δ. The chd1Δ strain in Additional filechd1Δ strain in Figure CHD1 by replacing the protein coding region of CHD1 with the His3MX6 cassette.The set2Δ[CHD1 stop codon, and 13XMyc at the C terminal of Chd1 was confirmed by PCR and immunoblot.All cells were cultured in YPD  media at 30°C to an A600 OD of 0.8 with shaking at 250 rpm. For heat shock, the mid-log cells were harvested, re-suspended in pre-warmed YPD media, incubated in 39°C water bath for 15 mins, treated with formaldehyde, and then stored at -80°C. To tag endogenous Chd1, a 13Myc-His3MX6 cassette was amplified from pFA6a-13Myc-His3MX6, and transformed into WT or set2Δ. The casset2Δ cells carrying endogenous 13XMyc tagged Chd1. Then, 30 μl of each extract was run on a 4 to 20% gradient SDS-polyacrylamide gel and transferred to a polyvinyl difluoride (PVDF) membrane. In order to confirm 13XMyc tagging of Chd1 and compare the levels of Chd1 expression between WT and set2Δ, we detected Chd1 using horseradish peroxidase (HRP)-conjugated c-Myc antibody . Levels of H3K36me3 in set2Δ were examined by probing with anti-Histone H3 (tri methyl K36) antibody , and GAPDH antibody  was used to visualize the loading control proteins.Whole cell extracts were prepared from 30 ml cultures of 0.8 OD WT and All 150-ml cell cultures were treated with formaldehyde to be a final concentration 1% for 15 min, then quenched with glycine to a final concentration of 125 mM for 5 min. The DNA-protein complexes were sheared by ultra-sound sonication, then incubated overnight with 100 μl of anti-Myc conjugated agarose beads , 8 μg of RNAPII Ser 5-P specific antibody , and 8 μg of RNAPII Ser 2-P specific antibody  to pull down Chd1, RNAPII Ser 5-P and, Ser 2-P, respectively. Then, for the RNAPII ChIP, 100 μl of pre-washed protein A beads were added and incubated for 4 hours. After serial wash steps, immunoprecipitated DNA was recovered with overnight incubation at 65°C water bath followed by ethanol precipitation. Subsequently, sequencing libraries were prepared using NEBNext ChIP-Seq Library Prep Master Mix Set  and Bioo multiplex adapter for Illumina , then sequenced on Illumina HiSeq 2000.We followed the mononucleosome isolation protocol described in. Brieflychd1Δ strains, we developed a novel analysis pipeline. MNase-seq has been widely used for mapping nucleosome occupancy, but quantifying differences in organization is challenging for several reasons. First, the variable strength of nucleosomal signals in MNase-seq complicate any quantitative comparisons; standard peak calling methods that are more suited for ChIP-seq data rely upon the ability to measure high, individual, and dispersed peaks. Second, the total number of nucleosomes detected is correlated with sequencing depth[Sequencing reads were mapped onto the sacCer3 reference genome using the Burrows-Wheeler Aligner  with default options. Wig filng depth, thus ding depth. HoweverThe Pearson correlation has been used to quantitatively compare MNase-seq profiles for levels of nucleosome occupancies at a given locus, 45. By For shapeDiff analysis, genomic regions between transcription start sites (TSS) and polyadenylation sites (PAS) were divided into bins of 10 bp, and reads were counted. Then, the counts were smoothed using the built-in spline function in R with default parameters (R version 3.0.2). For a given gene, the Pearson correlation coefficient was calculated for the smoothed counts between two samples. This process was iterated for every gene for which TSS and PAS coordinates were available.The ChIP-seq data from this study have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE56061. The MNase-seq data are also available from GEO as accession number GSE56095.A PDF document containing six supplementary figures.(PDF 519 KB)Additional file 1:


Mammalian transcriptomes contain thousands of long noncoding RNAs (lncRNAs). Some lncRNAs originate from intragenic enhancers which, when active, behave as alternative promoters producing transcripts that are processed using the canonical signals of their host gene. We have followed up this observation by analyzing intergenic lncRNAs to determine the extent to which they might also originate from intergenic enhancers.We integrated high-resolution maps of transcriptional initiation and transcription to annotate a conservative set of intergenic lncRNAs expressed in mouse erythroblasts. We subclassified intergenic lncRNAs according to chromatin status at transcriptional initiation regions, defined by relative levels of histone H3K4 mono- and trimethylation. These transcripts are almost evenly divided between those arising from enhancer-associated (elncRNA) or promoter-associated (plncRNA) elements. These two classes of 5′ capped and polyadenylated RNA transcripts are indistinguishable with regard to their length, number of exons or transcriptional orientation relative to their closest neighboring gene. Nevertheless, elncRNAs are more tissue-restricted, less highly expressed and less well conserved during evolution. Of considerable interest, we found that expression of elncRNAs, but not plncRNAs, is associated with enhanced expression of neighboring protein-coding genes during erythropoiesis.We have determined globally the sites of initiation of intergenic lncRNAs in erythroid cells, allowing us to distinguish two similarly abundant classes of transcripts. Different correlations between the levels of elncRNAs, plncRNAs and expression of neighboring genes suggest that functional lncRNAs from the two classes may play contrasting roles in regulating the transcript abundance of local or distal loci. Eukaryotic genomes are pervasively transcribed ,2 with eIn this study, for simplicity, we refer to intergenic lncRNAs as those that are transcribed by RNA-polymerase II, 5′ end capped and polyadenylated. Here we address two important, and incompletely answered, questions concerning the origins ) and classification of intergenic lncRNAs. First, what is the relative prevalence of promoter- and enhancer-associated transcripts within sets of transcripts that are annotated simply as being intergenic lncRNAs? Second, do differences in the chromatin status at intergenic lncRNA TIRs reflect their potential function?Histone modifications allow the distinction between different types of regulatory elements ,15. PromA second class of transcripts could be prevalent in current catalogues of intergenic lncRNAs, namely enhancer-associated noncoding RNAs (eRNAs) . TranscrTo address this question we generated new genome-wide maps of H3K4me3 and monomethylation of lysine 4 of histone H3 , deep poly(A) + RNA sequencing and nanoCAGE ,26 data cis-elements, trans-acting factors and epigenetic modifications associated with gene expression during the well-defined cellular stages of erythropoiesis have been studied extensively [The functional ensively -30. ThisP < 1 × 10-3; Materials and methods), number of these lncRNAs were also identified as being expressed in erythroblasts in an earlier experiment [We used nanoCAGE  to captuperiment . Differeperiment  were idehigh, 686 TIRs) or promoter-like   to identify regions enriched in H3K4me1 or H3K4me3 . A relatively high level of H3K4me1- over H3K4me3-modified chromatin is a well-established property of enhancers ,34,35 thP < 10-3). Of the top 15 enhancer-associated protein-coding transcripts, 12 correspond to enhancer-associated intragenic lncRNAs (meRNAs) identified in our previous study [As expected, most (92%) protein-coding transcripts initiate at promoter-like TIRs Figure A. The 56us study , includiNext we considered the set of 391 TIRs associated with lncRNA expression. Consistent with an earlier report , more thTo address whether differences in histone marks at their origin reflect differences in the properties of elncRNAs and plncRNAs, we next investigated their sequence features. The TIR sizes and transcript lengths of both classes of lncRNAs Figure A,B are sWe also determined the orientation of transcription of plncRNAs and elncRNAs relative to their closest protein-coding genes Figure D. Both eP < 0.002) than for plncRNAs (median density = 0.23) loci . Intergenic enhancers that are unidirectionally transcribed are rarely associated with CpG islands [P < 0.001, two-tailed Fisher’s exact test). The different origins of elncRNAs’ and plncRNAs’ TIRs suggest that these lncRNAs may have different patterns of expression and, if functional, may play different roles. During this manuscript’s preparation Schlesinger et al. [Next we investigated the DNA sequence underlying the TIRs of intergenic lncRNAs. It has previously been shown that transposable elements are enriched both at enhancer elements  and at t islands  and in ar et al.  also proP < 0.001).To determine how intergenic elncRNAs and plncRNAs are expressed throughout differentiation and development we used Cufflinks  to estimdneutral) [P < 1 × 10-14; Figure P = 0.35; Figure Evolutionary constraint should reflect conservation of function and therefore we assessed this for intergenic elncRNAs and plncRNAs by comparing the nucleotide substitution rates at their TIRs in rodents. We estimated these rates by comparing the substitutions in aligned mouse and rat TIR sequences with the corresponding rate at neighboring  sequence with matching G + C content  . The subP = 0.24; Figure P < 0.05; Figure per se that has been the subject of constraint during rodent evolution, we compared the substitution rates of plncRNA introns and exons. We found that plncRNA exons  of elncRNA or plncRNA loci. Consistent with the lack of detectable evolutionary constraint at their respective TIRs, we found that elncRNA loci accumulate substitutions at the same rate as neighboring neutrally evolving sequence  in the vicinity of protein-coding genes whose expression is higher (five-fold) in intermediate erythroblasts than in any other tissue. By contrast, no such enrichment was found for plncRNA loci . Equivalent results were obtained when the threshold of intermediate erythroblast relative to other tissues expression was set to 2- or 10-fold (data not shown).The levels of expression of intergenic lncRNAs in general correlate with the levels of transcription from adjacent protein-coding genes ,21,22,45To estimate the extent of the enhancement in protein-coding gene expression observed in association with neighboring lncRNA transcription we used publicly available transcriptome data from three stages of erythroid development to estimate the transcript levels of lncRNAs and protein-coding genes in progenitor (BFU-e), early erythroid (CFU-e) and