MYB and bHLH bind to two identifiable syntaxes present in target gene promoters, and the anthocyanin pathway is regulated as one unit as in other branches of the flavonoid network. cis regulatory architecture of the anthocyanin pathway genes targeted by the transcription factor (TF) complex including MYB, bHLH, and WDR was systematically analysed in one species and the findings extended to others. In Ipomoea purpurea, the IpMYB1-IpbHLH2-IpWDR1 (IpMBW) complex was found to be orthologous to the PAP1-GL3-TTG1 (AtPGT) complex of Arabidopsis thaliana, and interacted with a 7-bp MYB-recognizing element (MRE) and a 6-bp bHLH-recognizing element (BRE) at the proximal promoter region of the pathway genes. There was little transcription of the gene in the absence of the MRE or BRE. The cis elements identified experimentally converged on two syntaxes, ANCNNCC for MREs and CACN(A/C/T)(G/T) for BREs, and our bioinformatic analysis showed that these were present within anthocyanin gene promoters in at least 35 species, including both gymnosperms and angiosperms. For the anthocyanin pathway, IpMBW and AtPGT recognized the interspecific promoters of both early and later genes. In A. thaliana, the seed-specific TF complex  may regulate all the anthocyanin pathway genes, in addition to the proanthocyanidin-specific BAN. When multiple TF complexes in the anthocyanin pathway were compared, the cis architecture played a role larger than the TF complex in determining the variation in promoter activity. Collectively, a cis logic common to the pathway gene promoters was found, and this logic is essential for the trans factors to regulate the pathway.Cellular activities such as compound synthesis often require the transcriptional activation of an entire pathway; however, the molecular mechanisms underlying pathway activation have rarely been explained. Here, the  The MYB-bHLH-WDR (MBW) complex represents a mode of gene regulation not only for the anthocyanin pathway but also proanthocyanidin (PA) synthesis and epidermal cell differentiation  and C1   and C1 . For theng genes ; recentlArabidopsis GL3 and TTG1  may be pinpointed to an anthocyanin promoter through both bioinformatic and experimental approaches, and quantitative evaluation of contributions of TFs and cis elements to gene regulation is now achievable.In documented MBW complexes, specific residues were identified on maize C1 for its interaction with R (cis motifs of conspecific genes at the pathway level in I. purpurea Roth (common morning glory) and made parallel examinations of the homologous genes in Arabidopsis thaliana (L.) Heynh. as a reference. The I. purpurea genes included those encoding chalcone synthase D (CHS-D), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol reductase B (DFR-B), anthocyanidin synthase (ANS), UDP-glucose:flavonoid 3-O-glucosyltransferase (3GT), UDP-glucose:anthocyanidin 3-O-glucoside-2′-O-glucosyltransferase (3GGT), and the trans factors MYB1, bHLH2, and WDR1. Transcriptional activities of MBW complexes were subsequently compared between I. purpurea and Arabidopsis on interspecific and conspecific promoters. A bioinformatic analysis of homologues in multiple species was used to explore the generality of cis patterns . These extensive analyses revealed the features of the MYB- and bHLH- recognizing elements , patterns of MREs and BREs on the proximal promoters, and one cis logic for pathway regulation.In order to elucidate the molecular mechanism(s) of anthocyanin pathway regulation in the proximal regions, we analysed interactions of TFs and I. purpurea taken from a nationwide collection (Arabidopsis (tt3 mutant and the Columbia accession) were obtained from the Arabidopsis information resource (TAIR) and those of Petunia hybrida (R27) were a kind gift from Dr Quattrocchio . All seeds were germinated in the growth chamber.Seeds of I. purpurea (III6-4 and III6-9). Each plant was sampled at 2-h intervals starting 60h before flower opening. RNAs were reverse transcribed and quantifications of transcript copies followed the protocol reported by Supplementary Table S1).RNA samples were extracted in TRIzol (Life Technologies) from petals of IpCHS-D, IpF3′H, IpDFR-B, IpbHLH2, and IpWDR1) based on primers (Supplementary Table S1) whose designs followed the available sequences of conspecific or closely related targets (Supplementary Table S2). The promoter sequence of IpF3H was obtained by inverse PCR . Ipomoea TFs were reverse transcribed from RNAs extracted from fresh petals of I. purpurea. Arabidopsis TFs were similarly amplified from seedlings of the Columbia accession with the appropriate primers (Supplementary Table S1).Standard PCRs were applied to leaf genomic DNAs to amplify five gene promoters (ail-PCRs . HomologSupplementary Table S1) to replace its reporter gene, resulting in effector vectors. The reporter vectors were adapted from pJIT163-eGFP with the CaMV 35S promoters replaced by the proximal promoters and GFP replaced by the firefly luciferase (LUC) gene. The reference vector was also based on pJIT163-eGFP, with its GFP replaced by the renilla luciferase (RUC) gene (Ipomoea nil wdr1 mutant (seeds available on request) by particle bombardment (BioRad PDS-1000/He equipment). A construct mixture  was prepared for the plasmid cocktail, which was coated with 50mg ml–1 microparticles (Bio-Rad) at 2.0 μl per 1.0 μg plasmid DNA. The complex was then mixed with 2.5M CaCl2 and 0.1M spermidine in a ratio of 1:5:2. Other details followed The promoters were ligated into vectors to prepare for dual luciferase assays as described by IpMYB1, IpbHLH2, and IpWDR1 were integrated in frame into vectors pAD-GAL4 2.1 and pBD-GAL4 Cam of HybriZAP-2.1 (Stratagene), respectively, according to the manufacturer’s instructions. Constructs were introduced in pairs into the YRG-2 yeast strain (Stratagene). The transformants were plated onto SD media minus histidine, leucine, and tryptophan for identifying positive interactions. The strengths of TF interactions were then measured by β-galactosidase activities according to the protocol of the Matchmaker system (Clontech).The coding sequences of Escherichia coli strain BL21 (DE3) was incubated at 37°C in LB solution and treated as described (A complete coding sequence of MYB was integrated in frame into pMAL-c2G (New England Biolabs) to express a tagged protein consisting of maltose-binding protein (MBP) and MYB. A partial coding region of bHLH was similarly expressed. The vector-transformed Arabidopsis tt3 mutants were grown in the growth chamber for transgenic tests of the IpDFR-B promoter series. The coding regions of IpDFR-B were first subcloned into the SN1301 binary vector for the construction of SN1301-35S::DFR-Bcoding. Varying promoter regions  independently replaced the 35S promoter to generate series of SN130-proDFR::DFR-Bcoding vectors. These vectors were singly introduced into Agrobacterium strain GV3101 via the standard freeze-thaw protocol. Transformation of Arabidopsis followed a modified flower dip protocol  for observations. For 4–5-week old seedlings, treatment by dehydration (up to a week) and a low temperature (10–16°C) also induced anthocyanin accumulation. Anthocyanins extracted from representative seedlings were further examined by high performance liquid chromatography using cyanidin-3-O-glucoside (Extrasynthese) as the standard.Phenotypic complementation was observed in the T2 generation. Seeds were sterilized and cultivated on 1/2MS medium containing 5% sucrose, kept at 4°C in darkness for 2 days, and transferred to constant light conditions . We ended up with 159 sequences satisfying the search criteria, which provided the basis for mapping the natural distribution of known cis elements on anthocyanin gene promoters. The sequences included in the peak of the distribution were further characterized by two motifs identified via MEME software following the parameters of Multiple blastp searches in the NCBI database  identified protein homologues for each of the anthocyanin genes. The results were trimmed for redundancy following the protocol reported by t-tests were performed on comparisons between averages of experiments. Two-way ANOVAs were carried out after log transformation of the promoter activity. Pearson correlation coefficients were calculated with the significance levels corrected for multiple comparisons by the Dunn-Šidák method (taking the experiment-wise error rate α = 0.05). All tests were computed using SAS (ver. 9.0).Standard IpMYB1, IpbHLH2, and IpWDR1 are expressed in the corolla of I. purpurea . The transcript levels of the enzyme-coding  genes  were significantly correlated with those of regulatory genes  and among themselves (except between IpDFR-B and Ip3GT). The expression of IpF3′H was highly correlated with that of IpbHLH2 and IpWDR1, but not with that of IpMYB1. The quantitative data largely agreed with the perception that IpMYB1, IpbHLH2, and IpWDR1 regulated pathway transcription collaboratively. The features of the TFs were examined in two further experiments. One showed the subcellular localization of the three TFs. The vectors with fluorescent-tagged TFs were introduced into the onion epidermis , and the in vivo images indicated that IpbHLH2 and IpWDR1 were localized in both the nucleus and the cytoplasm, whereas IpMYB1 was mostly in the nucleus. This pattern agrees with the documented subcellular sites of their homologues , as expected.Although the regulatory genes purpurea , whetherI. purpurea, some pathway genes  were characterized through mutants or