The long non-coding RNA Paupar regulates the expression of both local and distal genes
2014; Springer Nature; Volume: 33; Issue: 4 Linguagem: Inglês
10.1002/embj.201386225
ISSN1460-2075
AutoresKeith W. Vance, Stephen N. Sansom, Stephen Lee, Vladislava Chalei, Lesheng Kong, Sarah Cooper, Peter L. Oliver, Chris P. Ponting,
Tópico(s)RNA modifications and cancer
ResumoArticle1 February 2014Open Access The long non-coding RNA Paupar regulates the expression of both local and distal genes Keith W Vance Corresponding Author Keith W Vance MRC Functional Genomics Unit, University of Oxford, Oxford, UK Search for more papers by this author Stephen N Sansom Stephen N Sansom CGAT, University of Oxford, Oxford, UK Search for more papers by this author Sheena Lee Sheena Lee Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Vladislava Chalei Vladislava Chalei MRC Functional Genomics Unit, University of Oxford, Oxford, UK Search for more papers by this author Lesheng Kong Lesheng Kong MRC Functional Genomics Unit, University of Oxford, Oxford, UK Search for more papers by this author Sarah E Cooper Sarah E Cooper Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Peter L Oliver Peter L Oliver MRC Functional Genomics Unit, University of Oxford, Oxford, UK Search for more papers by this author Chris P Ponting Corresponding Author Chris P Ponting MRC Functional Genomics Unit, University of Oxford, Oxford, UK CGAT, University of Oxford, Oxford, UK Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Keith W Vance Corresponding Author Keith W Vance MRC Functional Genomics Unit, University of Oxford, Oxford, UK Search for more papers by this author Stephen N Sansom Stephen N Sansom CGAT, University of Oxford, Oxford, UK Search for more papers by this author Sheena Lee Sheena Lee Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Vladislava Chalei Vladislava Chalei MRC Functional Genomics Unit, University of Oxford, Oxford, UK Search for more papers by this author Lesheng Kong Lesheng Kong MRC Functional Genomics Unit, University of Oxford, Oxford, UK Search for more papers by this author Sarah E Cooper Sarah E Cooper Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Peter L Oliver Peter L Oliver MRC Functional Genomics Unit, University of Oxford, Oxford, UK Search for more papers by this author Chris P Ponting Corresponding Author Chris P Ponting MRC Functional Genomics Unit, University of Oxford, Oxford, UK CGAT, University of Oxford, Oxford, UK Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Author Information Keith W Vance 1,‡, Stephen N Sansom2,‡, Sheena Lee3, Vladislava Chalei1, Lesheng Kong1, Sarah E Cooper4, Peter L Oliver1 and Chris P Ponting 1,2,3 1MRC Functional Genomics Unit, University of Oxford, Oxford, UK 2CGAT, University of Oxford, Oxford, UK 3Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK 4Department of Biochemistry, University of Oxford, Oxford, UK ‡These authors contributed equally to this work. *Corresponding author. Tel: +44 1865 282554; Fax: +44 1865 282849; E-mail: [email protected] *Corresponding author. Tel: +44 1865 282690; Fax: +44 1865 285862; E-mail: [email protected] The EMBO Journal (2014)33:296-311https://doi.org/10.1002/embj.201386225 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Although some long noncoding RNAs (lncRNAs) have been shown to regulate gene expression in cis, it remains unclear whether lncRNAs can directly regulate transcription in trans by interacting with chromatin genome-wide independently of their sites of synthesis. Here, we describe the genomically local and more distal functions of Paupar, a vertebrate-conserved and central nervous system-expressed lncRNA transcribed from a locus upstream of the gene encoding the PAX6 transcription factor. Knockdown of Paupar disrupts the normal cell cycle profile of neuroblastoma cells and induces neural differentiation. Paupar acts in a transcript-dependent manner both locally, to regulate Pax6, as well as distally by binding and regulating genes on multiple chromosomes, in part through physical association with PAX6 protein. Paupar binding sites are enriched near promoters and can function as transcriptional regulatory elements whose activity is modulated