The long non-coding RNA Paupar regulates the expression of both local and distal genes

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.

Full text

Article
The long non-coding RNA Paupar regulates the
expression of both local and distal genes
Keith W Vance
1,†,*
, Stephen N Sansom
2,†
, Sheena Lee
3
, Vladislava Chalei
1
, Lesheng Kong
1
,
Sarah E Cooper
4
, Peter L Oliver
1
& Chris P Ponting
1,2,3,**
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
,aswellasdistally
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 tran-
script 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.
Keywords CHART; CNS; lncRNA; PAX6; transcription
Subject Categories RNA Biology; Transcription
DOI 10.1002/embj.201386225 | Received 17 July 2013 | Revised 21 November
2013 | Accepted 22 November 2013
Introduction
The resolution of two key questions would greatly improve our
understanding of the functions of long noncoding RNAs (lncR-
NAs; ≥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 tran-
scription factor Dlx2 (Feng et al, 2006), and Hotair, a lncRNA tran-
scribed 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 tran-
scription 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
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
*Corresponding author. Tel: +44 1865 282554; Fax: +44 1865 282849; E-mail: Keith.Vance@dpag.ox.ac.uk
**Corresponding author. Tel: +44 1865 282690; Fax: +44 1865 285862; E-mail: Chris.Ponting@dpag.ox.ac.uk
†
These authors contributed equally to this work.
ª2014 The Authors This is an open access article under the terms of the Creative Commons Attribution License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
The EMBO Journal 1

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 associ-
ates 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 transcrip-
tional 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).
Paupar transcript is chromatin associated and co-expressed with
Pax
6
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 expres-
sion 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 [Sup-
plementary Fig S1B]) and Pax6, but not Pax6OS1 (Supplementary Fig
S1C), and are widely used as an in vitro model of neuronal differentia-
tion. 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 bio-
logical 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 knock-
down 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).
Paupar-regulated genes are significantly enriched in those
involved in cell cycle control, specifically DNA replication and mito-
sis, 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. Further-
more, Paupar overexpression induced dose-dependent changes in the
expression of six out of eleven Paupar-regulated genes tested (Supple-
mentary Fig S2C). The Paupar transcript therefore appears to function
as a large-scale regulator of gene expression in neural cells.
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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 differ-
entiation, 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 differen-
tiation, acting to maintain the self-renewal of N2A cells.
Paupar and Pax
6
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 knock-
down (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.
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 trans-
fection 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). Impor-
tantly, 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 regu-
lating 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 cooper-
ate 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 respon-
sive 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 chroma-
tin 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
Figure 1. Conservation and expression of Paupar.
A Schematic illustration of the mouse Pax6genomic 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 Pax6neuroretina 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=3replicates.
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 Idh1control which does not change significantly during
differentiation. Mean s.e., n=3.
H, I Paupar is a chromatin-associated transcript that functions to regulate Pax6expression. 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.
▸
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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
Figure 2.Paupar functions to regulate genes involved in cell cycle control and synaptic function.
AN2A 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.
BPaupar 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).
DPaupar 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.
EPaupar 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.
FPaupar knockdown cells display increased neurite outgrowth. Control and Paupar knockdown cells were imaged using bright field microscopy. Scale bar, 50 lm.
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.
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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).
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 oligo-
nucleotide, 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 gen-
ome, 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 experi-
ments (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
Figure 3.Paupar has both Pax6-dependent and -independent functions in transcriptional regulation.
APaupar knockdown leads to an increase in Pax6expression. N2A cells were transfected with two independent shRNA expression constructs targeting different regions
of the Paupar transcript. The levels of Paupar and the adjacent Pax6gene were quantified using qRT-PCR 3days 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.
B Cells were transfected with either a non-targeting control or a Pax6-targeting shRNA expression vector and PAX6protein levels were analysed by Western blotting
3days later. Lamin B1expression was used as a loading control.
CPax6knockdown resulted in statistically significant changes in the expression of 925 genes in N2A cells (10% FDR, Supplementary Table 2).