intermediate erythroblast (Terr119+) cells  MateriaB, the exP = 0.97; Figure P < 0.05; Figure We considered whether the abundance of elncRNAs and plncRNAs correlated with the levels of transcripts from adjacent protein-coding genes . Pairs of lncRNA loci and their closest neighboring protein-coding genes that both showed detectable expression in at least one erythropoietic stage were identified  (MateriaP = 0.27). To validate the results obtained from this genome-wide analysis we used quantitative PCR (qPCR) to investigate the association in expression between 10 protein-encoding RNAs and their genomically proximal 5 plncRNA or 5 elncRNA loci  or enhancers  based on the chromatin signatures of their TIRs. Unexpectedly, we found that approximately half of these intergenic lncRNAs are transcribed from enhancers rather than promoters. Importantly, it is the ratio between H3K4me1 and H3K4me3 chromatin marks that distinguishes enhancer- from promoter-like sequences: most TIRs have both chromatin marks  of the two classes of lncRNAs may have distinct roles. Importantly, the neutral, or near-neutral, evolution of elncRNAs and the evidence of constraint found for plncRNAs imply that the two lncRNA classes are distinct: a locus will rarely be an elncRNA in one tissue and a plncRNA in another. Enhancer-like lncRNA TIRs and sequences are poorly conserved during mammalian evolution, which is entirely consistent with previous reports of rapid turnover of DNA sequence at enhancers during evolution ,48. Thisnscripts . Due to By contrast, plncRNA promoters and the associated exons in their processed RNAs appear to have evolved under selective constraint, accumulating significantly fewer substitutions than neutral sequence, a signature of potential function. Constraint on plncRNA transcribed sequence occurs predominantly in the exons, indicating that it is the mature, spliced form, rather than the precursor molecule (or more simply the act of transcription) that mediates any plncRNA function. It should be borne in mind that plncRNA constraint is modest, only approximately 20% or approximately 5% of deleterious substitutions are predicted to have been purged from their TIRs or exons, respectively. The latter constraint suggests that either a small fraction of plncRNAs or a relatively small portion of their sequence has conserved  roles ,6-8.In summary, we have shown that the regulatory elements at the origin of intergenic lncRNAs, defined based on chromatin status, allow the distinction between two classes of transcripts that may have different biological functions. This categorization of previously indistinguishable lncRNAs will allow more specific genome-wide investigation of their properties, accelerating our understanding of their potential biological roles and the molecular mechanisms by which they operate.+) cells isolated from E12.5 mouse fetal livers. To enriched samples, cells were expanded in vitro for 4 days in StemPro media  supplemented with erythropoietin (1 U/ml), stem cell factor (50 ng/ml) and dexamethasone (1 μM) at 37°C, 5% CO2, followed by magnetic-activated cell sorting depletion of Ter119+ cells and FACS sorting of Ter119neg/CD44hi progenitor cells (CFUEs) - adapted from Chen et al.[+ terminally differentiated erythroid cells from the same culture, Ter119neg/CD44hi progenitor cells were FACS sorted as above and cultured StemPro media (Invitrogen) supplemented with erythropoietin (5 U/ml) without stem cell factor and dexamethasone for for 44 h .Biological material for the preparation of high-throughput sequencing libraries was obtained from primary erythroid cells isolated from spleens of phenylhydrazine-treated mice (C57BL/6)  using anen et al.. To obtaTotal RNA was extracted using Tri-reagent, DnaseI treated with Turbo DnaseI . Good quality RNA  was poly(A) selected using the PolyATract mRNA isolation system  according to the manufacturer’s instructions. Poly(A) + RNA was further depleted of globin transcripts using GlobinClear (Ambion). Libraries for RNA-Seq were prepared and sequenced (51 bp paired-end reads) using the standard Illumina protocol. Strand-specific NanoCAGE libraries from poly(A)-selected RNA were prepared and sequenced (100 bp paired-end reads) by Source Bioscience. .Chromatin immunoprecipitation on primary erythoid cells was performed as previously described  using thChIP-seq and DNAse I hypersensitive site (DHS)-seq data were aligned to the mm9 mouse genome build using bowtie (version 0.12.3) ,54 with de novo using Cufflinks (version 1.3.0) [Globin-depleted poly A-selected RNA sequencing reads were mapped to the mouse genome (mm9) with TopHat . Splice n 1.3.0)  with parn 1.3.0)  gene annPrior to analysis of the nanoCAGE data the first 21 bp of the read 1 fastq file was trimmed to remove the nanoCAGE specific primer. The resultant paired-end reads were aligned using TopHat (1.1.4b). Aligned reads were split into forward and reverse strands using Samtools . For visWe used globin-depleted poly(A) + selected nanoCAGE sequencing reads to annotate genome-wide transcriptional start sites and promoters as described elsewhere . BrieflyFor each transcript assembled using cufflinks, we identified reads supporting any of the 64,619 TIRs that overlapped (by >1 nucleotide) their associated transcribed sequence (exons). We excluded from our analysis TIRs that did not overlap (by >1 nucleotide) a DNAse I hypersensitive site region annotated as described above. We associated 14,689 transcripts to the remaining high confidence 11,131 TIRs. For single exonic transcripts only those with a putative TIR upstream of only one of the possible putative transcriptional starts were considered. For these transcripts the strand was imputed based on the strand information for their respective TIRs. This resulted in the annotation of 11,689 transcripts. We considered transcripts overlapping (>1 nucleotide) a protein-coding gene annotation (ENSEMBL build 68) as intragenic . The protein-coding potential of intergenic transcripts longer than 200 nucleotides was analyzed using CPC . IntergeNucleotide constraint between mouse and rat locus, exon, intron or TIR was estimated as described previously . PairwisMouse protein-coding transcript annotations were downloaded from ENSEMBL (build 68). Cufflinks (default parameters) was used to estimate the expression of protein-coding genes and lncRNAs using the poly(A)-selected RNA sequencing from intermediate erythroblast (Ter119+) and mapped reads from mouse ENCODE project that were publicly available  Additio.S) values for each tissue and each locus. TS is defined as the fractional expression of a locus in one tissue relative to the sum of its expression in all tissues. The maximum TS value (maxTS) for a locus thus provides an indicator of tissue specificity, with higher values reflecting more tissue-specific expression [We estimated the median expression and tissue specificity across all mouse tissue/cell lines   was usedWe downloaded total RNA sequencing reads  from the+) cells using TriReagent . RNA was DNase treated (Ambion), and reverse transcribed using SuperScript III (Invitrogen). Expression of each elncRNA/plncRNA and its nearest protein-coding gene was analyzed by qPCR using SYBR Green . Expression analysis was performed in three independent biological replicates, and normalized to expression of Rn18s . All reactions were performed in triplicate on each template using the StepOnePlus thermocycler (Applied Biosystems). The sequences of the primers used are available in Additional file Protein-coding genes and their closest neighboring five elncRNAs and five plncRNAs were selected from the total number of identified candidates in each class . RNA was extracted from primary mouse CFUEs and terminally differentiated  associated with intermediate erythroblast expressed transcripts.Click here for fileRelationship between TIRs and transcripts.Click here for fileThe number of tags supporting transcription initiation correlates with its expression. The logarithm of the number of reads supporting a given transcriptional initiation region  and the expression (log(FPKM), y-axis) of its associated transcripts are significantly correlated .Click here for fileContrasting epigenetic landscapes at transcriptional start sites of promoter or enhancer-associated lncRNAs in mouse intermediate erythroblasts. (A) All detected mouse erythroid DNAse I hypersensitive sites (DHSs) were sorted based on the difference in enrichment of H3K4me1 and H3K4me3. The same sort order was used for all panels displayed here, and levels of H3K4me3 and H3K4me1 are depicted as red and blue triangles, respectively. Each panel shows the distribution of signal in a 4 kb window centered on the middle of each DHS. The enhancer and promoter populations are demarcated by blue and red boxes, respectively. The active chromatin mark H3K27ac and NanoCage mapped sources of transcription are associated with both the enhancer and promoter populations, whereas the signal for the tissue-specific transcription factor Gata1 is predominately associated with the enhancer class as expected.  Chromatin profiles normalized for number of peaks associated with the mouse Ter119+ promoters and enhancers, respectively.  