by Paupar transcript levels. Our findings demonstrate that a lncRNA can function in trans at transcriptional regulatory elements distinct from its site of synthesis to control large-scale transcriptional programmes. Synopsis Paupar is a central nervous system-expressed lncRNA that regulates the balance between proliferation and neuronal differentiation. Paupar controls target gene expression by binding regulatory elements near promoters, thus offering the first example of lncRNA-mediated transcriptional regulation in trans. The conserved long non-coding RNA Paupar is a novel regulator of neural growth and differentiation. Paupar functions in trans to regulate the expression of genes important for neural stem cell fate. Paupar binds and modulates the activity of multiple transcriptional enhancer and repressor elements located across different chromosomes. Paupar and PAX6 directly interact and co-occupy a subset of Paupar binding sites. Introduction The resolution of two key questions would greatly improve our understanding of the functions of long noncoding RNAs (lncRNAs; ≥200 nucleotides). First, is it more often the RNA product or else the act of transcription that conveys lncRNA function? Second, is any given lncRNA more likely to control transcription locally (in the vicinity of its locus) or else more distally in the genome? A number of lncRNAs have been shown to regulate transcription of neighbouring genes on the same chromosome in an apparent cis-acting mechanism (Lai et al, 2013; Melo et al, 2013; Wang et al, 2011). These lncRNAs appear to function near their site of synthesis, in either an RNA-dependent manner to mediate looping onto the promoter regions of their transcriptional targets, or by using RNA-independent mechanisms to locally alter chromatin status. By contrast, lncRNA transcripts have also been proposed to regulate gene expression in trans, without influencing transcription of their genomically neighbouring genes (Guttman et al, 2011; Hung et al, 2011). Trans-acting lncRNAs include p53-induced lncRNAs involved in mediating the DNA damage response (Huarte et al, 2010; Hung et al, 2011), lncRNAs transcribed from within the promoters of cell cycle genes (Hung et al, 2011), lncRNAs that function in the control of pluripotency and lineage differentiation (Guttman et al, 2011) and those that are regulators of dosage compensation (Chu et al, 2011; Simon et al, 2011). Other examples include Evf-2 which binds and modulates the activity of the homeodomain containing transcription factor Dlx2 (Feng et al, 2006), and Hotair, a lncRNA transcribed from the HoxC locus, which regulates the activity of HoxD cluster genes in trans and interacts with chromatin at over 800 regions genome-wide (Chu et al, 2011; Rinn et al, 2007). LncRNAs therefore have the potential to interact with chromatin and specifically target multiple different loci genome-wide. LncRNA loci that are transcribed in the developing mouse central nervous system (CNS) show a preference to be located adjacent to transcription factor genes and thus may regulate their transcription (Ponjavic et al, 2009). Here, we investigate the transcriptional function of a CNS expressed, unspliced, and chromatin-associated intergenic lncRNA termed Paupar that is divergently transcribed 8.5 kb upstream of Pax6. Paupar was prioritised for detailed experimental investigation from among those we catalogued previously (Ponjavic et al, 2009) owing to the atypical evolutionary conservation of its sequence and transcription and because of its physical proximity to the transcription factor Pax6. Pax6 is required for eye and diencephalon specification and is known to control progenitor cell potency, progenitor cell proliferation, neuronal cell sub-type specification and spatial patterning in a dosage-sensitive manner (Georgala et al, 2011; Hill et al, 1991; Sansom et al, 2009; Shaham et al, 2012). Heterozygous human PAX6 mutations can result in aniridia and in a variety of structural brain abnormalities that closely resemble those seen in Small eye (sey) mice heterozygous for Pax6 mutations (Georgala et al, 2011; Hingorani et al, 2012). The proximity of the Paupar gene to Pax6 suggested to us that it