D Intersection of Pax6and Paupar targets reveals a significant (Fisher’s exact test) overlap approximately five times as large as expected by chance alone.
E Target genes for both Paupar and Pax6show correlated expression, with the majority being positively regulated by both factors. +, positive dependency; , negative
dependency.
F Enrichments of Gene Ontology categories in Pax6and Paupar target genes.
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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 Sox2and 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).
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focal peaks of 200–1000 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 loca-
tions. 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 transcrip-
tion 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 with PAX6 and
other neural transcription factors to target specific genomic regions
in a context-dependent manner.
Paupar binding sites are associated with genes and regulatory
regions of known stem cell and neural function
Using Gene Ontology assignments (Ashburner et al, 2000), we next
sought to characterise the set of genes associated with Paupar
binding locations (Fig 4G, Supplementary Table 6). This analysis
revealed enriched stem cell categories with peaks associated with
many key stem cell genes such as Sox2,Nanog,Hes1,Hes5,Rbpj
and Lif. We also found Paupar peaks to be enriched for neuronal
gene categories as well as in genes whose products are important
for the epigenetic regulation of gene expression. Intriguingly,
Paupar binding sites are also enriched in genes associated with
insulin secretion, a process in which Pax6 has an established role
(Gosmain et al, 2012).
To gain insight into the mechanism by which Paupar regulates
gene expression, we examined its binding in relation to known
functional regulatory regions. Firstly, we found that the majority of
Paupar binding sites overlap DNase I hypersensitivity (HS) sites and
cis-regulatory elements identified by the Mouse ENCODE Project
(Shen et al, 2012; Fig 5A, Supplementary Table 4). A large subset of
binding sites are significantly associated with neuronal DNase I HS
sites and a further subset are associated with DNase I HS sites found
in embryonic stem cells (Fig 5B and 5C). In agreement with the
enrichment of Paupar binding sites at 5′UTRs and promoters, we
also observed a set of binding sites associated with features charac-
teristic of transcriptional start sites including Pol II, predicted CpG
islands, and tri-methylation of histone H3 at lysine 4 (H3K4me3;
Fig 5D). Additionally, we saw significant overlaps with Ctcf-binding
regulatory elements and tissue-specific enhancers, defined using
ratios of H3K4 mono- and tri-methylation (Shen et al, 2012).
A subset of Paupar-regulated genes is also associated with
Paupar binding
To investigate the functional consequences of Paupar binding we
intersected the Paupar CHART-Seq peak-set with our microarray
analysis of Paupar and Pax6-mediated gene expression changes. This
identified 242 Paupar-bound and -regulated genes (5% FDR), repre-
senting likely direct transcriptional targets, and 254 Paupar-bound
and Pax6-regulated genes (10% FDR; Fig 5E). Hierarchical cluster-
ing of the 242 Paupar-bound and -regulated genes indicated that
while Paupar predominantly activates the expression of target
genes it can also function to repress certain loci (Fig 5F). In accor-
dance with this, genes associated with Paupar binding sites
showed significantly higher expression in N2A cells than other
genes (Supplementary Fig S6A, P<2×10
16
).
Notably, genes regulated by both Paupar and Pax6 showed a
small but significant enrichment for associated Paupar binding sites
(Supplementary Fig S6B). To dissect the relationship between gene
expression changes in the Paupar and Pax6 knockdown experiments
and the association of genes with Paupar binding further, we plotted
these observations (Fig 5G). First, we noted a moderate significant
correlation (0.20, P<2.2 ×10
16
) between all genes tested for
differential expression in the two knockdown experiments, and that
this correlation was stronger for genes also associated with Paupar
binding sites (0.31, P<2.2 ×10
16
). Secondly, we noted a distinct
group of genes that are significantly positively regulated by both
Paupar (5% FDR) and Pax6 (10% FDR) and associated with Paupar
binding (red circles, lower left Fig 5G). In line with the observed
intersection between genes regulated by Paupar and Pax6 (Fig 3),
these findings suggest that Paupar and Pax6 co-operate to regulate a
set of common target genes (red circles, Fig 5G) and further that
Paupar can act independently of Pax6 to directly regulate the
expression of a separate set of genes (red triangles).