mutant complementation ; similar information was also gathered for IpMYB1, IpbHLH2, and IpWDR1 to evaluate the TF regulatory capacity on the expression of each. By single and combinatory tests of the TFs as effectors in dual luciferase assays  of the ten promoter constructs could be activated strongly by IpMBW; however, the constructs hosting the IpWDR1 5′-noncoding regions responded only at low levels to IpMBW, and little promoter activity was found on those hosting various lengths of Ip3GT and IpMYB1 promoter sequences . The tests also showed that IpMYB1 could, alone, initiate a low level of transcription on the seven promoters (particularly F3H). By contrast, IpbHLH2 or IpWDR1 alone failed to activate observable promoter activities in the same setting. Only the collective presence of all three TF effectors generated considerable promoter activities  on the cloned 5′-noncoding regions of the anthocyanin genes and site-by-site tests on the predicted motifs  were adapted. In the initial case of IpDFR-B, three experimental approaches were taken for site-specific analysis, including dual luciferase assays performed on leaves of the wdr1 mutant of I. nil, gene transformations of the tt3 mutant of Arabidopsis, and electrophoretic mobility shift assays (EMSAs) with IpMYB1 and IpbHLH2. Starting by mutating predicted motifs  hosting a suspected motif  as mutations at the site (DFRB-M4) demolished the binding capacity of IpMYB1 .Though effective in validating d IpWDR1 , B. To ktructure . Likewisnd AtCHI , as withSupplementary Figure S3). Except in one case (IpbHLH2pro), where accidental mutations to pbHLHMRE2 caused an exceedingly high promoter activity, all other introduced mutations were mostly on targets, judging from their reduced promoter activities. To make sure that the TFs were not overexpressed to bias the detection of effective motifs, we compared 10-fold levels of particle bombardment in the assays for two genes including IpCHSDpro and IpF3′Hpro . Although lowered TF dosages led to reduced promoter activities , the patterns of the relative effects of mutated motifs were largely unchanged . To further make sure that the results of mutation tests were not biased by using leaves of I. purpurea, we compared the same promoters and effectors in the leaves of cultivated rice seedlings in transient assays. The results showed essentially the same patterns for the mutation effects  despite change in promoter strength. These experiments effectively validated applications of transient assays to assessing mutation effect of cis motifs.Complementing the EMSAs but in a cellular environment, dual luciferase assays showed that mutations correctly targeting MREs and BREs could reduce promoter activities . Putting all the information on cis motifs together, we found they converged on ANCNNCC for MREs and CACN(A/T/C)(G/T) for BREs , the pathway genes appear to have a tight motif organization in the proximal region of a promoter in common.Collectively, transient and EMSA results led to identification of the major  pathway , includifor BREs . The twoIpomoea genes as templates (cis layout pattern (CACNNG (N1–100)ANCNNCC) was taken as the search criterion for these sequences, as the pathway-based identification of cis motifs suggested a common layout of BRE and MRE (5′→3′) on the anthocyanin genes . Interestingly, the distance between the cis elements showed a peak at 6-bp apart  significantly weakened the promoter strength; nevertheless, about 12% of the promoter activity still remained even after the distance was increased further to 80-bp apart  and four sets of MBW complex from two species (Arabidopsis and I. purpurea). Three TF complexes  were from Arabidopsis, and one (IpMBW) from I. purpurea. We found that AtPGT, AtPET, and IpMBW could positively regulate all tested genes except AtF3′H, AtBAN and Ip3GT. In comparison, AtTTT also activated the same set of genes (except IpbHLH2), in addition to AtBAN , despite typical BRE and MRE sequences being located at –659 and –584 from the translation start site, respectively. Both Ip3GT and IpMYB1 were found lacking BREs in their proximal promoters , which explained their low promoter activities in the presence of IpMBW . In contrast, AtTTT could activate these genes at a relatively low level , we analysed the transient expression data in two-way ANOVAs. The analysis suggests that promoter identity is the leading factor for proximal promoter activity on the anthocyanin pathway, and the interactions between the TF complex and promoter (TF × promoter) plays a role as significant as the TF complex itself  were suggested  and AtBAN. The result agrees with reported regulation of AtTTT on AtCHS . At least three functional MREs were experimentally confirmed on each of IpCHI-226pro, IpF3H-211pro, IpF3′H-171pro, and IpDFRB-191pro . For instance, if one of the MREs was mutated on IpDFRB-191pro, no phenotypic change could be found on the seedlings transformed with the mutated construct. Although refractory in experimental validations of the cis elements involved, the redundancy in binding sites may provide dynamics as well as intricacy to the gene transcription. A full occupancy of the cis sites in a promoter region, for instance, may result in a higher level of gene transcription than is the case for partial occupancy of the cis sites.The pathway genes show multiple copies of each type of cis elements also mirror functional redundancy of TFs. Noticeably, AtTTT activates the same set of anthocyanin pathway genes as AtPGT and AtPET do, but possibly at a lower capacity , numbers of cis elements, spatial distance between cis elements, and the relative orientations of cis elements. These factors may significantly affect a promoter’s activity. For the anthocyanin pathway genes, the cis regulatory sites summarized by the syntax identified for MREs (ANCNNCC) overlap with several previously postulated motifs. For example, cctacc(n)7ct, described as an H-box . The same is also true for the syntax of BREs. It matches well with the previous G-box . The mutation tests of Bz2 (GST) promoter, suggesting that the BRE syntax holds for both monocots and dicots.Promoter architecture is believed to take a central position in transcription , but detan H-box , GGnGGnTan H-box . The intcis motifs over the anthocyanin pathway .Supplementary File S1. Bioinformatic survey with Perl scripts and instructions.The study was supported by National Science Foundation of China  and Chinese Academy of Sciences (KSCX2-YW-N-043).


Nkx2-5 genetically interacts with Xrn2 because Nkx2-5+/-Xrn2+/-, but neither +/-Nkx2-5nor +/-Xrn2, newborns exhibited a defect in ventricular septum formation, suggesting that the association between Nkx2-5 and Xrn2 is essential for heart development. Our results indicate that Nkx2-5 regulates not only the initiation but also the usage of poly(A) sites during heart development. Our findings suggest that tissue-specific transcription factors is involved in the regulation of APA.Transcription factors organize gene expression profiles by regulating promoter activity. However, the role of transcription factors after transcription initiation is poorly understood. Here, we show that the homeoprotein Nkx2-5 and the 5’-3’ exonuclease Xrn2 are involved in the regulation of alternative polyadenylation (APA) during mouse heart development. Nkx2-5 occupied not only the transcription start sites (TSSs) but also the downstream regions of genes, serving to connect these regions in primary embryonic cardiomyocytes (eCMs). Nkx2-5 deficiency affected Xrn2 binding to target loci and resulted in increases in RNA polymerase II (RNAPII) occupancy and in the expression of mRNAs with long 3’untranslated regions (3’ UTRs) from genes related to heart development. siRNA-mediated suppression of Nkx2-5 and Xrn2 led to heart looping anomaly. Moreover, DOI:http://dx.doi.org/10.7554/eLife.16030.001 About one in every hundred babies is born with problems that either affect the structure of the heart or how it works. These problems are known as congenital heart disease, and result when the development of the heart is disrupted. How the heart develops is determined by thousands of genes whose activity or “expression” must be precisely regulated. Proteins called transcription factors can control gene expression; therefore, researchers may discover new ways of treating congenital heart disease if they can understand how transcription factors work during normal heart development.To produce a protein, the information in a gene must first be “transcribed” to form a molecule of messenger RNA (mRNA). Not all of the mRNA sequence is subsequently “translated” to form the protein; this includes a stretch at the end of the mRNA called the 3’ untranslated region. The length of the 3’ untranslated region for a particular mRNA may vary depending on the type of cell it has been produced in, and this length can influence how efficiently the mRNA is translated to form a protein. However, it was not clear what changes the length of the 3’ untranslated region.Nimura et al. have now studied mice to investigate the role of a transcription factor called Nkx2-5, which was known to be important for heart development. This revealed that in addition to its expected role in starting the transcription of genes that are important for heart development, Nkx2-5 also controls the length of 3’ untranslated regions of certain mRNAs. To do so, Nkx2-5 binds to a protein called Xrn2 that stops transcription when the end of the gene is reached.Mouse embryos that lacked Nkx2-5 