may be involved in the spatiotemporal control of Pax6 expression and hence that it may be important for nervous system development and neurological disease. Our results demonstrate functions for Paupar in the control of growth and differentiation in neural cells. In addition to conveying these functions locally, via transcriptional regulation of Pax6, we unexpectedly discovered that Paupar also functions distally in trans to control neural gene expression on a large scale. We mapped genome-wide Paupar occupancy in N2A neuroblastoma cells and identified hundreds of genes that are both bound and regulated by Paupar. We discovered that the Paupar transcript physically associates with PAX6 protein and that Paupar and PAX6 co-occupy specific genomic binding sites. Our results also revealed that Paupar associates in trans with functional elements involved in transcriptional control and that the Paupar transcript can modulate these elements’ activity. Our data therefore demonstrate that a single lncRNA transcript can bind and regulate the activity of multiple transcriptional regulatory elements on different chromosomes distinct from its site of synthesis. Results Conserved Paupar genomic organisation and transcription RNA polymerase II-transcribed, CNS-expressed mouse lncRNAs tend to be evolutionarily constrained and to be preferentially located adjacent to transcriptional regulatory genes in the genome (Ponjavic et al, 2009, 2007). One of these lncRNAs (GenBank: AK032637), which we term Paupar (Pax6 Upstream Antisense RNA), is a single exon lncRNA transcribed from 8.5 kb upstream of the Pax6 gene in mouse which lies entirely within the first intron of Pax6os1, a previously defined non-coding Pax6 natural antisense transcript locus (Fig 1A; Alfano et al, 2005). Rapid amplification of cDNA ends (RACE) experiments in mouse neuroblastoma cells extended AK032637 by approximately 700 bp at the 5′ end revealing mouse Paupar to be a 3.48 kb transcript (Fig 1B). The Paupar locus contains two regions of high DNA sequence conservation across diverse vertebrates that unusually include fish and birds as well as mammals (Fig 1B). The first of these regions lies just 5′ upstream of the Paupar transcriptional start site and is likely to contain this transcript's promoter sequence. The second lies within the transcribed sequence and encompasses both a previously identified Pax6 neuroretina enhancer element (Plaza et al, 1999) and a region of the transcript that we predicted to contain a stem loop secondary structure (Ponjavic et al, 2009). The orthologous human transcript, transcribed from 8.6 kb upstream of the human PAX6 gene, was identified in foetal brain using RT-PCR and RACE and shows three regions of high sequence identity to its mouse orthologue (Fig 1C). Paupar transcripts are known from dog, as well as from more distantly related vertebrates, frog and zebrafish (Fig 1C). Paupar therefore is unusual in exhibiting higher degrees of sequence and transcriptional conservation than most lncRNA loci (Cabili et al, 2011; Marques & Ponting, 2009; Ulitsky et al, 2011). Figure 1. Conservation and expression of Paupar A. Schematic illustration of the mouse Pax6 genomic territory displaying coding and non-coding transcript structures (NCBI37/mm9). B. A detailed view of the mouse Paupar locus (red) indicating regions of vertebrate DNA sequence conservation and the location of sequence (blue) that, in human and quail, is a Pax6 neuroretina enhancer (Plaza et al, 1999). C. Conservation and relative sizes of identified Paupar transcripts in vertebrates. For human and mouse Paupar, transcript start sites (arrows) and transcript ends were confirmed by RACE (primer sequences in Supplementary Table 1). The identified orthologous ESTs from dog (DN871729), frog (CX414799, DN054151 and DN054152), and zebrafish (CT684153 and CT684154) are unlikely to represent full-length transcripts. Each of these Paupar orthologues displays conserved genomic location and transcriptional orientation relative to Pax6. D, E. Paupar is a brain-expressed lncRNA. Paupar (D) and Pax6 (E) expression levels were measured across a panel of adult mouse tissues using quantitative RT-PCR (qRT-PCR). Results are