Chromatin profiles normalized for number of peaks associated with the mouse Ter119+ plncRNAs and elncRNAs TIRs, showing their promoter and enhancer profiles, respectively. Color coding indicating each chromatin mark is shown below.Click here for fileClassification of TIRs.Click here for fileExamples of previously annotated meRNAs that were also identified in this study. High resolution maps of DNAse I (black), H3K4me1 (dark blue), H3K4me3 (light blue), Gata1 (red), nanoCAGE  and RNA-seq (orange) across mouse (A) Nprl3 (chr11:32123000 to 32175999), (B) Ccdc88c (chr12:102134000 to 102272999), (C) Inpp5d (chr1:89519000 to 89642999), (D) Dnttip1 (chr11:32045920 to 32245919) and (E) Hagh (chr17:24962000 to 25015999). Grey represents the regions previously annotated as enhancer-associated alternative first exons [Click here for fileGenomic coordinates of intermediate erythroblast expressed elncRNAs.Click here for fileGenomic coordinates of intermediate erythroblast expressed plncRNAs.Click here for fileDistance between pairs of intermediate erythroblast expressed transcripts. Distance in kilobase pairs between the transcriptional start sites of pairs of genomically neighboring protein-coding gene transcripts (grey); elncRNAs and neighboring protein coding genes (green); and, plncRNAs and neighboring protein-coding genes (blue). ***P < 0.001; NS, not significant.Click here for fileCumulative poly(A) depleted RNA sequencing reads around transcriptional start sites. Total number of poly(A)- RNA sequencing reads (y-axis) associated with the transcriptional start sites of protein-coding gene, plncRNA and elncRNA meta-genes’ transcriptional start sites  originating from the sense (blue) and antisense(red) direction. Arrow indicates direction of transcription.Click here for fileIntergenic lncRNA expression across different mouse tissues and cell types, including intermediate erythroblasts (Ter119+).Click here for fileExons and introns of elncRNAs have not been selectively constrained during rodent evolution. Mouse-rat nucleotide substitution rates for elncRNA exons, introns and neighboring putatively neutrally evolving sequence  are shown. NS, not significant.Click here for fileplncRNA but not elncRNA loci are selectively constrained over mammalian evolution. (A) Mouse-human nucleotide substitution rates for protein-coding (PC) transcriptional initiation regions (TIRs), lncRNA TIRs and neighboring putatively neutrally evolving sequence . (B) Mouse-human nucleotide substitution rates for transcribed loci of protein-coding genes (PC) and lncRNAs and for ARs. (C) Mouse-human nucleotide substitution rates for elncRNA and plncRNA TIRs. (D) Mouse-human nucleotide substitution rates for elncRNA and plncRNA transcribed loci. ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant.Click here for fileExpression of elncRNAs, plncRNAs and protein-coding genes through three stages of erythropoiesis. Percentage of loci with detectable expression in one (white), two (grey) or three (black) erythropoiesis stages; data from [ata from .Click here for fileFold-change in expression.Click here for filePrimer sequence.Click here for file


Residues 193 to 256 of ICP22 are sufficient for interaction with CDK9 and inhibition of pol II CTD Ser2 phosphorylation but do not interact with pol II. These results indicate that discrete regions of ICP22 interact with either CDK9 or pol II and that ICP22 interacts directly with CDK9 to inhibit expression of host cell genes.The Herpes Simplex Virus 1 (HSV-1)-encoded ICP22 protein plays an important role in viral infection and affects expression of host cell genes. ICP22 is known to reduce the global level of serine (Ser)2 phosphorylation of the Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 heptapeptide repeats comprising the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase (pol) II. Accordingly, ICP22 is thought to associate with and inhibit the activity of the positive-transcription elongation factor b (P-TEFb) pol II CTD Ser2 kinase. We show here that ICP22 causes loss of CTD Ser2 phosphorylation from pol II engaged in transcription of protein-coding genes following ectopic expression in HeLa cells and that recombinant ICP22 interacts with the CDK9 subunit of recombinant P-TEFb. ICP22 also interacts with pol II  Herpesviruses are significant human pathogens associated with a range of mild to severe diseases. Although Herpes Simplex Virus (HSV) infections are generally self-limiting in healthy individuals, they can cause inflammation of the brain and eye and pose a significant mortality risk to infants and immuno-compromised adults S1 gene) is required for efficient viral replication, infection and latency in cell and animal models and regulates expression of some viral late genes S1.5 is also expressed from the US1 gene S1.5 has been variously reported to initiate at an ATG within residues 90–92, 171–173 or 147–149 S1.5 have not yet been completely characterized. However, US1.5 is dispensable for both efficient acute virus replication and virus-induced chaperone-enriched (VICE) domain formation The HSV ICP22 protein  of the large subunit of RNA polymerase II (pol II) comprises multiple repeats of the consensus sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 and all three serines (Ser) undergo dynamic phosphorylation and dephosphorylation during transcription Here we show that ICP22 expression causes loss of phosphorylation of Ser2 and Tyr1 of the pol II CTD heptapeptide repeat without markedly affecting phosphorylation of Ser5 and Ser7. These results are in line with the effect of ICP22 expression in other systems. Chromatin immunoprecipitation (ChIP) analysis indicates that pol II association with host cell genes and CTD Ser2 phosphorylation on transcriptionally-engaged pol II are drastically affected by ectopic expression of either full-length ICP22 or a truncated version comprising the conserved domain from amino acids 193 to 256. GST-mediated pull-down assays using recombinant ICP22 and recombinant P-TEFb indicate that these two proteins interact directly and that residues 193 to 256 of ICP22 are sufficient for this interaction. Moreover, a distinct region of ICP22 is required for interaction with pol II in HeLa nuclear extracts. On the basis of these results, we propose that ICP22 interacts with P-TEFb directly and with pol II, either directly or indirectly, to downregulate expression of host cell genes and facilitate the viral life cycle.Full length ICP22 and the shorter form, Us1.5 are efficiently expressed from an expression vector containing the ICP22 open reading frame followed by three tandem Myc epitope tags after transient transfection into HeLa cells . An N-teThese results indicate that ICP22 specifically affects the global levels of phosphorylation of the serine at position 2 and the tyrosine at position 1 of the pol II CTD heptapeptide in HeLa cells.Amino acids 193 to 256 of ICP22 delimit a region conserved in the alpha-herpesviruses , which cICP22 expression is associated with a decrease in the steady-state level of RNA from a number of host cell genes The drastic loss of Ser2 phosphorylation from the CTD of pol II associated with the PLK2 gene suggests that the level of transfection of ICP22 is high and that Myc-ICP22 and Myc-193-256 are expressed in most cells. ICP22 is reported to localize in the nucleus in transfected and infected cells The level of Ser2 phosphorylation of the pol II CTD associated with primer pair 2 of the EIF2S3 gene is less affected than the same region of the PLK2 gene , suggestThe effects on pol II and pol II CTD Ser2 phosphorylation are similar to those caused by small molecule inhibitors of CDK9, like DRB ICP22 and 193–256 also cause a drop in the level of intronic RNA from the EIF2S3 gene, as measured by quantitative reverse transcription (qRT)PCR with primers specific for intron 2  of CDK9/Cyclin T1 involved in the interaction.Our ChIP studies of the PLK2, EIF2S3 and β-actin genes indicate that CTD Ser2 phosphorylation on transcribing pol II is drastically affected by expression of either full-length ICP22 or the truncated form comprising residues 193–256. Our data suggest that transcription elongation rather than initiation of transcription is affected, consistent with the role of P-TEFb in overcoming an early-elongation checkpoint by phosphorylating the pol II CTD, NELF and DSIF Our finding that ICP22 inhibits CTD phosphorylation at the site of transcription without affecting CDK9 recruitment contrasts with the finding that ICP22 reduces Cyclin T1 association with the HSV-1 α, β and γ promoters in HSV-1 infected cells in vivo function of ICP22. As residues 193–256 do not interact with pol II, but inhibit pol II CTD Ser2 phosphorylation and pol II levels, the interaction between ICP22 and pol II does not appear to be required for CDK9 inhibition.Finally, our pull-down studies have also revealed a hitherto-unsuspected interaction between ICP22 and pol II that is likely to be important for the On the basis of the data presented here, our current working model is that ICP22 is recruited to host cell genes where it directly associates with P-TEFb to inhibit the CDK9 kinase activity, which results in downregulation of expression of host cell genes at the level of transcription elongation . The levHeLa cells were grown in DMEM medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 2 mM L-glutamine at 37°C and 5% CO2.To generate Myc-ICP22 and Myc-193-256, three tandem Myc epitopes were cloned at the C terminus of the ICP22 coding region and residues 193–256 of ICP22 in pcDNA3. To make GST-ICP22 and GST-193-256, the coding regions were cloned into pGEX-6P-1. The identity of all plasmid constructs was verified by sequence analysis.6 cells transfected with pcDNA3, Myc-ICP22 or Myc-193-256 HeLa cells using TRIzol (Invitrogen) according to the manufacturer's instructions. Reverse-transcription (RT) was performed with 1 µg of RNA using random hexamers with the SuperScriptIII kit (Invitrogen) according to the manufacturer's instructions. cDNA was amplified by qRT-PCR using QuantiTect SYBR Green PCR (Qiagen) using a Rotor-Gene RG-3000 (Corbett Research). The sequences of primers are given in RNA was extracted from 6×10HeLa cells were transfected with pcDNA3, Myc-ICP22 and Myc-193-256 vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions before being subjected to ChIP analysis as detailed Western blotting was carried out as described previously Cells for immunofluorescence were plated into 6-well trays containing one coverslip per well and transfected with pcDNA3 or Myc-ICP22 plasmid DNA using Lipofectamine 2000 according to the manufacture's instructions. Cells were washed with PBS and fixed for 15 min at room temperature with 4% PFA/2% sucrose in 1xPBS. The samples were then blocked and incubated for 1 h with the primary antibody diluted 1∶100 . Following 3× washes with PBS +0,1% Triton, the secondary antibody  was added to the blocking solution and incubated for 30 minutes. Three additional washes were carried out before the coverslips were mounted . Samples were then examined with a Zeiss AxioObserver Z1 20× and images processed using Fiji Image J GST-ICP22, GST-193-256 and GST were purified from Escherichia coli (Rosetta cells) and immobilized on Sepharose 4B-gluthatione beads . Hela nuclear extract (NE) was produced as described For the GST-mediated pull-down of recombinant P-TEFb, the recombinant P-TEFb was produced as described File S1Contains figures S1–S4. Figure S1. Pol II levels are higher at the promoter than within the body of the PLK2 and EIF2S3 genes. Figure S2. Transient transfection of Myc- ICP22 in HeLa cells with Lipofectamine 2000 is efficient. Figure S3. ICP22 recapitulates the effect of DRB on pol II transcribing the β-actin gene. Figure S4. Ectopic expression of ICP22 or the 193–256 subdomain causes loss of intronic RNA.(PDF)Click here for additional data file.