produced mRNAs containing long 3’ untranslated regions from genes related to the development of the heart. Furthermore, suppressing the activity of both Nkx2-5 and Xrn2 resulted in the embryos developing heart defects.The findings of Nimura et al. suggest that transcription factors found in specific tissues are responsible for the different lengths of 3’ untranslated regions in mRNAs in different tissues. Furthermore, incorrectly regulating the length of these regions appears to be linked to the development of congenital heart disease. The next step is to understand exactly how the failure to correctly regulate the length of 3’ untranslated regions contributes to congenital heart disease.DOI:http://dx.doi.org/10.7554/eLife.16030.002 Transcription factors and chromatin regulators orchestrate the processes of heart development by positively and negatively regulating thousands of genes . Mutatioet al. demonstrated that multiple transcription factors activate certain cardiac enhancers without p300 . Furthermore, we discovered that Nkx2-5 is associated with both transcription start sites (TSSs) and downstream regions of genes and that Nkx2-5 controls APA in conjunction with the 5’-3’ exonuclease Xrn2. Simultaneously suppressing Nkx2-5 and Xrn2 caused heart-looping abnormalities. Moreover, Tnnt2  and Atp2a2  transcripts   Li et a. Embryonnscripts . We also5 hearts . These rTnnt2 and Atp2a2, which are highly expressed in eCMs. The looping between the TSSs and downstream regions of the genes (TSS-downstream looping) was dependent on Nkx2-5 but not on Gata4 or Tbx5   was incrown eCMs . Consistown eCMs . Althougd Atp2a2 . These fTo investigate whether mRNAs with long 3’ UTRs in Nkx2-5-knockdown eCMs are enriched for specific functional annotations, we performed Gene Ontology (GO) analysis ; 2009b. To elucidate the molecular mechanisms by which Nkx2-5 regulates APA, we examined the transcription termination factors associated with Nkx2-5 in the embryonic heart. We could detect an association between Nkx2-5 and the transcription termination factor Xrn2, which has 5’-3’ exonuclease activity  Figure . HoweverTnnt2 and Atp2a2, Xrn2 was repressed by transfection with a sequence-specific siRNA in eCMs -tailed mRNA showed that the knockdown of Nkx2-5 and Xrn2 significantly increased the expression of extended poly(A)-tailed mRNAs -tailed mRNAs. Northern blot analysis of ed mRNAs . Long 3’ed mRNAs . Consisted mRNAs . Next, wed mRNAs . FurtherTo demonstrate the coordinated functions of Nkx2-5 and Xrn2 in heart development, we introduced siRNAs directed against Nkx2-5 and Xrn2 along with GFP expression plasmids into early-head-fold-stage embryos  . These eXrn2, including exons1 and 2, which encode a part of the domain with enzymatic activity (+/-Xrn2+/-Nkx2-5 newborns by intercrossing +/-Nkx2-5 mice with two +/-Xrn2 mice (#2 and #3). Although neither +/-Nkx2-5nor +/-Xrn2 hearts showed a muscular ventricular septal defect (VSD) at postnatal day 0 (P0), we found a VSD in +/-Xrn2+/-Nkx2-5newborn hearts (n = 3 of 7)  . An atriormation . These rTnnt2 and Atp2a2, during heart development. The regulation of APA has been reported as one of the mechanisms for determining the length of the 3’ UTR , a cardiac sarcoplasmic reticulum Ca2+ pump that is essential for normal cardiac performance  as previously described, with minor modifications . Cas9 RN−1 micrococcal nuclease in 250 µl of a nuclear isolation buffer containing 400 mM NaCl, which was then diluted to 200 mM NaCl. The digested chromatin was immunoprecipitated with 15–50 µg of antibody. Only mono- and di-nucleosomes size DNA was used for construction of sequencing libraries. Sequencing libraries were prepared from two or more biological-replicate ChIP samples and from an input according to the instructions provided with the SOLiD Fragment Library Barcoding Kit . The libraries were sequenced with SOLiD 4. The resulting reads were mapped using BioScope software (Life Technologies) with the default configuration, combined biological replicates, and analyzed using Homer (http://jtreeview.sourceforge.net/).Nuclear extracts from four E12.5 embryonic hearts were used in each native ChIP experiment following a previously described protocol with minor modifications ; 2009. Ing Homer , CEAS  according to the manufacturer’s instructions. Strand-specific sequencing libraries from two biological replicate RNA samples were prepared according to the Life Technologies protocol as previously described (RNA was extracted from primary embryonic cardiomyocytes (from E12.5 hearts) and E9.5 hearts from escribed . Brieflyescribed ; 2012. TRefSeq genes were divided into three categories  according to the FPKM (fragments per kilobase of exon per million fragments mapped) of each gene in wild-type eCMs: High: FPKM > 500; Middle: FPKM 100–500; and Low: FPKM 10–100. The genes identified for analysis were either upregulated to more than a ln (fold change) of 0.2 or downregulated to less than a ln (fold change) of 0.2 compared with control siRNA-treated eCMs. The High, Middle, and Low categories included 90, 717, and 5516 genes, respectively.The expression of read-through RNA (FPKM > 0.5) was measured within 5 kb in eCMs and in Nkx2-5-deficient embryonic hearts using BioScope (Life Technologies). Genes with an average change in read-through RNA expression of more than a 1.4-fold compared with control siRNA-treated eCMs or wild-type littermate hearts were analyzed using DAVID. Genes for which the read-through RNA was expressed at less than 0.5-fold compared with controls were considered to have downregulated read-through RNA. Genes with expression within regions of a read-through RNA were removed from the analysis. The normalized read-through RNA tags were counted in 50-bp bins using CEAS, and the obtained values were then used to calculate the average expression in each bin.http://tools.genxpro.net/apadb/).The polyadenylation sites were obtained from APADB . Reverse transcription was performed with SuperScript III (Invitrogen) as previously described  and anal6 siRNA-transfected E12.5 primary cardiomyocytes were digested overnight at 37°C with EcoRI, BglII, or HindIII  in 100 µl of buffer with 0.4% NP-40 and complete EDTA-free protease inhibitors . The nuclei were ligated overnight with Ligation High (TOYOBO) and then extracted using phenol-chloroform. The primers were designed using the 3C Primer (http://dostielab.biochem.mcgill.ca/index.php) and Primer3 programs (http://frodo.wi.mit.edu/). BAC plasmids RP23-2E23 for Tnnt2 and RP23-128I8 for Atp2a2 were used as controls. PCR was performed using the THUNDERBIRD SYBR qPCR mix (TOYOBO) and a CFX384 thermocycler (BIO-RAD). The amplicons were separated on 2% agarose gels stained with ethidium bromide for visualization.Nuclei isolated from 10Tnnt2 and Atp2a2 mRNA were detected with 32P-labeled cDNA that contained a portion of the gene-body regions of Tnnt2 and Atp2a2. The intensities of the bands were quantitatively measured using a BAS5000 .Poly A-tailed mRNA was purified from total RNA that was extracted using TRIzol (Invitrogen) and the Ambion MicroPoly(A) Purist Kit (Ambion). The poly(A)-tailed mRNA (360 ng) was separated by 1% agarose gel electrophoresis and transferred to a Hybond-N+ nylon transfer membrane . Expression constructs containing HA-Nkx2-5, Flag-Xrn2, and their deletion mutants were cotransfected into C3H10T1/2 cells, purchased from ATCC, in 10 cm dishes as previously described . This ceE7.5 embryos were dissected from the uterus, and embryos at the early-head-fold stage were carefully selected. Liposomes were created by mixing 3.33 µM siRNAs directed against Nkx2-5, Xrn2, or control together with 53 ng/µl EGFP expression vector in 15 µl of OPTI-MEM with 3 µl of RNAiMAX (Invitrogen) diluted in 15 µl of OPTI-MEM. Liposomes were injected into heart fields as previously described . The embWhole-mount in situ hybridization was performed according to standard procedures  and probP values were calculated using the two-tailed t-test and the Tukey-Kramer HSD test using JMP, Fisher’s exact test was performed using http://aoki2.si.gunma-u.ac.jp/exact/fisher/getpar.html, and the two-sample Kolmogorov-Smirnov test was performed using R. p < 0.05 was considered statistically significant. The number of n shows biological replication.The data are presented as the mean ± s.e.m.  