presented relative to the average value of Gapdh and Tbp reference genes. Mean values ± standard error (s.e.) shown, n = 3 replicates. F, G. Similarly to Pax6, Paupar is up-regulated during neuronal differentiation of mouse ES cells. Neuronal differentiation of mouse ES cells was induced using RA. We determined the levels of Paupar (F) and Pax6 (G) using qRT-PCR. Results are expressed relative to an Idh1 control which does not change significantly during differentiation. Mean ± s.e., n = 3. H, I. Paupar is a chromatin-associated transcript that functions to regulate Pax6 expression. N2A cells were biochemically separated into cytoplasmic, nucleoplasm, 420 mM salt and chromatin fractions. The relative levels of Paupar (H) and a control mRNA (Tbp) (I) in each fraction were determined by qRT-PCR. Mean values ± s.e. of three independent experiments. RT, reverse transcriptase. Download figure Download PowerPoint Paupar transcript is chromatin associated and co-expressed with Pax6 in the neural lineages To begin our investigation of Paupar function we first characterised its expression profile and sub-cellular localisation. We found that mouse Paupar is most highly expressed in the adult brain (Fig 1D) and shows a clear correspondence in expression profile with Pax6 (Fig 1E). Notably, Paupar is expressed in the developing cerebellum in both the internal and external granular layers, where Pax6 is also strongly expressed (Supplementary Fig S1A). Given the apparent spatial co-expression of Paupar and Pax6, we then asked whether their expression is temporally coordinated during retinoic acid (RA)-induced differentiation of mouse E14 embryonic stem (ES) cells. While Paupar expression is undetectable in self-renewing ES cells, it is rapidly and transiently up-regulated after 1 day of RA treatment before increasing again at 4 days (Fig 1F), a profile similar to that observed for Pax6 (Fig 1G). Mouse neuro 2A (N2A) neuroblastoma cells express both Paupar (at an average level of approximately 15 copies per cell [Supplementary Fig S1B]) and Pax6, but not Pax6OS1 (Supplementary Fig S1C), and are widely used as an in vitro model of neuronal differentiation. In these cells, we found Paupar RNA (Fig 1H), but not a control mRNA (Tbp; Fig 1I), to be nuclear-enriched and located mainly in the chromatin fraction, and noted that ENCODE data show human Paupar to be similarly present in the nucleus and chromatin (ENCODE Project Consortium, 2012). Together, these data suggest that Paupar may act locally to regulate Pax6 expression or that it may regulate similar biological processes as Pax6. Paupar regulates neural gene expression To investigate the functional importance of the Paupar transcript we performed transcriptome profiling of Paupar knockdown in N2A cells. We reduced Paupar expression by approximately 52%, using transient transfection of a Paupar-targeting shRNA expression vector (Fig 2A), and verified that the chromatin-associated fraction of Paupar is depleted using this approach (Supplementary Fig S2A). Paupar knockdown resulted in statistically significant changes in the expression levels of 942 genes (False Discovery Rate [FDR] < 5%) compared to a non-targeting control (Supplementary Table 2); 654 (69%) of these genes were down-regulated and 288 (31%) were up-regulated (Fig 2B). Figure 2. Paupar functions to regulate genes involved in cell cycle control and synaptic function A. N2A cells were transfected with either a non-targeting control or a Paupar-targeting shRNA expression vector (sh408) and Paupar levels were determined by qRT-PCR 3 days later. B. Paupar knockdown induces statistically significant changes in the expression of 942 genes in N2A cells (5% FDR; Supplementary Table 2). C. Significant Gene Ontology annotation enrichments of Paupar-regulated genes (5% FDR, Supplementary Table 3). D. Paupar is important for normal S-phase progression and entry into mitosis. Mouse N2A cells were transfected with either a control or a Paupar-targeting shRNA expression vector. Three days later cells were fixed, stained with propidium iodide and the DNA content measured using flow cytometry. E. Paupar loss-of-function cell lines were generated by stable co-transfection