Paupar and PAX6co-occupy specific genomic binding sites
The identification of 71 Paupar and Pax6 co-regulated genes that are
also bound by Paupar in their regulatory regions (Fig 5E) together
with the discovery of the known PAX6 DNA binding motif from
Paupar bound sequences (Supplementary Fig S5) suggested a
functional interaction between PAX6 and Paupar. To investigate
this, we first tested for a physical association between PAX6 protein
and the Paupar transcript using UV cross-linking RNA immuno-
precipitation (UV-RIP) in N2A cells and detected a fourfold enrich-
ment of Paupar using an anti-PAX6 antibody compared to an
isotype control (Fig 6A). The Paupar-PAX6 interaction appears to be
specific as we observed no enrichment of a negative control U1
snRNA transcript.
We next measured PAX6 occupancy at a set of Paupar binding
sites within the regulatory regions of genes whose expression changes
in Paupar knockdown cells (Fig 6B). Using chromatin immunopre-
cipitation (ChIP), we found a strong enrichment of PAX6 at Paupar
binding sites within the regulatory regions of genes whose expression
changes significantly upon both Pax6 and Paupar knockdown. By
contrast, the majority of the assayed Paupar binding locations associ-
ated with genes that changed in expression upon Paupar but not Pax6
knockdown showed little enrichment for PAX6 (Fig 6B).
Several lncRNAs titrate DNA binding transcription factors away
from their genomic targets (Hung et al, 2011; Rapicavoli et al, 2013;
Sun et al, 2013). We therefore examined PAX6 occupancy at a
number of Paupar binding sites following Paupar knockdown and
discovered that Paupar depletion does not significantly affect PAX6
chromatin occupancy at the regions tested (Supplementary Fig S7).
In a similar manner, the androgen receptor (AR)-associated
lncRNAs Prncr1 and Pcgem1 do not affect AR DNA binding but
instead function to recruit transcriptional co-factors to DNA bound
The EMBO Journal Paupar,atrans-acting lncRNA Keith W Vance et al
The EMBO Journal ª2014 The Authors
8

Figure 5. Association of Paupar binding sites with regulatory regions.
A The majority of Paupar binding sites overlap with DNase I HS sites and transcriptional regulatory elements as identified by the ENCODE project (selected tissues;
Supplementary Table 5).
B Hierarchical clustering of HS sites intersecting Paupar binding sites (light yellow) reveals large clusters of HS sites identified in neural tissues and embryonic stem
cells that are bound by Paupar in N2A cells.
C Fold enrichment of Paupar CHART-Seq-DNase I HS site associations.
D Hierarchical clustering of cis-regulatory elements and Paupar peaks show groups of Paupar peaks associated with promoter-like features (H3K4me3, Pol II binding,
CpG island predictions), peaks associated with Ctcf and peaks associated with tissue-specific enhancer elements.
E The intersection of genes proximal (<1 Mb) to Paupar peaks and genes changing expression upon Paupar (5% FDR) and Pax6 (10% FDR) knockdown.
F Heatmap displaying expression changes in the 242 Paupar bound and regulated genes from (E).
G Analysis of Paupar-associated genes and changes in expression from Paupar and Pax6knockdown array experiments reveals a set of genes positively regulated by
both Paupar and Pax6and directly bound by Paupar (red circles, lower left). Correlations (cor) are significant at P<2×10
16
.
Data information: Asterisks (C, D) indicate significance at 5% FDR (Benjamini-Hochberg).
Keith W Vance et al Paupar,atrans-acting lncRNA The EMBO Journal
ª2014 The Authors The EMBO Journal 9

AR (Yang et al, 2013). Taken together, these findings indicate that
PAX6 and Paupar specifically co-occupy a subset of Paupar binding
sites associated with genes whose expression change upon Pax6 and
Paupar knockdown, and that PAX6 protein plays a role in targeting
Paupar to the genome.