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by James Manley as the Senior editor and three reviewers, including Maurice Swanson (Reviewer #3) and Nick Proudfoot (Reviewer #1), who is a member of our Board of Reviewing Editors.Thank you for submitting your article "Regulation of alternative polyadenylation by Nkx2-5 and Xrn2 during heart development" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.This paper describes an interesting data set showing that the cardiac development associated transcription factor Nkx2-5 plays an important role in regulating mRNA 3' end formation for a number of more highly expressed cardiac genes.I am pleased to tell you that all three consider the paper interesting and worthy of publication provided fairly extensive additional data can be added to strengthen the paper. I list below the key points that need to be addressed in revision.1) The effect of Nkx2-5 or Xrn2 knock down produces apparently only relatively small effects on alternative polyA site (APA) selection (1.5 to 2 fold). However, this data is based on total RNA-seq analysis so will not directly measure the selective use of PAS as it will also be affected by RNA stability differences between mRNA with different 3'UTRs . More convincing APA data would come from for instance RNA-seq on chromatin fractionated RNA to look at nascent transcripts. This type of data would also allow them to determine if Nkx2-5 plays a role in regulating alternative splicing.Tnnt2 as well as the analysis of additional Nkx2-5 sensitive genes. Also a RIP approach might produce more or complementary convincing data.2) The evidence that Nkx2-5 is required for Xrn2 recruitment to gene 3' ends based on ChIP is a bit marginal. More primer pairs are needed across Tnnt2 and Atp2a2 needs more primers across the gene to convincingly show a promoter to PAS region interaction that is specifically sensitive to loss of Nkx2-5. What are the control lanes in 3) The 3C analysis on 4) The manuscript’s text and figures need improved clarity and clearer annotation. pA site usage and transcription termination should be distinguished. The positions of annotated PAS should be clearly shown on all figures5) A summary figure of interacting regions between Nkx2-5 and Xrn2 would be helpful in 6)  1) The effect of Nkx2-5 or Xrn2 knock down produces apparently only relatively small effects on alternative polyA site (APA) selection (1.5 to 2 fold). However, this data is based on total RNA-seq analysis so will not directly measure the selective use of PAS as it will also be affected by RNA stability differences between mRNA with different 3'UTRs . More convincing APA data would come from for instance RNA-seq on chromatin fractionated RNA to look at nascent transcripts. This type of data would also allow them to determine if Nkx2-5 plays a role in regulating alternative splicing.Tnnt2 and Atp2a2 on chromatin-fractioned RNA by RNA-seq and qPCR  The evidence that Nkx2-5 is required for Xrn2 recruitment to gene 3' ends based on ChIP is a bit marginal. More primer pairs are needed across Tnnt2 as well as the analysis of additional Nkx2-5 sensitive genes. Also a RIP approach might produce more or complementary convincing data.Tnnt2 was decreased in Nkx2-5-knockdown eCMs, we examined the primer for amplifying the exon 10 of Tnnt2 in Myl7 gene is one of Nkx2-5 sensitive genes  The 3C analysis on Tnnt2 and Atp2a2 needs more primers across the gene to convincingly show a promoter to PAS region interaction that is specifically sensitive to loss of Nkx2-5. What are the control lanes in Tnnt2 gene and the primer for 32.3 kb from the promoter at Atp2a2 gene. The results show that knockdowns of Nkx2-5, Tbx5, and Gata4 do not affect the interaction between the promoter and 14.0 kb region at Tnnt2 gene and the promoter and 32.3 kb region at Atp2a2 gene. At the controls in In 4) The manuscript’s text and figures need improved clarity and clearer annotation. pA site usage and transcription termination should be distinguished. The positions of annotated PAS should be clearly shown on all figuresTnni1 gene. We changed the description as per your kind suggestions.We put the positions of pA sites on “Our results indicate that Nkx2-5 regulates not only the initiation but also the usage of poly(A) sites during heart development.”“and defects in these factors increase the level of RNA with long 3’UTR due to the dysregulation of RNAPII termination .”5) A summary figure of interacting regions between Nkx2-5 and Xrn2 would be helpful in Thank you for the kind suggestion. We added schematic presentation of deletion mutants and summary of interacting regions between Nkx2-5 and Xrn2 in 6) We added the EtBr-staining gels and northern blots of b-actin as loading controls for the Northern blots at


Prefoldin is a cochaperone, present in all eukaryotes, that cooperates with the chaperonin CCT. It is known mainly for its functional relevance in the cytoplasmic folding of actin and tubulin monomers during cytoskeleton assembly. However, both canonical and prefoldin-like subunits of this heterohexameric complex have also been found in the nucleus, and are functionally connected with nuclear processes in yeast and metazoa. Plant prefoldin has also been detected in the nucleus and physically associated with a gene regulator. In this review, we summarize the information available on the involvement of prefoldin in nuclear phenomena, place special emphasis on gene transcription, and discuss the possibility of a global coordination between gene regulation and cytoplasmic dynamics mediated by prefoldin. A large set of molecular chaperones and auxiliary factors are involved in this phenomenon (reviewed in ). Prefola and Prefoldin is not present in Eubacteria, but it is present in Archaea , suggestfigure 1b). Recognition of actin and tubulin by prefoldin involves specific interactions between certain domains of the target proteins [Saccharomyces cerevisiae results in impaired cytoskeleton functions [in vitro [Cytoskeleton components are the best-known targets of eukaryotic prefoldin . This cafigure 1proteins  and the proteins . The so-proteins . Interesproteins . Consistunctions . These cin vitro .Caenorhabditis elegans and Drosophila results in embryonic lethality. In both cases, prefoldin-defective cells show low tubulin levels and several cytoskeleton abnormalities [mgr flies, which are mutated in the gene encoding PFDN3, exhibit circular mitotic figures and loss of meiotic spindle integrity [Arabidopsis, PFDN3, PFDN5 and PFDN6 are required for normal microtubule dynamics and organization [While none of the prefoldin subunits is essential for yeast viability, the mutation of prefoldin genes or the depletion of their products in malities ,19. In tmalities  and prevmalities . mgr flinization , whereasnization ,23.C. elegans [in vitro, which is consistent with its upregulation in a murine model for Alzheimer's disease [All these pieces of evidence have shaped the eukaryotic prefoldin concept as a highly specialized cochaperone for actin and tubulin folding. This specialization would be consistent with the more severe phenotype of CCT knockdown than the prefoldin one in  elegans . However elegans . This ro elegans . Human p disease  and the  disease .c), although no evident sequence similarity exists between canonical and non-canonical prefoldin polypeptides. The prefoldin-like complex participates in the cytoplasmic assembly of RNA polymerase II [The involvement of prefoldin subunits in the cytoplasmic assembly of some non-cytoskeletal complexes has also been well established. In this case, mammalian PFDN2 and PFDN6 form a complex together with UXT, RPB5, WDR92/Monad, PDRG1 and URI . In thisfigure 1erase II , and in erase II . In botherase II . The laterase II ). Functierase II . URI is erase II  and is ierase II .α-class prefoldin-like subunit UXT is also located in human centrosomes, associates with γ-tubulin and its overexpression disrupts the centrosome structure . This fi3.All the phenomena related to prefoldin that we have examined so far in this review take place in the cytoplasm. However, both the canonical and prefoldin-like subunits have also been found in the nucleus. Proteomic analyses of URI nuclear interactors indicate that all the components of the R2TP/prefoldin-like complex, including the canonical prefoldin subunits PFDN2 and PFDN6, are found in the nucleus of prostate cells, and shuttle between the nucleus and the cytoplasm, probably together with RNA pol II . The shoNicotiana benthamiana and Arabidopsis thaliana leaf cells [Canonical plant prefoldin, at least its subunits PFDN5 and PFDN6, is also found in the nucleus of af cells . Its nucaf cells . Human Paf cells . In turnaf cells . Localizaf cells .In addition to their cytoplasmic localization, all yeast canonical prefoldin subunits can be detected in the nucleus. Moreover, they accumulate in the nucleus when the nuclear export systems based on Xpo1 and Mex67 become genetically inactivated . So, froArabidopsis [C. elegans [Localization of prefoldin in the nucleus may be the result of the regulation of its cytoplasmic functions by means of a cytoplasm-exclusion mechanism, as has been demonstrated in bidopsis . In addi elegans . A clear elegans . PFDN3 b elegans  and medi elegans   and enhancing its transcriptional activation ,63. UXT A different prefoldin-like subunit, URI, can repress AR-mediated transcription by interacting physically with UXT in the chromatin context . URI seea). This inhibitory effect of PFDN5 on c-Myc offers a chance to modulate this transcription factor; a good example of it is the ability of the hepatitis C virus ARFP/F protein to enhance the gene transactivation activity of c-Myc by interfering in its interaction with PFDN5 [In addition to the degradation of HIV integrase, the best-characterized contribution of a prefoldin subunit to transcription is the action of PFDN5/MM-1 as a co-repressor of the E-box-dependent transactivation activity of c-Myc . The MM-figure 3th PFDN5 .Figure b). The monoubiquitination of PFDN5 by Rabring7, a Rab7-binding and RING finger-containing protein, stimulates this second role of PFDN5 in the control of c-Myc [b). In addition, PFDN5 and the Egr-1 repressor bind and downregulate the promoter of the wnt4 gene. Because the c-Myc gene is the target of the Wnt–β-catenin pathway, PFDN5 also inhibits the expression of c-Myc by this indirect mechanism [c).The negative action of PFDN5 on the c-Myc function also takes place at two other levels. It favours c-Myc degradation by recruiting the ubiquitin ligase Skp2–ElonginC–ElonginB–Cullin2 complex, and driving it to the proteasome via the 26S subunit Rpt3  figure b. The moof c-Myc  (figure echanism  (figure Saccharomyces