Echinococcus spp. and Taenia solium) have very few mobile genetic elements (MGEs) in their genome, despite lacking a canonical PIWI pathway. The MGEs of these parasites are virtually unexplored, and nothing is known about their expression and silencing. In this work, we report the discovery of a novel family of small nonautonomous long terminal repeat retrotransposons  which we have named ta-TRIM (taeniid TRIM). ta-TRIMs are only the second family of TRIM elements discovered in animals, and are likely the result of convergent reductive evolution in different taxonomic groups. These elements originated at the base of the taeniid tree and have expanded during taeniid diversification, including after the divergence of closely related species such as Echinococcus multilocularis and Echinococcus granulosus. They are massively expressed in larval stages, from a small proportion of full-length copies and from isolated terminal repeats that show transcriptional read-through into downstream regions, generating novel noncoding RNAs and transcriptional fusions to coding genes. In E. multilocularis, ta-TRIMs are specifically expressed in the germinative cells (the somatic stem cells) during asexual reproduction of metacestode larvae. This would provide a developmental mechanism for insertion of ta-TRIMs into cells that will eventually generate the adult germ line. Future studies of active and inactive ta-TRIM elements could give the first clues on MGE silencing mechanisms in cestodes.Taeniid cestodes (including the human parasites  Mobile genetic elements (MGEs) have the capacity to replicate within the genome of their host. This gives them a selective advantage over other genetic elements, resulting in their ability to increase their frequency in natural populations . In thisLong terminal repeat (LTR) retrotransposons are a large group of MGEs present in almost all eukaryotes, including animals (metazoans) . These ePogonomyrmex barbatus; In addition to these functional retrotransposons, other elements have been described that lack most or all of the coding sequences, but which are still mobilized by the machinery of functional (autonomous) elements . These nBecause of the deleterious effect of MGEs, the hosts have developed effective mechanisms to suppress their expression and expansion. This is particularly important in the germ line of animals, as replication of MGEs in these cells would lead to their accumulation in the next generation. The PIWI pathway is a conserved metazoan mechanism that silences MGEs in the germ line . PIWI ispiwi homologs in their neoblasts   is highly expressed in neoblasts, and it has been proposed that this gene could perform similar functions to those of piwi in other organisms , and expression is also seen in postmitotic, differentiated cells, so it is unclear whether the functions of these argonaute genes are shared between cestodes and trematodes (E. multilocularis is very low (∼2% of the genome), as is for other related cestodes, indicating that a highly effective mechanism of MGE suppression may be at play  . In the rganisms . We haveocularis . In cestocularis . In contematodes . However at play .ta-TRIMs]) that is specific for E. multilocularis and other related taeniid cestodes. A fraction of these TRIMs have escaped silencing and are massively expressed in the E. multilocularis germinative cells, constituting their best molecular marker to date. These TRIMs have expanded during the evolution and divergence of taeniids, and may still be mobilizing in some species. In E. multilocularis, these elements may now be inactive for retrotransposition, but have left in their wake a substantial reshaping of the host’s transcriptome, as they are the source of TRIM transcripts and of fusion transcripts to coding genes and novel noncoding RNAs.In this work, we report the discovery of a novel group of TRIM elements . The draft genome of the planarian Schmidtea mediterranea was obtained from SmedGD (smedgd.neuro.utah.edu) .Genomic assemblies from crostoma  as well  mansoni  were dowtah.edu) . ExpressEchinococcus spp. ESTs and genomes using as a query the previously described Cluster A of long noncoding RNAs (lncRNAs) of E. granulosus  from full length sequences using hmmer2.3 (www.hmmer.org), and used them to search for additional ta-TRIM elements. Because of the divergence between ta-TRIM sequences in different taeniid species, the manually identified ta-TRIM sequences from each species were used for new BLASTN searches. A list of full-length elements was extracted and manually curated for each species from these BLASTN results, which was used for analysis of TSD sites, LTR divergence, and comparison of synteny between species . A separate list containing all fragments longer than 800 bp from each species was compiled for phylogenetic analyses.Initially, we performed BLASTN searches of anulosus . Once thta-TRIMs that are transcribed, we identified ESTs of E. multilocularis with similarity to ta-TRIMs  and mapped them to the E. multilocularis genome by BLASTN, eliminating all hits with less than 99% identity to the genome or smaller than 50 bp, as well as those which mapped to more than one region in the genome with greater than 99% identity. The resulting loci, plus a duplicated genomic locus which was supported from 3′ rapid amplification of cDNA ends (RACE) experiments, were collected and manually analyzed to determine whether they were full-length ta-TRIMS or solo-LTRs, whether transcription was likely initiated from within an LTR, and whether there was downstream transcriptional read-through into neighboring intergenic regions or coding genes. An identical analysis was performed for E. granulosus, except that the identity threshold was decreased to 95%, due to the draft quality of the assembly.In order to indentify E. multilocularis tRNA genes was generated from the genomic assembly with tRNAscan-SE (LeutRNA genes was identified as having complementarity for eight or more bases in this region.A list of Ascan-SE . The 3′ E. multilocularis by TBLASTN using as queries the sequences of Pol proteins of LTR retrotransposons from Drosophila melanogaster and S. mansoni (lennie elements). The LTRs, PBS, and PPT were manually identified from these sequences.We searched for autonomous LTR retrotransposons in  mansoni . Althougta-TRIMs, RNA sequencing (RNA-Seq) data sets from In order to determine the transcriptional activity of ta-TRIM elements and fragments longer than 800 bases from all species was performed with ClustalW  hits with less than 80% identity upstream or downstream of the ta-TRIM were discarded, and the retrieved sequences were aligned with ClustalW and manually inspected. The same procedure was performed when comparing T. solium and T. asiatica.When comparing ta-TRIMs for each taeniid species were analyzed using the program GENECONV  under a K2P+G model, was calculated using MEGA 5.0  were performed with 2 μl of cDNA per reaction using KOD polymerase (Millipore). For 3′-RACE of ta-TRIMs, a seminested RT-PCR approach was performed, using degenerate forward nested primers for the U5 region of several ta-TRIMs  and the reverse primer AAGCAGTGGTATCAACGCAGAGTAC. For confirming the transcriptional fusions between solo-LTRs and downstream coding genes, the following primer combinations were used: locus 8, TTCGTCTTCTTTCGTCTTCAGAGAG and GCATCCTTGATCGAAGTTTGGG (fusion to EmuJ_000118100); locus 21, CTTTTGTACTTTGAGTTAGCCCCTTGTAC and CCATGGCGAAATCGACCAC (fusion to EmuJ_000465100); locus 60, CCTTGTACCTAGCTAAGAGGGCTGAC and CGACGTAGGCACTCAAGCAAG (fusion to EmuJ_001025350); locus 2 , CCGAGTATTGTGTCTTCTTTCGTCTTC and CGGAATGACATTTGGCAAAGTC (fusion to EmuJ_000054900). All products of the expected size were gel-purified, cloned into pJET1.2 (Thermo-Scientific), and several clones were sequenced for each product.Reverse transcription was performed with 700 ng of total RNA from in vitro cultured s larvae  using PrE. multilocularis larvae was performed with a digoxigenin-labeled antisense probe, corresponding to the region interior to the LTRs of the ta-TRIM locus 39 of E. multilocularis , as described . Three independent experiments with different laboratory isolates were performed. These isolates were GH09 and Ingrid/10, obtained from accidental infections of Old World Monkeys in a breeding exclosure . In vitro 5-Ethynyl-2′-deoxyuridine labeling  and detection were performed as previously described (Fluorescent whole-mount in situ hybridization (WMISH) of in vitro cultured escribed . Primersxclosure , and MS1escribed .Echinococcus spp., the metacestode larvae develop as fluid-filled cysts in which numerous protoscoleces, the infective form for the definitive host, develop from the cyst wall by asexual budding. Previously, an lncRNA of approximately 900 bases with similarity to the E. granulosus repeat element EgRep , and which we have denominated ta-TRIMs. These characteristics include: 1) Two LTRs of approximately 198 bp, starting with 5′-TG-3′ and finishing with 5′-CA-3′ ; 2) a PBS with nine bases of complementarity to the 3′-end of an EchinococcusLeutRNA, positioned at four bases from the end of the 5′-LTR; and 3) a PPT of 15 bases, located three bases upstream of the 3′-LTR , as well as nontaeniid cestodes and other flatworms . We could clearly find related ta-TRIM sequences in all Taenia spp., which were approximately 50% identical after alignment to the Echinococcus ta-TRIMs . This comprised the conserved LTRs and also parts of the region between LTRs, including the crucial PBS and PPT. Furthermore, TSD sequences could usually be found flanking the LTRs of the full-length ta-TRIMs in these species . In contrast, no similar sequences could be detected in other flatworms, or in GenBank. Because the analyzed species of Taenia and Echinococcus cover all of the major lineages of taeniid cestodes , or into partial elements. At least 142 full-length elements can be found in E. multilocularis, but many have substitutions in presumably important positions such as the 5′-CA-3′ motif at the LTRs, or within the PBS and PPT, resulting in only 19 perfect elements  . In addition, a total of 1,939 loci in the genome show significant similarity by BLASTN and HMM searches to the LTR regions in the E. multilocularis genome (with hits located less than 1 kb apart counted as single hits). Pairwise divergence between full-length copies was between 4% and more than 60%, not taking into account linked copies (less than 30 kb distance) with less than 1% divergence. These numbers may change as newer versions of the E. multilocularis assembly are released, but are likely to be close to the real numbers given the high quality of the current version , with no detectable compositional bias in the surrounding regions. No chromosomal regions showed an overrepresentation of ta-TRIMs except for a few examples of tandem repeats. Remarkably, these closely located ta-TRIMs have highly similar sequences , suggesting that these tandem elements were generated by unequal crossing over, and not because of a bias of the integration process (data not shown). Among the partial ta-TRIM sequences, many consisted of “solo-LTRs,” that is, isolated LTR elements. Solo-LTRs are a common derivative of LTR-retrotransposons, and are thought to be originated from unequal crossing-over between LTRs of a single element . In particular, although lacking TSDs, it seems that the EgRep element previously described for E. granulosus by ta-TRIM element, embedded within a larger, sparsely repeated sequence lacking any other distinctive elements (data not shown). In E. granulosus, there are at least 1,183 loci with similarity to ta-TRIM elements. In the case of T. solium, the total number of copies is difficult to assess given the fragmentary nature of the genomic assembly, but at least 827 loci show significant similarity to ta-TRIMs, including 24 full-length elements, of which 5 were perfect (using the same definitions as for E. multilocularis). Only a superficial analysis of the number of ta-TRIMs was performed for the unpublished provisional drafts of T. asiatica and T. taeniaeformis. Results in T. asiatica were similar to T. solium, and at least three perfect ta-TRIM elements could be found. In contrast, in T. taeniaeformis, although some almost complete ta-TRIMs could be found, they were divergent and had substitutions in key positions, suggesting that they are pseudoelements.Similar results were found in the ta-TRIMs showed high similarity between E. granulosus and E. multilocularis, as well as between T. solium and T. asiatica (>90% for the most similar copies), but had much lower similarity when comparing other species pairs (approximately 50%). Phylogenetic analysis of ta-TRIMs from all the analyzed species shows that the elements of Echinococcus spp. form a well-supported clade, as do those of T. asiatica plus T. solium, and those of T. taeniaeformis (A). Furthermore, most T. asiatica elements form a monophyletic clade. This topology suggests that massive independent expansions of ta-TRIM elements occurred in each of the main taeniid lineages, as well as in T. asiatica. Alternatively, the reciprocal monophyly between the elements of each species could be the consequence of extensive gene conversion between all of the copies of each genome. We searched for evidence of ongoing gene conversion between copies of ta-TRIMs in each species using the program GENECONV . The low levels of gene conversion suggest that the first hypothesis is correct. However, we cannot discard the possibility that gene conversion was more extensive in the past, during the early expansion of ta-TRIMs.Strikingly, aeformis A. FurtheGENECONV , 1999. Nta-TRIM integration events after the splitting of E. multilocularis and E. granulosus, a relatively recent event that was estimated to have occurred between 2.5 and 9.2 Ma (E. multilocularis (at least 2 kb upstream and downstream of each ta-TRIM) with the homologous regions of E. granulosus (B). In three of seven comparisons, strong evidence of integration after the divergence of both species could be found, as the ta-TRIM is precisely lacking in the equivalent