Paupar modulates in trans the activity of transcriptional
regulatory elements of neuro-developmental genes
We next tested whether Paupar CHART-Seq peaks function as tran-
scriptional regulatory elements. To do this, we first selected 11
Paupar binding sites, ranging from 170 bp to 2.5 kb in length,
located within the regulatory regions of genes controlling neural
growth and differentiation (E2f2,E2f7 [2 peaks], Cdc6,Cdkn2c,
Kdm7a [2 peaks], Sox1,Sox2,Hoxa1,Hes1) and cloned each upstream
of a heterologous SV40 promoter in a pGL3 luciferase vector. All of
these genes, except Sox2 whose expression is undetectable in N2A
cells, showed evidence of being regulated by Paupar using microarray
profiling. The transcriptional activity of these constructs was then
compared to that of the SV40 promoter alone following transient
transfection in N2A cells. One (E2f2BS) out of six focal peaks of
<600 bp in length strongly activated (3.9-fold) the SV40 promoter, a
further three displayed a small, but significant (1.3–1.7-fold), increase
in SV40 promoter activity (Fig 6C), while four out of five broad peaks
>1 kbp (Fig 6D) reproducibly repressed the SV40 promoter (1.6–1.8-
fold). Consistent with the observed enrichment of Paupar
Figure 6.Paupar functions in trans to modulate the activity of neurodevelopmental gene transcriptional regulatory elements.
APaupar interacts with PAX6 protein in N2A cells. Nuclear extracts were prepared from UV cross-linked cells and immuno-precip itated using either anti-PAX6 or
control IgG antibodies. Associated RNAs were purified and the levels of Paupar and control U1 snRNA detected in each UV-RIP using qRT-PCR. Results are
expressed as fold enrichment relative to an isotype IgG control antibody.
B PAX6 and Paupar co-occupy a specific set of genomic binding sites. ChIP assays were performed in N2A cells using either an antibody against PAX6 or an isotype-
specific control. The indicated DNA fragments were amplified using qPCR. Fold enrichment was calculated as 2
DDCt
(IP/IgG).
C, D Paupar binding sites act as transcriptional regulatory elements. N2A cells were transfected with the indicated reporter constructs in a luciferase assay. Luciferase
activity was compared to that of the empty SV40 promoter construct.
E–GPaupar transcript modulates the transcriptional activity of its binding sites in trans. Luciferase reporters were co-transfected into N2A cells together with either a
non-targeting control or two independent Paupar targeting shRNA expression vectors. Paupar depletion was confirmed using qRT-PCR. For these reporter assays, a
Renilla expression vector was used as a transfection control and the total amount of DNA transfected in each case was made equal.
Data information: Results are presented as mean values s.e., n=3(A–D) or n=4(E–G); ***P<0.001, **P<0.01, *P<0.05, one-tailed t-test, unequal variance.
The EMBO Journal Paupar,atrans-acting lncRNA Keith W Vance et al
The EMBO Journal ª2014 The Authors
10

CHART-Seq peaks at neuronal DNase I HS sites and cis-regulatory
elements (Fig 5) these results indicate that Paupar binding sites
function in transcriptional control and can operate as both transcrip-
tional enhancers and repressors.