prefoldin, however, seems to play a transcriptional role that involves at least the concerted action of PFDN1, PFDN2, PFDN5 and PFDN6 [The above-described interactions indicate a role of prefoldin subunits in the regulation of the transactivation capacity or stability of gene-specific transcription factors. With the exception of its contribution to HIV expression, the transcriptional roles of prefoldin have been described for single subunits. Although the presence of other prefoldin subunits cannot be ruled out, the mechanisms described so far do not involve the action of prefoldin complexes. nd PFDN6  (figure nd PFDN6 . In agrend PFDN6 . This prnd PFDN6 ,68. Intend PFDN6 . Moreovend PFDN6 , and depnd PFDN6 . Taken a5.To date, we have shown that the scientific literature contains a significant amount of information on the presence of canonical and prefoldin-like subunits in the nucleus, and their functional involvement in gene transcription. The examples that we cite demonstrate that, from yeast to mammals, a given prefoldin subunit can play cytoplasmic and nuclear roles in the same organism. Moreover, a set of yeast canonical subunits plays concerted actions in both cytoplasmic cytoskeleton assembly and nuclear gene transcription. This fact might merely be the result of a functional divergence during the evolutionary transition from archaea to eukaryotes. Alternatively, the cytoplasmic and nuclear functions of prefoldin might be coupled in such a way as to favour a global coordination of gene expression and cytoskeleton dynamics. This hypothetical coordination is particularly expected in those situations where cells are challenged by stimuli that require a specific genomic response and, at the same time, the reorganization of cytoplasmic organelles. Although this speculative model is far from having been demonstrated, there are some experimental hints that are compatible with the notion of prefoldin as a global cell regulator.Drosophila cells, its stabilization does not require prefoldin either, although it does when ectopic β-tubulin is expressed [The cytoskeletal function of prefoldin is not essential for the housekeeping assembly of microtubules or actin filaments. This is the reason why yeast prefoldin genes can be deleted without compromising cell viability . Similarxpressed . This inxpressed .One of the most drastic cytoplasmic reorganizations takes place during B-lymphocyte activation by antigens, when they differentiate into highly secretory plasma cells. PFDN1 knockout mice are severely affected in this process. Interestingly, the mammalian PFDN6 gene is located in the centromeric portion of the class II region of the major histocompatibility complex , and itsArabidopsis thaliana. The plant controls anisotropic cell elongation by a mechanism that finely coordinates the abundance of DELLA proteins in the nucleus with the subcellular localization of prefoldin [Arabidopsis, it is possible that prefoldin participates not only in the cytoplasmic part of this regulated process, but also in the transcriptional response.So far, the prefoldin role in lymphocyte activation has not been connected to any nuclear event, but there is one example where the cytoplasmic function of prefoldin is linked to the nucleus. This is the case of organ growth by cell expansion in the model plant refoldin . When enrefoldin . DELLAs The coordinated action in cytoskeleton dynamics and transcription would be facilitated if prefoldin acted on similar targets in these two processes. It is well known that the main cytoplasmic targets of prefoldin are actin and tubulin monomers. It is conceivable that actin, tubulin or both might also be nuclear targets of prefoldin. The transcriptional characterization of yeast prefoldin mutants, using chemical inhibitors of cytoskeleton assembly, indicates that the role of prefoldin in chromatin dynamics during transcription elongation is not mediated by actin or tubulin polymerization . Yet theThe existence of such mechanisms of coordination between cytoplasmic dynamics and genome regulation, and the potential involvement of prefoldin and other protein-folding factors in them, is a challenging field for contemporary biology.


The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. C. elegans gene sig-7 encodes a nuclear cyclophilin, a highly conserved protein family characterized by the presence of both a peptidyl isomerase domain and an RNA-recognition motif. Studies on sig-7 homologs in plants and fission yeast have shown these proteins to interact with RNA Polymerase II, and indicate they regulate the phosphorylation status of its C-terminal domain. We show that SIG-7 activity is essential for both efficient co-transcriptional splicing and normal RNA Polymerase II elongation and may provide an important link between the two processes.mRNA splicing can occur co-transcriptionally; i.e., splicing occurs as the RNA emerges from the RNA Polymerase II holoenzyme during transcription elongation. Recent studies suggest that defective splicing can cause defective transcription elongation, suggesting an interdependency of the two mechanisms. The  Transcription by RNA Polymerase II (Pol II) is a highly regulated process involving coordination of multiple processes that together modulate the level of gene expression and its temporal and spatial control –4. EpigeA connection between the modifications of the CTD and mRNA splicing has long been observed. It has been shown that the association of SR (Serine/Arginine-rich) splicing factors with the CTD requires phosphorylation of Ser2 of the heptapeptide repeat –28. It hcis-trans isomerization of prolines in the context of Ser/Thr, such as those found in the Pol II CTD heptapeptide repeats, and the activity of these PPIs is affected by the phosphorylation of Ser/Thr , KW2230: sig-7(n5037) I/hT2 [bli-4(e937) let-?(q782) qIs48], PD7271: pha-1(e2123ts) III; ccEx(pBK48.1::pC1), KW2309: sig-7(n5037)I; ckIs33II (below).Strains: wild type N2 (Bristol), KW1317: sig-7 gene prevented PCR-based cloning of the whole gene. We used a fosmid clone (construct ID: 15087717651452437 A06) containing sig-7 engineered with a 3’ GFP::3XFLAG tag obtained from the TransgeneOme project . ResuspRNAi-treated embryos were resuspended in 4X volume of RIPA buffer  and 2X volume of glass beads , and homogenized using a Mini Beadbeater-16  for 1 min 3 times at 4°C. The homogenized embryos were incubated at 4°C on a rotator for 30 min and further processed in a Bioruptor sonicator  at high setting for 10 min to fragment chromatin. The final lysates were centrifuged at 13,000g for 10 min at 4°C, and the supernatants were collected. The protein concentration was determined using the Bradford reagent . Supernatants were mixed with an equal volume of 2X SDS-PAGE sample buffer and denatured for 5 min at 95°C, and equal protein amounts were loaded and run on a 4–20% precast SDS-PAGE gel  and transferred to PVDF membrane. Transferred proteins were blocked in 5% milk PBST for 1 hour, incubated with primary antibody overnight, and washed 3 times with 1X PBST for 10 min each. After incubation with secondary antibody for 2 hours at room temperature, the blot was washed 3 times with 1X PBST for 10 min each. The washed membrane was incubated with chemiluminescence reagent  for 5 min, and protein bands were visualized with autoradiography film . The primary antibodies used are the following: anti-FLAG , anti-Actin , H14 , H5 , anti-AMA-1 , 8WG16 , anti-H3K36me3 , anti-H3K79me2 , anti-H3K4me3  and anti-H3 . The following secondary antibodies were used: Goat Anti-Rabbit IgG, HRP-conjugated , Goat Anti-Mouse IgG, Peroxidase-conjugated , AffiniPure Goat Anti-Mouse IgM, Peroxidase-conjugated .sig-7(RNAi) or ama-1(RNAi) experiments were then compared to the transcript levels of L4440 controls to generate ΔΔCT plots of relative transcript levels. The averages of two technical replicates from two biological samples were plotted with standard deviation.Embryos collected after RNAi exposure were washed with M9, and pelleted embryos were resuspended in Trizol , snap frozen in liquid nitrogen, and subjected to 3 freeze/thaw cycles. 62μl of chloroform was added and mixed thoroughly by shaking 10 times and spun down for 15 min at 4°C. Nucleic acids were precipitated with 0.3M acetic acid in 100% isopropanol and resuspended in 100μl of nuclease-free water. Total RNA was purified using RNeasy kit  as per the manufacturers’ instructions. cDNA was synthesized from 1μg of purified total RNA using iScript select cDNA synthesis kit . 50ng of cDNA was used for qPCR using SsoFast reagent  on CFX96 Real-Time system . The transcript levels of genes analyzed were first normalized to 18S RNA for each sample, and the normalized transcript levels of either Total RNAs were purified as described for qRT-PCR and sent to Axeq Asia  for transcriptome sequencing. 