region in E. granulosus, and at that position a sequence can be found that is identical or has only one mismatch to the flanking TSD of the E. multilocularis ta-TRIM . With the same strategy, we searched for integration events after the splitting of T. solium and T. asiatica, and from five regions that could be compared between these assemblies, three examples showed strong evidence of integration after the splitting of these species . Using less restrictive criteria (searching only 300 bp surrounding each ta-TRIM and disregarding whether the TSD is present or not) we could find 12 examples suggesting insertion of ta-TRIMs in E. multilocularis after the divergence of E. granulosus, and 2 analogous examples suggesting insertions that are specific for E. granulosus.We searched for evidence of the occurrence of d 9.2 Ma . For thianulosus B. In thrta-TRIM can be used to estimate the time of integration . Furtheta-TRIMs with intact LTRs, for one species of each main taeniid lineage . In E. multilocularis, divergence values between LTRs were between 6.1% and 22.9% (n = 21), whereas in T. solium they were between 2.2% and 14.9% (n = 25), and in T. taeniaeformis between 19% and 27% (n = 3). The distribution of the divergence values of LTRs was significantly different between all species pairs . Assuming equal substitution rates for all lineages, this implies that the waves of retrotransposition occurred at different time points for each lineage, and the relatively large divergence in E. multilocularis and T. taeniaeformis LTRs further suggests that in these species ta-TRIMs may be no longer active.We thus analyzed the divergence between 5′- and 3′-LTRs for full-length E. vogeli and E. oligarthrus were proposed to have split during the great American biotic interchange, 3 Ma , it is apparent that most insertions must have occurred after the divergence of both lineages. In contrast, divergence in introns between E. multilocularis and E. granulosus  is of the same magnitude as between the most similar LTRs in E. multilocularis, suggesting that the last insertions in E. multilocularis occurred approximately at the same time as these species diverged.There is no fossil record for taeniids, but the neotropical sister species ge, 3 Ma . Unfortuge, 3 Ma , we obtage, 3 Ma  in two aetazoans . By applta-TRIM elements after the divergence of the main lineages of Taeniidae, as well as after the splitting of E. multilocularis and E. granulosus, and after the splitting of T. solium and T. asiatica. These elements may still be active (or may have been active until very recently) in T. solium, whereas in E. multilocularis and T. taeniaeformis, it seems that they do not mobilize any longer.In summary, we found evidence for retrotransposition of E. multilocularis by searching for a short distance (<20 kb) between BLASTN searches for ta-TRIMs and TBLASTN searches for RTs (data not shown). As an alternative, we characterized some of the most complete LTR-retrotransposons in the E. multilocularis genome, and compared their sequence with the E. multilocularis ta-TRIMs. We found one family of LTR-retrotransposons (which we have dubbed lennie) that has characteristics suggesting that it may have fulfilled this role , which is coincident with the length of the TSDs for lennie elements and their derived solo-LTRs . Although we could observe some expression of lennie elements from ESTs and RNA-Seq (data not shown), we were unable to detect intact copies of lennie in the E. multilocularis genome . This suggests that although lennie elements are transcriptionally active, they are probably unable to complete the retrotransposition cycle. If lennie was the element mobilizing ta-TRIMs in trans, then the lack of intact lennie elements could explain the absence of recent ta-TRIM retrotransposition events in E. multilocularis.So far, no specific retrotransposons have been proposed to be responsible for the mobilization of TRIMs in any species , probablhis role . In parthis role  are idenon cycle , any autIN group . Most TSTaenia spp., we were unable to determine whether intact lennie elements are present in these species. However, in all the analyzed Taenia spp., the TSDs of ta-TRIMs were five bases long . This suggests that the element mobilizing the ta-TRIMs may have been different in Taenia spp.Due to the draft status of the genome assemblies of ta-TRIMs were noticed in E. granulosus due to their massive expression . In T. solium, a similarly high proportion of ESTs in GenBank showed similarity to ta-TRIMs, .Originally, the transcripts of E. multilocularis  we searched for ta-TRIM-like transcripts by BLASTN and mapped them to the genome of E. multilocularis under stringent requirements (single mapping position in the genome with greater than 99% identity to the EST). In this way, we identified 73 different loci with similarity to ta-TRIMs and with strong evidence of transcriptional activity, which we then manually curated. For 63 loci, transcription was apparently originated from within an LTR , strongly indicating that it was generated from an LTR promoter and not by read-through from upstream genes.By using diverse EST libraries of ta-TRIMs, which in most cases showed several deletions and substitutions of presumably important sites, but which nonetheless had two LTRs (A). Alternative cis-splicing events were sometimes detected within the ta-TRIM for these loci, and were confirmed for one locus by 3′-RACE (A). No transcriptional read-through was detected downstream of the U5 region of the 3′-LTR for full-length ta-TRIMs. The other 38 loci corresponded to transcription initiation from solo-LTRs, and these showed two main distinct behaviors. In some cases, solo-LTRs generated internal transcripts, from the U3/R limit to the R/U5 limit, indicating that in the absence of ta-TRIM sequences downstream to the LTR, short “abortive” transcripts are generated . These are reminiscent of short transcripts originating from mutant retroviruses in which polyadenylation is no longer repressed in the 5′-LTR . In five loci, the solo-LTR promoter initiated transcripts that were fused to a downstream coding gene, generating an alternative isoform under the control of the solo-LTR . Control RT-PCRs in which the forward primer was located ≤200 bp upstream of the solo-LTRs, as well as in the U3 region within the LTR, gave no amplification, providing strong confirmatory evidence that the transcript is initiated within the solo-LTR (data not shown). For most loci, analysis of RNA-Seq data suggested that the new LTR promoter is a minor alternative one, but in the case of locus 8 (shown in B) it seems to be the main or only promoter (data not shown).We further confirmed by RT-PCR these transcriptional fusions for three of four investigated loci . No ta-TRIM or solo-LTR was found for the other two cases in E. granulosus (loci 8 and 21), and because the loci are collinear except at the precise position of the solo-LTR, this suggests that a ta-TRIM was inserted and reduced into a solo-LTR after the divergence of both species (fig. 5F and data not shown). The E. granulosus ortholog of locus 21 is a trans-spliced gene , suggesting that it competes for this site with the spliced-leader.Very interestingly, three of the loci producing fused transcripts between solo-LTRs and coding genes were found to differ between d leader  at the 5E. granulosus, which are however more difficult to map precisely given the draft status of the genomic assembly. Among these ESTs we could also find one example of a transcriptional fusion between an LTR and a downstream gene .We also analyzed ESTs from ta-TRIMs and derived solo-LTRs are massively transcribed in E. granulosus, E. multilocularis, and T. solium. In E. multilocularis, we provide strong evidence for the generation of novel lncRNA and alternative isoforms for coding genes from dispersed solo-LTRs. Some of these ta-TRIM elements are absent in the corresponding regions in E. granulosus, suggesting that these differences were generated during or after their speciation.In summary, some E. multilocularis larval and adult stages , dormant protoscoleces and activated protoscoleces (activated by pepsin and low pH treatment), and primary cell cultures that are undergoing metacestode regeneration . As a rta-TRIM copy. Remarkably, ta-TRIMs are very unequally expressed: A few appear transcriptionally active, whereas the majority is not transcribed . Indeed, 11% of all ta-TRIMs generate over 90% of uniquely mapping reads. The different ta-TRIM copies seem to be either always expressed or always silent across life stages , suggesting that silencing of ta-TRIM elements can be stably inherited . This is further supported by the fact that these data sets were obtained from different E. multilocularis isolates, suggesting that the expression levels of individual ta-TRIM copies are stably maintained in different lineages. These transcriptionally active ta-TRIMs are approximately evenly distributed across the genome . There is a highly significant correlation of the expression levels of individual ta-TRIMs as determined from EST and from RNA-Seq data , confirming that the variation observed among ta-TRIMs is not an artifact of the mapping algorithm or the selected parameters.Using highly conservative settings , we determined the expression levels for each ta-TRIM probe to analyze at the cellular level the expression of ta-TRIMs during the development of the metacestode larva of E. multilocularis. Within the metacestode cysts, the cells are organized as a thin layer , which has dispersed germinative cells (stem cells). These germinative cells are the only proliferating cell type in the larva, whereas all differentiated cells are postmitotic . Furthermore, they were in average 23% of all cells in the germinal layer , which corresponds well to the proportion of germinative cells as estimated by morphology  . All these results strongly indicate that ta-TRIMs are specifically expressed in the germinative cells, and only very low or null levels can be seen in other cells. Furthermore, ta-TRIMs appear to be expressed in almost all of the proliferating germinative cells, given the high colocalization of ta-TRIMs with EdU incorporation.Finally, we performed WMISH with a tmitotic . The ta-nucleoli ) . They massively accumulate during the early budding process that leads to the formation of brood capsules , as well as during early and intermediate protoscolex formation, except at the apical most region where no proliferation is observed (fig. 7C and D). At the last stages of development, expression is restricted to ever decreasing cell numbers in the posterior body, whereas in the scolex (the head of the protoscolex with the attachment organs) expression is only observed in the proliferative region at the base of the suckers (fig. 7E–G). In summary, during the development of the metacestode larva, the ta-TRIM WMISH signal is strongly and specifically observed in the germinative cells.Throughout the development of protoscoleces from the germinal layer, the ve cells  and contve cells B–G. Theyproceeds ; fig. 7B suckers  . This is strong evidence against horizontal transference of these elements, and suggests that independent expansions of ta-TRIMs occurred in each lineage. Further support for this comes from the fact that the estimated insertion dates are significantly different for ta-TRIMs of each species investigated (C), and that specific insertions occurred after the divergence of closely related species (B). However, we cannot rule out the possibility that the phylogenetic pattern is caused by extensive gene conversion between ta-TRIM elements in each genome. In general, ectopic gene conversion would not be expected to maintain sequence similarity for such a large number of elements widespread throughout the chromosomes   and with INT (the ends of the LTRs), and that most of the specificity is achieved by the specific packaging of the TRIM RNA into the VLP particle . Furthermore, it would be expected that competition with highly expressed ta-TRIM elements could drive the extinction of an autonomous retrotransposon group such as lennie, as the rate of their own retrotransposition decreases and the existing copies accumulate deleterious substitutions  , few paiparticle . Packagiparticle  and therocularis . The absitutions . In TaenIN group . This imta-TRIMs are transcribed at very high levels . Furthermore, the presence of solo-LTRs has a clear effect in the expression of downstream sequences in E. multilocularis, resulting in many new lncRNA, and in alternative promoters for coding genes , and it is likely that many more examples will be found with larger data sets, given the number of positions  with similarity to ta-TRIMs in the genome. Usage of retrotransposons as alternative promoters and as a source of new lncRNA has been described for many individual examples, and also at a large global scale for model organisms such as humans, mice, and Drosophila (trans-splicing acceptor site (C) provides a novel and simple evolutionary mechanism by which LTR retrotransposons can be “exapted” as alternative promoters. In this model, the pre-existing trans-splicing acceptor site (as found in E. granulosus) is spliced to an appropriate splice-donor site originating from the new LTR. This donor site may be there by mere chance, as it does not need to be a particularly strong one . This is because splicing has a “donor-first” syntax, in which a cis-splicing site efficiently out-competes the spliced-leader for a downstream splicing acceptor site  could indicate the existence of a different small subpopulation of germinative cells, or could be the result of fluctuating silencing of these elements in the germinative cells. At this point, nothing is known about the mechanism by which cestodes silence MGEs in the absence of a canonical PIWI pathway  would allow the expansion and transmission of ta-TRIMs in the genome of the next generation. At this point, we do not know whether ta-TRIMs are also expressed in the germ line in the gonads of the adult stage. However, the analysis of published RNA-Seq data .