of shRNA expression plasmids against either Paupar or a non-targeting control and a hygromycin expression vector for selection. qRT-PCR analysis confirms the generation of two clonal cell lines expressing reduced levels of Paupar. Mean values ± s.e. F. Paupar knockdown cells display increased neurite outgrowth. Control and Paupar knockdown cells were imaged using bright field microscopy. Scale bar, 50 μm. G. Quantification of neurite outgrowth. Cells with one or more neurites of length greater than twice the cell body diameter were scored as positive. Average values ± s.e., n = 3. A total of 100–200 cells were counted in each case. H, I. The relative levels of the neuronal differentiation marker Tubb3 (H) and Pax6 (I) were quantified in Paupar knockdown and control cells using qRT-PCR. Samples were normalised using Gapdh and are presented relative to expression in control cells (set arbitrarily to 1). Mean values ± s.e., n = 3. Download figure Download PowerPoint Paupar-regulated genes are significantly enriched in those involved in cell cycle control, specifically DNA replication and mitosis, those playing a role in synaptic function, and those modifying chromatin and chromosome organisation (Fig 2C, Supplementary Table 3). To validate the changes in expression observed from the microarrays, we performed qRT-PCR for 12 Paupar-regulated genes with two additional Paupar-targeting shRNA expression constructs (Supplementary Fig S2B). We observed consistent changes for all 12 genes and saw changes in expression commensurate with the strength of Paupar knockdown indicating that transcript level changes are specific and are not likely to result from off-target effects. Furthermore, Paupar overexpression induced dose-dependent changes in the expression of six out of eleven Paupar-regulated genes tested (Supplementary Fig S2C). The Paupar transcript therefore appears to function as a large-scale regulator of gene expression in neural cells. Paupar regulates neural growth and differentiation in N2A cells We next investigated the role of Paupar in cell cycle control by assaying the effect of Paupar knock-down on the proliferation of N2A cells. Paupar knockdown cells accumulate in S and G2 phases (Fig 2D) indicating that this transcript is necessary for normal passage through S phase and entry into mitosis. Taken together with the temporally regulated expression of Paupar during neural differentiation, these data indicate that Paupar may be important for the control of neural progenitor cell proliferation and differentiation. To further investigate this hypothesis we generated stable Paupar loss-of-function N2A cell lines and analysed the role of the Paupar transcript in neural differentiation. We isolated and expanded two independent clones in which Paupar levels had been reduced by 50–60% (Fig 2E). Strikingly, the Paupar knockdown clones showed a clear increase in neurite outgrowth, a well-characterised feature of neuronal differentiation, compared to a non-targeting control line (Fig 2F and G). Additionally, the Paupar knockdown clones also showed increased levels of the neuronal differentiation marker Tubb3 (encoding tubulin beta-3 chain) and a moderate reduction in Pax6 which is known to be down-regulated upon neuronal differentiation (Fig 2H and I). Together, these results indicate that Paupar regulates gene expression programmes that control neural growth and differentiation, acting to maintain the self-renewal of N2A cells. Paupar and Pax6 have both common and distinct transcriptional targets Given the known roles of Pax6 in controlling neural stem cell fate, we next sought to further investigate the effect of Paupar RNA reduction on the expression of Pax6. While we observed a small decrease in Pax6 transcript levels following stable Paupar knockdown (Fig 2I), this finding cannot be interpreted unambiguously given that neural progenitor cells can down-regulate Pax6 as they become neurogenic (Hsieh & Yang, 2009) and that Pax6 is known to auto-regulate its own expression (Aota et al, 2003; Manuel et al, 2007). We therefore reduced Paupar levels with two distinct shRNAs transfected into N2A cells (Fig 3A) and used qRT-PCR to measure acutely