1μg of total RNA was used as starting material, and a sequencing library was prepared using TruSeq Stranded Total RNA Sample Prep Kit after treatment with Ribo-Zero (Human/Mouse/Rat) for rDNA depletion. Library QC was performed using Tapestation D1000 Screen Tape (Agilent) and quantified using KAPA Library Quantification Kit (for Illumina platforms). Clusters were generated by HiSeq PE (Paired-End) Cluster Kit v3 cBot, and sequencing was done on a HiSeq2000 with 100bp paired-ends using TruSeq SBS v3-HS kit reagents.C. elegans (ce10) reference genome using TopHat2 version 0.5. TopHat2 internally uses Bowtie2 to map the reads. Mapping results were used to identify splice junctions between exons. Cufflinks version 2.1.1 was used to assemble transcripts and estimate their abundance. The transcript assembly outputs from Cufflinks were merged into a unified list of transcripts using Cuffmerge. Cuffdiff version 2.0.2 was then used to quantify gene and transcript expression levels and test them for significant differences. Default parameters were utilized for all steps. This analysis was done in part by the Emory Integrated Genomics Core (EIGC), which is subsidized by the Emory University School of Medicine and is one of the Emory Integrated Core Facilities. For RNA-seq reads were quality-checked using FastQC version 0.5.2 to ensure per-base sequence quality, per sequence quality scores, per base sequence content, per base GC content, per sequence GC content, per base N content, sequence length distribution, sequence duplication levels, kmer-content, and that over-represented sequences were within accepted norms. FastQ Quality Trimmer version 1.0 was used to trim reads with less than optimal quality scores. The DE analysis protocol outlined in Trapnell et. al. was used to perform the DE analysis . The quaet al. with the following modifications [Chromatin immunoprecipitation (ChIP) was done as described in Ercan ications . 1) Wormications , 102. DNet al. or in Wang et al., and were intersected with the strict maternal gene class in Baugh et al.; any genes expressed in any of the somatic SAGE expression data of Meissner 2009 were removed [et al. or the germline-enriched set of Reinke et al. [sig-7(RNAi) [Gene set definitions used were as published in . Briefly removed , 103–105e et al. . Embryo-e et al. , 107. The et al. . MACS2 w-7(RNAi) . The broProtein sequences of SIG-7 homologues were obtained from the NCBI protein database. The alignment of homologues was generated with ClustalW2 . The conS1 Figsig-7(cc629) adults exhibit a highly penetrant protruding vulva  and lower penetrance multiple vulvae . C) Adult hermaphrodites are sterile due to failure to switch from spermatogenesis to oogenesis (masculinization of germline or Mog phenotype), as shown in a DAPI stained whole mount ovary. The dotted line and arrow shows the direction of meiotic progression. The excessive sperm accumulation is outlined. Other phenotypes observed in the cc629 allele include molting and seam cell defects and enhancement of germline tumors (not shown). D) RNAi depletion of sig-7 from hermaphrodites starting at the L3 stage resulted in ~95% embryonic lethality  among their progeny, with rare survivors exhibiting the defects described above. Scale bar = 20um.A,B) Homozygous (TIF)Click here for additional data file.S2 Figsig-7 gene with exons (red boxes) and introns (black lines), the position of the GFP::3XFLAG tag, and the genomic region used in the rescuing transgene construct (dotted line with arrowheads). B) The n5037 deletion and cc629 splice acceptor mutant alleles are illustrated. C) The domain structure of SIG-7 is illustrated; all three domains are also found in SIG-7 orthologs listed in (D). No homolog has been identified in budding yeast.A) The CEOP1492 operon (blue), the (TIF)Click here for additional data file.S3 FigProtein sequences were aligned using ClustalW2 (110). The degree of conservation is noted with different symbols in the bottom row of each alignment. The cyclophilin-type peptidyl-prolyl cis-trans isomerase domain (PPI) and RNA Recognition Motif (RRM) were identified using the ScanProsite web-based tool and are indicated with black solid lines , 112. Th(TIF)Click here for additional data file.S4 Figsig-7(n5037) allele was rescued with a SIG-7::GFP::3XFLAG fusion protein. The GFP expression patterns in live animals are shown. A-D) SIG-7::GFP::3XFLAG is nuclear and observed in all tissues and at all stages, including early embryos (A), late embryos (B), adult somatic cells , and germ cells . Small speckles in A,C,D are auto-fluorescent gut granules. The protein is associated with DNA in all germ cells except in transcriptionally inactive diakinesis-stage oocytes, where it becomes dispersed in the nucleoplasm (insets in D). The protein is also associated with DNA in male germ cells at all stages, with faint nuclear signal observed in mature sperm (not shown).The (TIF)Click here for additional data file.S5 FigLysates from embryos expressing SIG-7::GFP::3XFLAG were divided into equal aliquots and incubated with antibodies specific for RNA Pol II , GFP (Lane 4), and the FLAG epitope (Lane 5), and the immunoprecipitates were analyzed by western blots probed with anti-Pol II Ser-5P , anti-FLAG  and anti-Actin (bottom). The lysate in Lane 2 was incubated with 100ug of RNase A for 30min at RT prior to the IP procedure.(TIF)Click here for additional data file.S6 FigThe abundance of germline-specific transcripts was measured by qRT-PCR in RNAi embryos  and adults Click here for additional data file.S7 FigEmbryos collected from RNAi experiments were fixed and stained with DAPI. The approximate numbers of nuclei in the embryos in a field were binned as indicated. (TIF)Click here for additional data file.S8 Figsig-7(RNAi) embryos , 607 and 205 genes, respectively, were among those classified by Baugh et al. as Strictly maternal, Strictly Embryonic, or Maternal/Embryonic [Of the 1522 down-regulated genes and 389 upregulated in mbryonic . The per(TIF)Click here for additional data file.S9 Figsig-7(RNAi) reads, which are reduced compared to L4440 controls, is scaled to the exon reads in L4440. An “outron” is the RNA segment removed by trans-splicing of nascent transcript; its sequence corresponds to that between the TSS (Transcription Start Site) and SL1 (Splice Leader 1 acceptor site) of trans-spliced genes. Exons (blue boxes) and introns (black solid lines) are shown under each RNA-seq profile. The relative levels of introns and outrons (indicated with asterisks) compared to exons are significantly higher in sig-7(RNAi) compared to L4440 control, reflecting persistence of primary transcripts. B) Average log2 read coverage per gene for exons and introns in sig-7(RNAi) vs L4440 is shown. Genes up- and downregulated in sig-7(RNAi) compared to L4440 by cuffdiff analysis are shown in red and green, respectively. Exon levels change in the manner expected for mis-regulated genes, while intron levels remain relatively unchanged.A) Examples of zygotically expressed genes with splicing defects. The number of aligned reads generated by Tophat are indicated on the left. The y-axis of the (TIF)Click here for additional data file.S10 Figsig-7(RNAi) embryos, and the read density profiles were compared with the RNA-seq results from the experiments described in sig-7(RNAi) are associated with a transcription defect.Genome-wide RNA Pol II (anti-AMA-1 antibody) ChIP-seq was performed in L4440 control RNAi and (TIF)Click here for additional data file.S11 Figsig-7(RNAi) embryos relative to control L4440 embryos were classified and compared as in sig-7(RNAi) embryos. B) Genes expressed in the germline show an increase in RNA Pol II occupancy in sig-7(RNAi) embryos.Genes with RNA Pol II occupancy changes in (TIF)Click here for additional data file.S12 Figsig-7(RNAi) (red). A) Genes were categorized as in Figs Metagene displays of RNA-seq reads, plotted from 1kb upstream and downstream of the annotated Transcription Start Sites (TSS) and Transcription End Sites , for different classes of genes in L4440 RNAi control (blue) and (TIF)Click here for additional data file.S1 Tablesig-7(RNAi) in RNA reads corresponding to C. elegans homologs of known splicing factors is shown.The log2 fold change after (TIFF)Click here for additional data file.S2 Table(XLS)Click here for additional data file.


The phenotypic properties of embryonic and adult stem cells are largely mediated by epigenetic modifications which control stemness, multipotentiality and fate specification. In this study, our objective was to identify the epigenetic mechanism regulating the growth and differentiation of human cardiac stem cells (hCSCs).Discarded surgical specimens were enzymatically digested and c-kit-positive hCSCs were sorted by FACS.Genome-wide analysis of histone modifications documented that adult hCSCs are characterized by a bivalent chromatin configuration similar to that of embryonic stem cells with repressive and activating marks at lysine residues of histones H3 and H4. The high levels of di- (H3K4me2) and tri-methylation (H3K4me3) at lysine 4 of histone H3 prompted us to test the function of histone methyltransferase Smyd3, which is downregulated by the non-functional mutation in the c-kit receptor, in hCSCs. Inhibition of Smyd3 by siRNA strategy decreased the fraction (60%) of cycling hCSCs and increased apoptosis (2.5-fold). Attenuation of hCSC growth was coupled with a decline in the expression of telomerase, hTERT, and the myocyte specific transcription factors hNkx2.5 and hGata6. By immunolabeling, Smyd3 co-localized with hTERT, hNkx2.5 and hGATA6 in hCSC nuclei. Conversely, transfection of hCSCs with a plasmid carrying Smyd3 was coupled with upregulation of hTERT and hNkx2.5. Chromatin immunoprecipitation assay show that Smyd3 binds to hTERT, hGata6 and hNkx2.5 promoter regions that contains the putative binding sites for Smyd3. Moreover, the mutations at the Smyd3 DNA binding sites, decreased the promoter activity of hTERT and hGata6. Smyd3 siRNA treatment decreased the reporter activity of luciferase reporter plasmids containing hTERT.Our results show that Smyd3 represents a crucial epigenetic modulator of hCSC commitment to the myocyte lineage.


GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks  on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the  Drug response varies between individuals and can be caused by genetic factors. Nucleotide variation in gene regulatory elements can have a significant effect on drug response, but due to the difficulty in identifying these elements, they remain understudied. Here, we used various genomic assays to analyze human liver cells treated with or without the antibiotic rifampin and identified drug-induced regulatory elements genome-wide. The testing of numerous active promoters in human liver cells showed only a few to be induced by rifampin treatment. A similar analysis of enhancers found several of them to be induced by the drug. Nucleotide variants in one of these enhancers were found to alter its activity. Combined, this work identifies numerous novel gene regulatory elements that can be activated due to drug response and thus provides candidate sequences in the human genome where nucleotide variation can lead to differences in drug response. It also provides a universally applicable method to detect these elements for other drugs. Adverse reactions to drug treatment constitute a substantial health problem that is a leading cause of morbidity and mortality in hospitalized patients The vast majority of pharmaceutical compounds are metabolized by the cytochrome P450 family (CYP) of enzymes. Of these, CYP3A4 is the most abundantly expressed in sites of drug disposition in the liver NR1I2), which is expressed predominantly in the liver and intestine in vivo, although PXR response elements have been identified in the putative CYP3A4 promoter cis-regulatory elements in vitro. Additional PXR responsive enhancers have been found for other CYPs CYP3A4 is one of many targets of the nuclear receptor PXR  and ChIP-seq with antibodies for PXR and three different enhancer marks on primary human hepatocytes treated with rifampin or vehicle control. These included the E1A binding protein p300 (EP300/p300) which has been used to identify functional enhancers in vitro and in vivo enzyme and transporter induction studies during drug development (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm292362.pdf). Ten genes were induced greater than two-fold  and only one was downregulated (CYP26B1). CYP3A4 was by far the most differentially expressed gene with a >1,000 fold induction (>10 PCR cycles difference), followed by the other members of the CYP3A family  . The resCYP3A4, similar to our qPCR results. Of the eleven differentially expressed genes identified by qPCR, seven (64%) were also found to be differentially expressed by RNA-seq. It is worth noting that two  of the four genes that didn't replicate in the RNA-seq data, showed a non-statistically significant induction by rifampin in our RNA-seq.Our RNA-seq analyses found several differentially expressed genes, the majority of which are known to be involved in drug response. The number of differentially expressed genes using a p-value cutoff, after adjustment for multiple testing less than or equal to 0.05, was 157 . AmongstWe observed a massive recruitment of PXR binding across the genome following rifampin treatment. PXR-bound DNA fragments clustered into 1,158 discrete peaks with DMSO treated cells versus 6,302 after treatment with rifampin , with onThe binding of p300 was also more extensive after rifampin treatment, with 13,811 peaks compared to 10,253 in DMSO-treated cells . There wIn contrast to PXR and p300, the distribution of histone marks was relatively stable, with about 49,000 enriched islands of H3K4me1 activity and about 40,000 H3K27ac islands in both treatments . We alsoWe next looked at overlaps between the different ChIP-seq peaks. Amongst the 6,302 PXR rifampin treated peaks 1,037 (16%) overlapped p300 and around half overlapped histone marks . This was similar for PXR peaks in the DMSO treated cells . For p30ACTB), a strong constitutive promoter that should not be induced by rifampin and 2) The CYP3A4 proximal promoter, which is known to be induced by rifampin. We tested the 227 promoters in HepG2 cell lines co-transfected with human PXR and treated with rifampin or vehicle control (DMSO). Out of the 227 tested promoters, 179 were found to be functional promoters (>2 fold luciferase activity above empty vector) in the DMSO treated cells  in a similar assay, only this time without co-transfecting human PXR, and found only 2 of them to be induced by rifampin (>2 fold increase in promoter activity upon rifampin treatment) and at much lower levels  CYP3A4 (CLEM4) CYP3A7, was additionally identified intergenically between CYP3A7 and CYP3A4CYP3A4 locus is one of the few in which we observed a substantial difference in rifampin-induced enrichment of the H3K4me1 and H3K27ac marks.−9; binominal fold enrichment), originating from Pathway Commons (http://www.pathwaycommons.org), was ‘xenobiotics’  gene ontology enrichment terms for drug catabolic processes and other terms fitting with drug response (www.qiagen.com/ingenuity) and found enrichment for the PXR/RXR Activation Canonical pathway  Forty-two RIRs residing near drug-associated genes as manually determined by the literature. 2) Seven rifampin-induced PXR ChIP-seq peaks harboring SNPs that are in linkage disequilibrium (LD) with pharmacogenomics or drug related genome-wide association study (GWAS) lead SNPs, termed GWAS linked peaks (GLPs) . PreviouApoE liver enhancer CYP3A4 promoter-enhancer combination (p3A4-362(7836/7208ins) Candidate enhancer sequences were cloned into the pGL4.23 (Promega) enhancer assay vector, which contains a minimal promoter followed by the luciferase reporter gene. Since the peaks (or islands) enriched for the two histone marks are relatively long  showed significant reporter expression levels versus the empty vector (≥2 two fold) in either condition: 15 RIRs and 4 GLPs . Among t2C locus  and contCYP3A7 XREM (GSTA) locus near GSTA2    and was 2C locus , which hart site . UGT1A eWe determined common haplotypes in all five sequences using the phased 1000 Genomes data . Common By carrying out RNA-seq and ChIP-seq on rifampin and DMSO treated human hepatocytes, we have uncovered numerous drug-dependent regulatory elements. We observed that promoters bearing a rifampin-dependent signature were largely unable to independently induce the expression of a reporter upon rifampin treatment. This result suggested that other gene regulatory elements, such as enhancers, could constitute the predominant group of target sequences that are activated by this drug. An analysis of nucleotide variation in these enhancers showed that specific variants could affect enhancer activity, raising the possibility that nucleotide variants in enhancers could contribute to pharmacogenomic phenotypes more broadly.CYP3A4, fitting with its role as the most abundantly expressed gene in sites of drug disposition in the liver Our RNA-seq analyses that combined eight different individuals identified 157 differentially expressed genes, using a p-value cutoff that adjusted for multiple testing less than or equal to 0.05 . Many ofCYP3A locus, which was radically altered by rifampin treatment. For the most part, the regulatory hotspots that we identified in this region are consistent with established literature. It is worth noting, however, that we saw almost equal recruitment of PXR and p300 to the XREMs that putatively regulate CYP3A4 and CYP3A7, despite the fact that we observed substantially less CYP3A7 mRNA induction by qPCR and RNA-seq.Similar studies have been conducted to detect environmentally induced regulatory elements CYP3A4 locus). The fact that we observed more rifampin-induced peaks for H3K27ac is consistent with its hypothesized role in marking active enhancers. Combined, these results suggest that while these two marks are incredibly stable in the face of massive changes in PXR/p300 binding, there are still measurable drug-induced changes in chromatin structure.We did not observe large changes in the assayed histone marks, H3K4me1 and H3K27ac. This could be attributed to these sites being poised to be activated by various xenobiotic responses, though H3K27ac has been previously shown to mark active enhancers in vitro in PXR-transfected HepG2 cells, both our negative and positive controls showed the expected results , have been shown to have important roles in catalyzing carcinogenic substrates and nucleotide variation in them has been shown to be associated with cancer We identified several functional enhancers that were rifampin-increased or rifampin-induced whose location was near pharmacogenomic-associated variants. One of these elements is RIR46, which resides near GSTA2, a Phase II enzyme involved in the detoxification of numerous drugs It is worth noting that there are several caveats to our study. Our ChIP-seq experiments only analyzed hepatocytes from a single donor at a single time point (24 hours post treatment), selected for being commonplace in rifampin induction studies. It is possible that there are enhancers that play a role much earlier than 24 hours post-treatment. To test regulatory sequences in reporter assays, it was also necessary to clone smaller fragments from each RIR bearing the PXR and p300 peaks. It is possible that we missed flanking sequences that were essential for enhancer function. Furthermore, because we had a limited amount of primary hepatocytes from the same donor, our reporter assays were carried out in PXR-transfected HepG2 cells, which could result in false negatives. Finally, we employed the pGL4.23 vector, which is a commonly used enhancer assay vector, but possesses a very short TATA-box containing minimal promoter that may not be compatible with all of the enhancer sequences tested.Promoter regions upstream of drug metabolizing enzymes and transporters are presumed to be the major targets of xenobiotic activators of PXR, such as rifampin. Our work challenges this conception and strongly supports the idea that the direct targets could be enhancer elements, which may subsequently interact with promoters to enhance gene transcription. Combined, our