The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases. AMPK regulates the metabolism and so drugs that activate AMPK might have potential for the treatment of metabolic disease. Here, the authors report the structure of AMPK bound to an activating compound, revealing two binding sites and indicating that dual therapy might be a good drug strategy. Once phosphorylated on α-Thr172 AMPK is further activated by AMP, and AMP sustains AMPK signalling by inhibiting dephosphorylation of α-pThr172 (ref. 167AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase responsible for regulating metabolism in response to energy supply and demand. During metabolic stress the cellular AMP/ATP ratio increases leading to activation of AMPK, which in turn switches off energy-consuming anabolic pathways and switches on catabolic pathways to restore ATP levels. The AMPK αβγ heterotrimer comprises a catalytic α-subunit associated with β and γ regulatory subunits . Adenine2172 ref. . AMP sti172 ref. . The α-r172 ref. 6 is requThere has been keen interest in developing AMPK-activating drugs for potential therapeutic use in treating metabolic diseases including type 2 diabetes, obesity and cardiovascular disease. A number of small-molecule activators have been identified8Another potent small-molecule allosteric activator of AMPK, 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid , was identified by screening an AMP mimetic libraryWe have determined the crystal structure of the C2: AMPK complex, revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results raise the possibility that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.We found that C2 activates both AMPK γ1 and γ2 complexes  to a similar degree as AMP, whereas neither C2 nor AMP-activated γ3-containing complexes (α1β1γ3 and α1β2γ3) . Lack of18Since ATP concentration was previously shown to alter AMP dependence−1 mg−1) was co-incubated with combinations of AMP/A769662 (≈671 nmol min−1 mg−1 or≈1100-fold) and C2/A769662 (≈636 nmol min−1 mg−1 or ≈1040-fold), but not with CaMKK2-treated AMPK α1 (−1 mg−1) co-incubated with AMP/A769662 or C2/A769662 was relatively insensitive to dual ligand activation  and solved to an equivalent resolution of 3.02 Å) revealed strong electron density for both AMP-bound heterotrimers in the asymmetric unitOur AMP-bound heterotrimer closely resembles the previously published AMP-bound α2β1γ1 structure (PDB 4CFE)16The two C2 molecules occupy the interface between the CBS-binding sites 1, 3 and 4, with the phosphate group of C2 occupying a similar position to that of AMP-bound phosphate group in sites 1 and 4 . For thiIn both sites the C2 molecule makes six protein-mediated hydrogen bond contacts, while C2 in γ-pSite-4 makes an additional π-stacking interaction with γ-His298 . All of 15−1 mg−1 lysate for γ-T89E and γ-H298E, respectively) in the absence of C2 and AMP compared with γ1 WT (1.4±0.1 nmol min−1 mg−1 lysate), and were insensitive to further activation by either C2 or AMP  in COS7 mammalian cells and measured their sensitivity to C2 or AMP. We initially found that all the γ1 mutants displayed increased but variable Thr172 phosphorylation and basal activities compared with the wild-type (WT) γ1-containing AMPK complexes. We therefore co-expressed the mutants with the phospho-mimetic AMPK α1(T172D), which is more amenable to studying the effect of these γ mutations on allosteric activation 521. C2 a52 or AMP . It is p2 or AMP , while o2 or AMP .et al.67A superposition of the two AMPK heterotrimers reveals two major side-chain conformational changes between AMP and C2 binding. Firstly, the imidazole ring of the aforementioned γ-His298 has flipped 180° and rotated at the Cβ>90° ∼ 6.0 Å) to form a π-stacking interaction with C2 in γ-pSite-4 while simultaneously blocking site 3, mimicking ATP-bound AMPK (PDB 4EAK)1.0 Å to fSecondary structure elements of αRIM2 are crucial for the isoform specificity of C2. Structural comparison of α1 and α2 isoform αRIM2/γ-subunit interactions reveal the α-helical α1RIM2 (PDB 4CFH)In this study we showed that two topographically disparate drug sites on AMPK, one located at the classic α-kinase domain/β-CBM interface (ADaM site) and the other located within the solvent-accessible core of the γ-subunit (γ-pSite-1/γ-pSite-4), can be exploited to achieve synergistic activation of unphosphorylated α1-AMPK independent of AMP and upstream phosphorylation events. This provides a major advance to previous demonstrations of AMPK synergistic activation that have been dependent on AMP. Importantly, identification of C2-binding sites on the γ-subunit represents an entirely unexplored dimension for future rational drug design.Antibodies for pan AMPK α (#2793), FLAG (#2368) and HA (#2367); phosphospecific antibodies for α-pThr172 (#2535) and β-pSer108 (#4181), and λ-phosphatase (#P0753) were from Cell Signaling Technology. β1 antibody (#ab58175) and A769662 (#ab120335) were from Abcam. Anti-rabbit and anti-mouse IgG secondary antibodies fluorescently labelled with IR680 or IR800 dye, respectively, were from LI-COR Biosciences. Glutathione Sepharose 4B was from GE Life Sciences. FLAG peptide (DYKDDDK) was provided by GL Biochem (Shanghai), purified by reversed-phase chromatography and stored as lyophilized powder. All other reagents were purchased from Sigma.Escherichia coli-expressed AMPK preparations were mass verified by time-of-flight (TOF) mass spectrometry. Heterotrimeric human AMPK His6-α2β1γ1 was expressed in E. coli strain Rosetta (DE3) using the pETDuetTM-1 expression system (Novagen) as previously describedTM-1 multiple cloning sites (MCS) 1 (BamH1/Not1) and 2 (MfeI/XhoI), respectively, resulting in incorporation of an N-terminal hexahistidine tag onto α2. cDNA for β1 was inserted into pRSFDuetTM-1 MCS1 (NcoI/ BamHI). Heterotrimeric human AMPK α2/α1 RIM chimaera (α2(1-347)/α1(349-401)/α2(397-end) β1γ1)E. coli strain Rosetta (DE3) using the pETDuetTM-1 and pCOLADuetTM (Novagen). cDNAs for α2/1 chimaera and γ1were sequentially inserted into pETDUETTM-1 multiple cloning sites (MCS) 1 (BamHI/Not1) and 2 (MfeI/XhoI), respectively, resulting in incorporation of an N-terminal hexahistidine tag onto α2/1 chimaera. cDNA for β1 was inserted into pCOLADuetTM-1 MCS1 (NcoI/ BamHI).All mutants were generated using QuikChange Site-Directed Mutagenesis Kits (Stratagene), and constructs were sequence verified. D-1-thiogalactopyranoside, before overnight incubation. Cells were lysed using a precooled EmulsiFlex-C5 homogenizer (Avestin) and AMPK purified using Nickel Sepharose and size exclusion chromatography in 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2 mM Tris(2-carboxyethyl)phosphine buffer. AMPK was phosphorylated by incubation with CaMKK2 . Protein was re-purified using size exclusion chromatography, concentrated to ∼10 mg ml−1 and flash frozen in liquid nitrogen before storage at −80 °C phosphine).Expression cultures were grown in Luria-Bertani broth and induced at 16 °C with 0.25 mM isopropyl β-−1) was mixed with a twofold molar excess of C2, AMP and S108tide (synthetic peptide: β1(102-117) R115-117, NH2-KLPLTRSHNNFVARRR-COOH) and a 1:1 molar ratio of Staurosporine and A769662, while full-length phosphorylated RIM swap chimaera (α2 1-347/α1 349-401/α2 397-end β1γ1)−1) was mixed with a sixfold molar excess of C2, twofold molar excess of S108tide and a 1:1 molar ratio of Staurosporine and A769662. Crystals from both protein preparations were grown following the previously described method2, 1.0% glucose, 0.001% cocamidopropyl betaine and 0.1 M imidazole (pH 6.2). Protein crystals were incubated with reservoir solution containing an additional 5% glycerol, 5% PEG 400, 5% MPD, 5% sucrose, 5% sorbitol and 5% ethylene glycol for∼2 min before flash-cooling in liquid nitrogen. Data were collected at the Australian Synchrotron MX2 beamline, Melbourne, Australia. Data were processed and integrated using XDShttps://www.globalphasing.com/buster/)https://www.pymol.org/). Data collection and refinement statistics are listed in Full-length myristoylated and phosphorylated α2β1γ1 (4 mg ml2) or CaMKK2   prior to extensive washes with buffer A and elution in buffer A supplemented with 20 mM glutathione. The levels of α-pThr172 and β-pSer108 of treated AMPK complexes were assessed by immunoblot as described previouslyAll mutants were generated using QuikChange Site-Directed Mutagenesis Kits (Stratagene), and constructs were sequence verified. Heterotrimeric AMPK was expressed in COS7 mammalian cells by transient transfection, using FuGENE HD (Promega) transfection reagent according to manufacturer's protocol. Transfected constructs included N-terminal GST-α1 and α2 fusions2-HMRSAMSGLHLVKRR-COOH), 5 mM MgCl2, 200 μM [γ-32P]-ATP were incubated AMPK for 10 min at 30 °C in the absence or presence of various C2 concentrations. AMP (100 μM) and/or A769662 (20 μM) were included as appropriate positive controls. After 10 min, phosphotransferase activity was quenched by spotting onto P81 ion-exchange chromatography paper  followed by repeated washes in 1% H3PO4 . Statistical tests were conducted using analysis of variance (ANOVA). The values for EC50 and fold stimulation were calculated based on the equation: Activity=Basal+−Basal × [C2])/((EC50)+[C2])). Curve fitting was performed using KaleidaGraph software.AMPK activity assay was conducted as described previouslyPO4 ref. . The levAccession codes: Coordinates and structure factors have been deposited in the protein data bank with the accession codes 4ZHX and 5EZV.How to cite this article: Langendorf, C. G. et al. Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding. Nat. Commun. 7:10912 doi: 10.1038/ncomms10912 (2016).Supplementary Figures 1-4, Supplementary Table 1 and Supplementary Notes 1-3.