induced changes in Pax6 expression. Transient reduction in Paupar RNA levels up-regulated Pax6 in a dose-dependent manner: a maximum 54% reduction in Paupar levels resulted in an 80% increase in Pax6 expression (Fig 3A). These observations agreed with a small (1.2-fold), yet genome-wide non-significant, increase in Pax6 expression detected on the Paupar knock-down microarrays. Figure 3. Paupar has both Pax6-dependent and -independent functions in transcriptional regulation Paupar knockdown leads to an increase in Pax6 expression. N2A cells were transfected with two independent shRNA expression constructs targeting different regions of the Paupar transcript. The levels of Paupar and the adjacent Pax6 gene were quantified using qRT-PCR 3 days later. Samples were normalised using Gapdh and the results are presented relative to a non-targeting control (set at 1). Mean values ± s.e., n = 4, one-tailed t-test, unequal variance. Cells were transfected with either a non-targeting control or a Pax6-targeting shRNA expression vector and PAX6 protein levels were analysed by Western blotting 3 days later. Lamin B1 expression was used as a loading control. Pax6 knockdown resulted in statistically significant changes in the expression of 925 genes in N2A cells (10% FDR, Supplementary Table 2). Intersection of Pax6 and Paupar targets reveals a significant (Fisher's exact test) overlap approximately five times as large as expected by chance alone. Target genes for both Paupar and Pax6 show correlated expression, with the majority being positively regulated by both factors. +, positive dependency; −, negative dependency. Enrichments of Gene Ontology categories in Pax6 and Paupar target genes. Download figure Download PowerPoint Given the ability of Paupar to regulate Pax6 expression, we sought to determine the extent to which this could explain the Paupar transcriptional response. Reduction of PAX6 protein levels in N2A cells by approximately 70%, through the transient transfection of a Pax6-targeting shRNA expression vector (Fig 3B), resulted in statistically significant expression level changes in 925 genes (FDR < 10%; Fig 3C and Supplementary Table 2). Importantly, we noted no change in the levels of Paupar transcript upon Pax6 knockdown (Supplementary Fig S3). Genes changing in expression, as expected from the role of PAX6 as a key neuro-developmental transcription factor, were enriched for genes involved in neurogenesis and transcription factor binding (Fig 3F, Supplementary Table 3). The set of genes showing significant expression changes in both Paupar and Pax6 knock-downs was 5.1-fold greater than expected by chance (P < 2.2 × 10−16; Fig 3D), consistent with Paupar regulating Pax6 expression. A large majority of these genes showed positively correlated changes in expression for both Paupar and Pax6 knock-down (Fig 3E) indicating that while Paupar may repress Pax6 transcription, Paupar RNA and PAX6 protein cooperate to coordinate the expression of a common set of target genes. However, notwithstanding the significant overlap between genes regulated by Pax6 and Paupar, a large majority of Paupar responsive genes are not significantly altered by Pax6 knockdown suggesting that Paupar may also possess Pax6-independent trans-acting functions. Notably, genes regulated by Paupar but not by Pax6 are enriched for regulators of cell cycle control and chromatin organisation, while genes whose expression are controlled by both Paupar and Pax6 include many with synaptic functions (Fig 3F, Supplementary Table 3). Genome-wide binding profile of the Paupar lncRNA in N2A cells We next investigated whether Paupar might function as a trans-acting transcriptional regulator by binding to genomic locations distal to its own locus. We first mapped the genome-wide binding profile of Paupar using the recently described Capture Hybridisation Analysis of RNA Targets (CHART)-Seq technique (Simon, 2013; Simon et al, 2011) in N2A cells. This approach uses anti-sense oligonucleotides to purify target lncRNAs and their associated chromatin complexes and thus identifies both direct and indirect genomic associations. We mapped accessible regions of the Paupar transcript based on RNase H