results show that ChIP-seq in combination with drug treatment has a large potential in identifying novel regions in the genome associated with drug response. These regions can provide exceptional candidates for the detection of nucleotide variants associated with inter-individual differences in drug response. Our methodology could easily be adapted to other drugs/target/tissue combinations. With whole genome datasets becoming more widely used as a clinical toolbox, the ability to highlight these important drug response regions in the genome is of extreme importance.Cryopreserved human hepatocytes from a 19 year old Caucasian male donor with no history of medications  were thawed in CHRM recovery media  and cultured in CHPM plating media  on 6-well collagen-coated plates (Life Technologies). After 6 hours, the media was swapped with maintenance media, consisting of phenol-free Williams E media containing culture incubation supplements  and 0.01% DMSO or 10 µM rifampin (Sigma) for 24 hours. The rifampin dose (10 µM for 24 hours) was used based on previously reported assays achieving high induction rates in human hepatocytes 2 First Strand Kit (Qiagen). Gene expression levels for 84 genes of interest was determined using the “Drug Metabolism: Phase I Enzymes” RT2 Profiler PCR Array (Qiagen). Five housekeeping genes  were used to control for loading. Fold induction was calculated using the ΔΔCt method Cultured hepatocytes were treated with DMSO or rifampin for 24 hours in triplicate. The cells were then washed with PBS, and lysed directly with Buffer RLT from the RNAeasy mini kit (Qiagen) with the on-column DNase digestion step. One µg of total RNA was used to generate cDNA using the RTTotal RNA was acquired as described for qPCR for two replicates each of DMSO and rifampin treated hepatocytes. Libraries were made with ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicenter). Briefly, 3–5 ug of total RNA were subjected to rRNA removal using Ribo-Zero Magnetic Kit (Epicenter) prior to library construction. 5 ng of the rRNA-depleted sample was fragmented enzymatically and annealed with random hexamer to create the first strand of cDNA. Upon removal of the RNA template transcript by RNase, Terminal-Tagging Oligo (TTO), a known 5′-sequence tag, a 3′-random sequence, and a terminally blocked 3′ end to prevent priming of DNA synthesis, was added to create cDNA with known sequence tags at their 5′ and 3′ ends for directionality. Upon purification, adaptors with barcodes were added to cDNA fragments and enriched by 15 cycles of PCR. Sequencing was carried out on an Illumina HiSeq. The resulting reads were demultiplexed and aligned to the human genome (hg19) using TopHat v2.0.10 Twelve million cells (an entire 6 well plate) per immunoprecipitation were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. The remainder of the ChIP-seq protocol was carried out by Active Motif Inc., as follows. Chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300–500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield.An aliquot of chromatin (30 ug) was pre-cleared with protein G agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 ug of antibody against PXR , 4 ug of antibody against p300 , 20 ug of antibody against H3K27ac  and 5 ug antibody against H3K4me1 . Due to the limited amount of cryopreserved human hepatocytes from this donor, we elected to do various enhancer-associated ChIP-seq marks instead of additional replicates. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.ChIP and input DNAs were prepared for amplification by converting overhangs into phosphorylated blunt ends and adding an adenine to the 3′ ends. Illumina adaptors were added and the library was size-selected (175–225 bp) on an agarose gel. The adaptor-ligated libraries were amplified for 18 cycles. The resulting amplified DNAs were purified, quantified, and tested by qPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. Amplified DNA libraries were sequenced on the Illumina Genome Analyzer II.The 36-nt sequence reads identified by the Sequencing Service were mapped to the genome using the ELAND (Illumina). Default settings were used: only tags that map uniquely, have no more than 2 mismatches, and that pass quality control filtering were considered.−7 was used for MACS and an FDR of 0.01% was used as a cutoff for SICER, and the remaining parameters set to the default. For the alternate analysis presented in −10.PXR and p300 peaks were called against input using MACS version 1.4 http://matplotlib.org) to plot.For the analysis of the distribution of PXR ChIP-seq peaks following rifampin treatment, we used knownGene from the UCSC Genome Browser to define the transcription start site (TSS) as the center and pybedtools 5′-GCTCGCTAGCCTCGAG-3′ and 5′CGCCGAGGCCAGATCT-3′) complementary to the sequence flanking the BglII and XhoI sites in the pGL4.23 vector (Promega). Primers were designed to encompass the PXR/p300 ChIP-seq peak plus up to 500 bp of sequence on either side of the peak. PCR products were cleaned using the QIAquick PCR Purification Kit (Qiagen) and cloned into BglII and XhoI digested pGL4.23 using the Infusion HD cloning system (Clontech). Haplotypes were generated either by PCR amplification of DNA from various ethnic individuals or by site-directed mutagenesis using mutant primers amplified by PfuUltra High Fidelity DNA polymerase (Agilent) followed by DpnI digestion of the parental DNA (New England Biolabs) and transformation into competent cells. Sequences were verified by Sanger sequencing using RVPrimer3 (5′-CTAGCAAAATAGfGCTGTCCC-3′) and a custom reverse primer (5′-TCTTCCATGGTGGCTTTACC-3′).Selected promoters were obtained from the LightSwitch Promoter Collection (SwitchGear Genomics) and sequence verified in the pLightSwitch_Prom vector. Candidate enhancers were amplified from human genomic DNA (Roche) using oligonucleotides designed in Primer3 with 16 bp overhangs , we identified a larger set of SNPs in linkage disequilibrium with those SNPs (r2>0.8) in the race/ethnicity corresponding to the pharmacogenetics study. Ten rifampin-induced PXR peaks overlapped at least one of these GWAS-LD SNPs  vector), 10 ng of pGL4.74, 21.5 ul Opti-MEM, 100 ng of reporter construct, and 0.48 ul of Lipofectamine LTX reagent (Life Technologies). After 18 hours of incubation at 37 degrees Celsius, the cells were washed with 150 ul Opti-MEM and the media was replaced with 100 ul of Opti-MEM supplemented with 10 uM Rifampin or 0.1% DMSO. After 24 hours of incubation, the cells were washed with PBS and the promoter assay cells were placed at −80 degrees Celsius for 30 minutes to lyse and the enhancer assayed cells were lysed with 25 ul of Passive Lysis Buffer (Promega). All LightSwitch promoter vectors were measured with the LightSwitch Luciferase Assay reagents (SwitchGear Genomics) following the manufacturers protocol. For enhancers, reporter activity was measured using the Dual-Luciferase Reporter Assay System (Promega). Both assays were measured on a microplate luminometer (Promega).HepG2 cells (ATCC) were maintained in D-MEM (Life Technologies) supplemented with FBS (JRS Scientific), Penicillin-Streptomycin and Glutamine (UCSF Cell Culture Facility). On the day of transfection, the cells were trypsinized, washed, and diluted to a density of 1 million cells/ml in Opti-MEM I Reduced Serum Media (Life Technologies). Ninety thousand cells and 125,000 cells were added to each well of a 96-well clear bottom black tissue culture plate (Greiner Bio-One) containing the transfection mixture for the promoter and enhancer assay respectively. The transfection mixture consisted of 50 ng of human PXR .Figure S1Distribution of PXR ChIP-seq peaks following rifampin treatment. (A) All genes. (B) Differentially expressed genes only.(PDF)Click here for additional data file.Figure S2CYP2A6* represents the CYP2A13 gene which is upregulated.Ingenuity Pathway Analysis of genes near rifampin induced regions (RIRs) shows genes involved in “PXR/RXR Activation”. Upregulated genes are colored in red and downregulated genes are colored in green as determined through either qPCR or RNA-seq. Fold differences are shown as a log2 ratio below each one of the differentially expressed genes. (PDF)Click here for additional data file.Figure S3CYP2C locus showing both the RNA-seq and ChIP-seq results for DMSO (blue) and rifampin (red) treated hepatocytes. GLP1 and GLP2 are depicted by green lines above the CYP2C genes. (B) Common SNPs in GLP1 and GLP2.GLP1 and GLP2. (A)(TIF)Click here for additional data file.Figure S4GSTA locus showing both the RNA-seq and ChIP-seq results for DMSO (blue) and rifampin (red) treated hepatocytes. The cloned RIR46 fragment is depicted by a black rectangle upstream to GSTA2.(TIF)Click here for additional data file.Figure S5UGT1A locus showing both the RNA-seq and ChIP-seq results for DMSO (blue) and rifampin (red) treated hepatocytes. GLP5 is depicted by a green line above the UGT1A genes. (B) The location of common SNPs within GLP5.GLP5. (A)(TIF)Click here for additional data file.Table S1qPCR results for 84 genes known to be involved in drug response and RNA-seq results following vehicle or rifampin treatment.(XLSX)Click here for additional data file.Table S2ChIP-seq summary.(XLSX)Click here for additional data file.Table S3Promoter assay results.(XLSX)Click here for additional data file.Table S4GREAT analysis of rifampin induced regions (RIRs).(XLSX)Click here for additional data file.Table S5Ingenuity Pathway Analysis of genes near rifampin induced regions (RIRs).(XLS)Click here for additional data file.Table S6GWAS linked peaks (GLPs).(XLSX)Click here for additional data file.Table S7Enhancer assay results.(XLSX)Click here for additional data file.Table S8The various common haplotypes selected for differential enhancer assays, haplotype enhancer assays results and ANOVA analyses.(XLSX)Click here for additional data file.Table S9H3K4me1 and H3K27ac islands identified by alternate analysis in which the rifampin-treated sample was compared directly to the DMSO treatment as reference.(XLSX)Click here for additional data file.