Comparative analysis of 1,587 bp region comprising promoter , exon-I (53 bp), and partial intron-I (116 bp) revealed 35 nucleotide substitutions  and 4 Indels. Within promoter, 15 variations at positions −1399 (A > G), −1288 (G > A), −1259 (T > C), −1158 (T > C), −1016 (A > T), −941 (T > G), −778 (C > T), −610 (G > A), −536 (A > G), −521 (A > G), −330 (A > C), −214 (A > G), −205 (A > T), −206 (C > A), and −175 (A > G) were located within the potential transcription factor binding sites (TFBSs), namely, NF-κE1/c-Myc, GATA-1, GATA-1/NF-E, Oct-1/POU3F2, MEF-2/YY1, GATA-1, AP-1, POU1F1a/GR, TMF, GAL4, YY1/Oct-1, HNF-1, GRalpha/AR, GRalpha/AR, and AP-1, respectively. Seventy-four percent (26/35) of the observed SNPs were novel to Indian cattle and 11 of these novel SNPs were located within one or more TFBSs. Collectively, these might influence the binding affinity towards their respective nuclear TFs thus modulating the level of transcripts in milk and affecting overall protein composition. The study provides information on several distinct variations across indicine and taurine αS1-CN regulatory domains.Regulatory region of milk protein alpha S1-casein ( Amongst these, B and C, differing in amino acid substitution (Glu/Gly) at position 192 of the mature protein, are the most common. The variant C has been reported to be common in zebu breeds, while other rare variants like A, D, and F have only been reported in European cattle [αS1-CN, mutations in the promoter region have been reported to influence the protein-coding efficiency of the relevant structural gene by changing the binding affinity towards their respective nuclear transcription factors (TFs) [αS1-CN5′-flanking region (αS1-CN5′) have also been associated with economically important traits like longevity and somatic cell scores in different taurine breeds [αS1-CN5′ has been widely studied in several species like cattle involving mainly B. taurus [αS1-CN5′ in B. taurus and few zebu cattle [αS1-CN5′ among Indian cattle breeds. The Indian native (B. indicus) breeds are adapted to diverse climatic conditions and range from good milch (of dairy merit) animals to extreme draught types. These breeds are known for their survival under inadequate feeding and low-input production practices naturally. Further, due to evolutionary divergence, B. indicus and B. taurus are expected to have variations in the candidate genes related to dairy traits. Keeping in view the scanty information available in Indian native cattle breeds, the present study was aimed to (i) sequence the full-lengthαS1-CN5′ in 13 Indian zebu breeds; (ii) search for putative TFs based on the indicine sequence (sequence specific to Indian zebu cattle); (iii) check if detected polymorphisms lie within identified TFBSs; (iv) identify variations within indicine breeds and their comparison with taurine breeds; and (v) identify homologies in the regulatory domains as well as phylogenetic relationship forαS1-CN5′ from different mammalian species. Bovine caseins are distinguished into four protein fractions, namely, alpha S1-casein, alpha S2-casein, beta-casein, and kappa-casein encoded by genes:n cattle , 4. The n cattle  and thisn cattle , 6. Moren cattle  either irs (TFs) , 9 and ce breeds , 10, 11.. taurus , yak [15. taurus , goat [1. taurus , and she. taurus . In contu cattle , 19, no αS1-CN5′ and to determine the variants/haplotypes among Indian zebu cattle, blood samples of 19 unrelated animals from 13 breeds from diverse agroclimatic zones were collected from their respective native breeding tracts. The selected breeds and their respective sample sizes (given in parenthesis) represented dairy {Gir (1), Tharparkar (2), Rathi (2), Red Sindhi (2), and Sahiwal (2)}, draught {Amritmahal (1), Kangayam (1), and Red Kandhari (2)}, and dual purpose {Deoni (1), Gaolao (1), Hariana (2), Kankrej (1), and Mewati (1)}. Genomic DNA was isolated by enzymatic Proteinase-K digestion  followed by standard phenol-chloroform extraction procedure [TM  stained 0.6% agarose gel and was quantified through NanoView .For characterization ofrocedure . The quaαS1-CN-F1 (5′-CCAATCCAGATATTGAACCTGC-3′) andαS1-CN-R1 (5′-ATAGGAAAGTACCAATACTTGC-3′) were used to amplify a fragment of 1,639 bp including promoter, exon-I, and intron-I region ofαS1-CN. The primers were designed through PRIMER2 software using cow genomic sequence data available at ENSEMBL database (BTAU 4.0:6). PCR reaction was performed in 25 μL reaction mixture containing 100-150 ng of genomic DNA, 5 p mole of each primer, 200 μM of dNTPs mix, 1X PCR buffer, 1.5 mM MgCl2, and 1.5 unit of Taq DNA polymerase  and was carried out in Mastercycler ep Gradient thermal cycler  using thermal cycling conditions as initial denaturation at 95°C for 2 min, followed by 30 cycles at 95°C for 60 sec, 59°C for 45 sec and 72°C for 2.30 min with a final extension at 72°C for 10 min. The amplicons were electrophoresed through 1.2% SafeView stained agarose gel and were visualized under UV transilluminator. Primer pairsExo-CIP) enzymatic treatment and used to sequenceαS1-CN5′ region using forward primerαS1-CN-F1 and three additional internal primers  designed on the 1,639 bp amplicon. Sequencing reactions were performed with BigDye v3.1 cycle Sequencing Kit in an ABI PRISM 3130 Genetic Analyzer . The resulting sequences were aligned and polymorphic sites identified from sequence comparison were confirmed through manual inspection. The transcriptional factor binding sites were identified using TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess/), MATCH [http://www.gene-regulation.com/cgi-bin/pub/programs/match/bin/match.cgi/), TRANSFAC [http://www.biobase-international.com/), AliBaba2.1 Search engine [http://www.gene-regulation.com/pub/programs/alibaba2/index.html), and from the literature [αS1-CN5′ from major milk producing mammalian species  were extracted from GenBank and Ensemble databases. Molecular Evolutionary Genetic Analysis (MEGA) software version 5.0 [The PCR product from each sample was purified using Exonuclaese I/Calf Intestinal Phosphatase , MATCH  . Apart from consensus sequences of CAAT box and TATA box, the promoter region contained dense array of potential transcriptional factor binding domains as AP (activator protein), AR (androgen receptor), AREB6 (Atp1a1 Regulatory Element Binding protein 6), CAAT (CAAT box), C/EBP (CCAAT/enhancer binding protein), Cart-1 (cartilage homeoprotein 1), c-Myb (cartilage homeoprotein 1), c-ReI (Nuclear Factor kappa B) c-Rel, ER (estrogen receptor), GATA-1 (GATA-binding factor 1), Gfi-1 (Zinc finger protein Gfi-1), GR (Glucocorticoid receptor), HNF-1 (Hepatocyte Nuclear Factor), MEF-2 (myocyte enhancer factor 2A), MGF/MPBF (mammary gland factor), NF-E (Nuclear Factor-E), Nkx2-5 (Homeobox protein Nkx-2.5), Oct-1 (Octamer-binding factor), POU1F1a ), POU2F1a , POU3F2 , PR (progesterone receptor), Sox-5 (SRY-related HMG-box gene 5), Sp1 (Specificity Protein 1), TBP (TATA-box-binding protein), TFIID (Transcription factor IID), TMF (TATA element modulatory factor), and YY1 (Ying-Yang factor). Binding of most of the transcription factors to their respective sites was associated with basal, tissue specific developmental, and cis-trans gene regulation [αS1-CN gene  and 13 (43.59%) were transitions and transversions, respectively. The transition/transversion rate ratios were αS1-CN5′. Within promoter region, 36 nucleotide substitutions and 4 consecutive Indels (at −224 to −221 (TTGT>- - - -) with respect to transcriptional start site) were observed  observed withinobserved . Throughobserved . αS1-CN5′ were found to be novel as they have not been reported in any other cattle breeds  to be most polymorphic with 62% (24/39) of the observed variations, whereas Gaolao  with 8% (3/39) variations was least polymorphic. Across different utility categories , dairy breeds showing all the observed variations  were the most polymorphic followed by draught  and dual  purpose breeds. None of the variations were specific to breed utility category. Amongst Indian zebu cattle; 26 out of 39 variations (67%) observed withine breeds . For theαS1-CN5′ did not affect any known TFBS. Across the variations at DNA binding domains, −1259  (T > C) is located within the GATA-1 and NF-E exhibited maximum frequency (0.63).Among 36 variations observed in the promoter region; 15 were located within the putative TFBS influencing the binding affinity of their respective TFs, thus possibly affecting the gene transcript. Further, in intron-I, variation at position 82 (T > A) was also located within Gfi-1 TFBS . Many ofαS1-CN5′ among major livestock species of dairy purpose  revealed divergence at binding domain for several ubiquitous TFs and motifs specific to mammary gland and hormone receptors. Amongst the 31 different TFB elements annotated for Indian zebu cattle, 12 showed variations (Sequence comparison ofriations . These vriations . B. taurus (99.3%), followed by B. grunniens (99.1%), Bubalus bubalis (97.2%), Ovis aries (94.8%), Capra hircus (95.5%), Canis lupus familiaris (60%), Gorilla gorilla (54.3%), Macaca mulatta (54.2%), Pongo abelii (54.5%), Homo sapiens (54.8%), Pan troglodytes (54.7%), and Rattus norvegicus (48.6%). The analysis revealed Indian native cattle to be closest to B. taurus followed by yak and buffalo and most distant from Equus caballus (43.8%). Phylogenetic relationship based on UPGMA with 5,000 bootstrap replicates forαS1-CN5′ among 15 different mammalian species revealed four major groups. Ruminants from Bovidae family  were grouped together; members of Hominidae family  and monkey formed another group; rat and mouse from Muridae family were clustered together, while, horse from Equidae family was distinctly separated  breeds has been provided in number of studies [Due to close linkage of four casein genes, regulatory domains of one casein gene might influence the other caseins as well in addition to the respective casein . It is p caseins . Furthervariable  and thes studies , 9, 10. αS1-CN5′ among Indian zebu cattle (B. indicus) revealed a dense region of binding sites for tissue-specific factors, hormone receptors, and ubiquitary transcription factors with few overlapping binding sites. Overall 39 variations identified in Indian zebu cattle breed indicated high variability ofαS1-CN5′. The polymorphic nature ofαS1-CN5′ has also been reported by Schild and Geldermann [B. indicus and three B. taurus breeds from Cameroon and Nigeria. Out of these 17 variations, 13 were similar to those observed for Indian zebu cattle in the present study, while 4 variations at positions −728 (− > T), −733 (T > C), −774 (C > T), and −820 (G > A) were unique to B. taurus (B. taurus the variation −728 (− > T) was genotyped using SspI restriction site and results suggested significant association of heterozygous genotype (− > T) with average higher α-S1 protein content in taurine breeds [αS1-CN5′ variation −728 (− > T) with variation inαS1-CN5' −1084 C > T and −186 T > C andβ-CN5′ (−109 C > G) [B. indicus) breeds and all animals were homozygous for T allele (TT). Another important variation at −175 (A > G) located within the binding site of ubiquitously