sensitivity and designed four biotinylated DNA oligonucleotides, complementary to these regions (Supplementary Fig S4). For a control oligonucleotide, we used a sequence corresponding to Escherichia coli LacZ which is absent from the mouse genome. Paupar probes showed strong enrichment (17-fold) of the Paupar transcript compared to the LacZ control (Fig 4A), and did not enrich for negative control transcripts, Malat1, a nuclear lncRNA, or Gapdh mRNA. As expected from physical association of a nascent transcript with its site of synthesis, we observed specific enrichment of Paupar at its DNA locus (Supplementary Fig S4). Figure 4. CHART-Seq analysis of Paupar genomic binding sites A. Specific enrichment of Paupar RNA using oligonucleotides complementary to accessible regions of Paupar, as determined by RNase H mapping (see Supplementary Fig S4), compared to the LacZ control. Mean value ± s.e., n = 4. B. Sequencing of Paupar-bound DNA (RNase H elution) reveals peaks of Paupar binding, including those at the promoter of E2f2, upstream of Sox2 and downstream of Hes1. C–E. Peaks were called by comparing with sequences both from control CHART-seq experiments and from input DNA. Here we only consider the 2,849 peaks common to both comparisons (C, and Supplementary Table 4). Paupar peaks are broadly distributed across the mouse genome (D) but are particularly enriched in 5′ UTRs and gene promoters (E). Red arrowheads in (D) indicate the position of the Paupar locus. Asterisks in (E) indicate significance at 5% FDR (Benjamini-Hochberg). F. The width distribution of Paupar binding peaks. G. Representative categories from Gene Ontology analysis of genes associated with Paupar binding sites reveal enrichments for stem cell and neuronal categories amongst others (Supplementary Table 6). Download figure Download PowerPoint Following the CHART-seq protocol, we used RNase H elution to recover genomic DNA associated with endogenous Paupar transcripts and genomic DNA associated with the control oligonucleotide, sequencing replicate samples using the Illumina HiSeq system. Using the paired-end peak caller MACS2 (Zhang et al, 2008), we identified Paupar binding sites in comparison both to DNA recovered using the control LacZ oligonucleotide and to input DNA from N2A cells. We discovered thousands of peaks across the genome, for example at the transcriptional start site (TSS) of E2f2, and at sites upstream of Sox2 and downstream of Hes1 (Fig 4B) and defined Paupar binding sites as those peaks found in comparison to both input DNA and the control oligonucleotide samples (Fig 4C). Paupar occupancy at nine candidate binding sites was validated using CHART-qPCR in two further independent experiments (Supplementary Fig S4). These 2,849 candidate Paupar binding sites (Supplementary Table 4) are generally widely distributed across the genome, show a significant three-fold depletion on the X chromosome (P < 0.001 by genome-wide simulation accounting for mappability and GC biases (Heger et al (2013); Supplementary Table 5; Fig 4D), and occur preferentially within the promoters and 5′ UTRs of protein-coding genes (Fig 4E). Candidate Paupar binding sites range from narrower focal peaks of 200–1,000 bp, similar to those previously described for Hotair and Terc lncRNAs (Chu et al, 2011), to broader genomic regions of up to 9.5 kb (Fig 4F). We examined the sequence of the Paupar binding locations for clues as to its genomic targeting. Using a local alignment approach (see Materials and Methods), we did not find evidence for sequence complementarity between Paupar and its binding locations. However, de novo motif discovery (Supplementary Fig S5) identified a motif closely resembling a known PAX6 DNA binding motif in 9.2% of the top 500 scoring Paupar binding locations (Supplementary Fig S5C). Further analysis of Paupar CHART-Seq peaks for the presence of known vertebrate transcription factor motifs revealed enrichment of motifs for several neural transcription factors (Supplementary Table 7). Together, these results suggest that Paupar is not targeted to the genome through direct RNA-DNA interactions but that instead it interacts wi
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