expressed AP-1 TF [B. taurus and Indian native cattle breeds. The particular variation was genotyped across 62 Simmental and 80 German Holstein cows by Kuss et al. [αS1-CN protein content. Conversely, none of the animals in our study showed heterozygous AG genotype at position −175, as all were either homozygous with GG or AA genotype. These findings clearly demonstrate the nucleotide divergence in the regulatory region ofαS1-CN5′ across Indian and taurine cattle breeds.In the present study, sequence characterization ofldermann  with 17 ldermann  while an. taurus . Among te breeds , 30. Thi AP-1 TF  showed ds et al.  and reflcis-acting elements, observed variation c536 A > G was positioned at TMF binding domain which represses activation of TATA box and 82 T > A (intornic region variation) within the Gfi1 TFBS (Supplementary Table S3). Some of the variations were located within the binding sites for TFs with activator and the repressor activity was; −1016 A > T and −330 A > C within ubiquitously distributed YY1 TFBS; −610 G > A, −207 A > T and −206 C > A within GR TFBS while; −778 C > T and −175 A > G within AP-1 TFBS. AP-1 is a known transcriptional activator, but few studies also suggest its role as repressor forαS1-CN5′ [Out of 39 variations observed in the present study, 16 were located within putative TFBS, some of which are ubiquitously expressed and involved in regulation of tissue-specific gene expression. The variations located within transcriptional activators included −1158 T > C and −330 A > C, positioned in the potential binding sites for ubiquitously expressed Oct-1 that could possibly change the transcriptional mechanism. The −1158 T > C also overlaps with POU3F2 TFBS specific to nervous system and binds Oct-1 . Additionally, variations at −207 A > T and −206 C > A overlapping with the binding domain for GRalpha were located within AR that mediates androgen-specific gene regulation. Eleven out of the sixteen above-discussed variations occurring within important putative TFBSs are specific to Indian zebu cattle and have not been observed in any other breed>species. The remaining five variations  are common with B. taurus  might be responsible for difference at production level. As regulation of gene expression is under multifactorial control, there is a need to focus on haplotypes rather than individual variations.The variations located within other important TFBS included −1399 A > G and −1259 T > C, located within Nuclear TFs   which ar. taurus . The effαS1-CN gene, wherein such information was lacking. The novel variations found in Indian cattle breeds may be responsible for differential content of milk components as compared to taurine breeds. This study needs to be extended further in combination with protein coding gene polymorphism (intragenic haplotypes) to evaluate effects of promoter polymorphism on milk production traits. The ability to link sequence variability to dairy traits in context of other members of casein family using SNP chip or other tools could be important. This would lead to efficient utilization of resources like Indian native cattle impacting the socioeconomic structure of large population in India. The present study generates the knowledge related to variations in naturally evolved Indian cattle breeds within regulatory region ofαS1-CN5′ using MATCH and TESS software, list of haplotypes for αS1-CN5′ among Indian cattle and predictive role of various putative regulatory domains affected due to polymorphismSupplementary material contains information for identification of potential TFBSs within


Popdc2 gene as an example, we also show that a significant proportion of target genes for NKX2-5 contain a binding motif recognized by NKX2-5, which overlaps with a binding site for MEIS1. Binding of the two factors to such overlapping sites is mutually exclusive, and this provides a simple regulatory mechanism for spatial and temporal synchronization of a common pool of targets between NKX2-5 and MEIS1.The homeobox transcription factors NKX2-5 and MEIS1 are essential for vertebrate heart development and normal physiology of the adult heart. We show that, during cardiac differentiation, the two transcription factors have partially overlapping expression patterns, with the result that as cardiac progenitors from the anterior heart field differentiate and migrate into the cardiac outflow tract, they sequentially experience high levels of MEIS1 and then increasing levels of NKX2-5. Using the   •NKX2-5 shares a DNA-binding site with MEIS1•Popdc2 enhancerMEIS1 and NKX2-5 successively bind a •Successive binding by MEIS1 and NKX2-5 is a general mechanism of regulation•NKX2-5 represses fast troponin isoforms in the atria Dupays et al. use a combination of ChIP-seq and RNA-seq to identify the direct targets of the homeodomain transcription factor NKX2-5 during cardiac development. They propose a mechanism of transcriptional regulation during cardiac differentiation by successive binding of the two homeodomain transcription factors MEIS1 and NKX2-5. NKX2-5 gene cause congenital heart disease; haploinsufficiency results in a spectrum of congenital heart disease of varying phenotypic penetrance, which is mirrored in mouse models  recently has been used to identify NKX2-5-binding sites in both cultured cell lines and adult heart ; but, deHere, we have combined genome-wide ChIP followed by massively parallel DNA sequencing (ChIP-seq) and whole-transcriptome RNA sequencing (RNA-seq) using embryonic hearts from a mouse line that shows hypomorphic expression of NKX2-5 and models human cardiomyopathy. This has enabled us to identify direct targets of the NKX2-5 transcription factor during heart development in vivo. We have found that a large proportion of cardiac enhancers binding NKX2-5 have an overlapping binding site for the homeobox transcription factor, MEIS1, indicating a regulatory interaction between these two proteins. Our data suggest that the combination of shared transcriptional targets, differential DNA-binding affinities, and overlapping expression domains provides a mechanism for spatial and temporal synchronization of a common pool of targets between NKX2-5 and MEIS1.We performed ChIP with embryonic day (E)11.5 mouse embryo hearts using an NKX2-5 antibody followed by massive, parallel sequencing (ChIP-seq) to identify 2,610 regions enriched for that factor . Of thesBioinformatic analysis identified 3,313 genes flanking the NKX2-5-binding loci (the two nearest genes for each region enriched in a maximum distance of 1 Mb were considered), and we performed a computational analysis for enrichment of gene ontology (GO) terms associated with those genes. This identified significant enrichment of biological process related to muscle structure development and cardiac chamber development A, in agrTo assess whether the NKX2-5-binding loci are likely to identify transcriptional enhancer regions, we compared them with those enriched for binding of P300 in the heart at the same stage . P300 ishttp://dmdd.org.uk; Only a small proportion of putative targets have been the subject of published studies using gene ablation that could reveal cardiac defects. However, several already have been constitutively targeted as part of the EUCOMM/KOMP initiative  and subjSequence analysis of the 25 bp flanking the summit of each NKX2-5-binding region was undertaken, using a de novo motif-binding program to identify conserved DNA motifs. As expected, the most common of these was a consensus NKX2-5-binding site  , overlapping at this stage with the expression domain of Nkx2-5. At E9.5, expression of both Nkx2-5 and Meis1 persists strongly in the AHF while levels of Meis1 in the distal OFT are reduced , we confirmed that both proteins can indeed bind in vitro to the de novo motif identified from ChIP-seq B. By comOur EMSA results indicate that binding of NKX2-5 and MEIS1 on the de novo motif is mutually exclusive. Consistent with this, in a luciferase assay, individually both NKX2-5 and MEIS1 were able to transactivate reporter expression via the de novo sequence, but we found no evidence of synergistic activation by the two factors D.Popdc2 gene itself . Moreover, Popdc2 RNA expression was downregulated in Nkx2-5-null hearts at E9.0 compared with controls  also were identified by MEIS binding in the first branchial arches. A similar overlap was evident with the second branchial arches  due to failure of heart development  that reflects atrial and atrioventricular nodal conduction  Act 1986 and with the approval of the MRC National Institute of Medical Research Ethical Review Panel. Genotyping of the transgenic mouse lines eviously .Please refer to the Libraries were prepared with 10 ng immunoprecipitated DNA or input. Samples were sequenced on the Genome Analyzer IIx platform, and ChIP-seq reads were aligned to the mm9 genome assembly using BOWTIE. Peak calling was done using MACS1.4. Please refer to the All enriched GO categories were determined using the programs GeneCoDis3  and GREAMotif distribution and overlap of the different NKX2-5 motifs among the identified peaks were done with FIMO using default parameters and the matrices identified with MEME.GSE13614) used to analyze working myocardium genes versus atrio-ventricular genes were analyzed using Limma at Geo2r (http://www.ncbi.nlm.nih.gov/geo/geo2r/). The p values were adjusted for multiple testing using the Benjamin and Hochberg test, and transcripts were identified as differentially expressed if the adjusted p value was <0.01.Expression array data . After genotyping, three control and three hypomorphic samples were selected for total RNA extraction using Trizol and purified on RNeasy columns (QIAGEN). Total RNA was converted to a library of templates suitable for high-throughput DNA sequencing using the Truseq kit (Illumina). Templates were sequenced using the Illumina Genome Analyzer IIx platform. Sequence reads were aligned to the mm9 genome with TopHat. Mapped reads were analyzed using the Avadis NGS package .Recombinant proteins were synthesized using a coupled transcription-translation system (Promega TnT T7/SP6 kit) and incubated with 0.5 ng radiolabelled oligonucleotide probe in binding buffer  at room temperature for 10 min. For competition experiments, the excess of unlabelled oligonucleotide probes was added and incubated for 10 min prior to the addition of radiolabelled oligonucleotide probes. Binding reactions were analyzed on 6% 0.5× Tris-borate-EDTA (TBE) polyacrylamide gels electrophoresed at 150 V; gels were dried prior to detection of signal by autoradiography.RNA in situ hybridizations were performed as described previously . Tnnt3, Immunohistochemistry was performed as described previously  with NKXEmbryos were embedded and processed for imaging as described previously .Mouse DNA fragments were tested in vivo using the tol2 system . BrieflyThe Popdc2 region tested previously in fish was subcloned in an hsp68-LacZ reporter gene. Transient transgenic mouse embryos were generated by the Procedural Services Section at The Francis Crick Institute Mill Hill Laboratory by standard pronuclear microinjection techniques.