Finally, we examined whether the Paupar transcript plays a
trans-acting role in modulating the transcriptional response of its
genomic binding sites. For this we chose to investigate the
dependence on the Paupar transcript of the transcriptional activity
of the strong E2f2 enhancer element as well as the weak E2f7 acti-
vating region and the repressor elements associated with the Sox2,
Hes1 and Hoxa1 genes. To achieve this, reporters were co-transfected
into N2A cells along with either a non-targeting control or two inde-
pendent Paupar-targeting shRNA expression vectors in a luciferase
assay. Strikingly, depletion of the endogenous Paupar transcript led
to a dose-dependent increase in the enhancer activity of the
SV40-E2f2BS reporter (Fig 6E and F). In accordance with this, E2f2
displayed one of the greatest expression changes (1.9-fold up-regulation;
Supplementary Table 2) in our transcriptome profiling of Paupar
knockdown cells. The activity of the SV40-E2f7BS reporter was not
altered by Paupar loss of function in this assay. Furthermore,
Paupar depletion suppressed the ability of both the SV40-Sox2BS
and SV40-Hes1BS reporters to function as transcriptional repressors,
in a manner dependent on the levels of the Paupar transcript, while
the repressive function of the SV40-Hoxa1BS reporter was not
altered (Fig 6G). These findings demonstrate a transcriptional regu-
latory function for the Paupar transcript at its genomic binding sites
in trans and reveal that a lncRNA can function at transcriptional
regulatory regions on different chromosomes distinct from its site of
synthesis to control target gene expression.
Discussion
The extent by which lncRNAs contribute to genome function
remains unclear. Here, we have used shRNA mediated knockdown,
microarray profiling, genome-wide mapping and reporter assays to
detail the RNA-dependent mode of action of Paupar, a chromatin-
associated CNS expressed lncRNA transcript, in the regulation of
gene transcription. Our findings that the Paupar transcript acts both
locally, to regulate Pax6 expression, and distally in a transcript- and
Pax6-dependent manner, reveal that lncRNAs can be functionally
complex and will not always fall exclusively into cis-ortrans-acting
categories. Our data indicate that the Paupar transcriptional
response is driven by at least three contributing components: down-
stream gene expression changes arising from Paupar regulation of
Pax6 expression; Pax6- and Paupar-dependent trans-acting gene
expression changes likely mediated through a physical interaction
between Paupar and PAX6 protein at Paupar associated loci; and
trans-acting Pax6-independent Paupar functions at many bound
transcriptional regulatory regions genome-wide.
Consistent with a role for Paupar in regulating Pax6 expression,
the Paupar locus has characteristics of a transcriptional enhancer. It
spans both a previously defined Pax6 neuroretina specific enhancer
conserved between human and quail (Plaza et al, 1999) and a
known N2A cell DNase I HS site (McBride et al, 2011). Furthermore,
ENCODE data indicate its locus to be marked by a high ratio of
histone H3K4me1 compared to H3K4me3, with high levels of
H3K27me3 and an absence of H3K27ac in the mouse E14.5 brain.
Different classes of enhancer-associated cis-acting ncRNAs have
been described to date. Enhancer RNAs (eRNAs) are relatively short
polymerase II-transcribed, predominantly non-polyadenylated,
bidirectional transcripts, first identified at neuronal enhancers (Kim
et al, 2010) while enhancer-like lncRNAs are strand-specific, poly-
adenylated transcripts (De Santa et al, 2010; Orom et al, 2010)
which in these respects are more similar to Paupar. The chromatin
signature of the Paupar locus, along with the finding that Paupar
regulates Pax6 expression, implies that Paupar may play an
important role in nervous system development. Furthermore, the
widespread effect on over 900 genes when Paupar transcript levels
were reduced by 52% may be associated with the haploinsufficiency
and dosage-sensitivity of Pax6 (Georgala et al, 2011).
We show that Paupar associates with approximately 2800 sites
in the genome and, where tested, these regions operate as transcrip-
tional regulatory elements whose activity can be modulated by
Paupar transcript levels. Our data are consistent with a model in
which Paupar is indirectly targeted to the genome through RNA-
protein interactions with multiple different neural transcription
factors including PAX6. In accordance with this, we discovered a
motif resembling a known PAX6 DNA binding motif within approxi-
mately 9% of the 500 top-scoring Paupar bound sequences and
showed that Paupar and PAX6 co-occupy specific genomic sites
within the regulatory regions of genes whose expression change
upon both Pax6 and Paupar knockdown. Furthermore, our data
show that Paupar does not affect PAX6 chromatin occupancy and
suggest that Paupar may regulate the association of PAX6 with its
transcriptional cofactors to control target gene expression.
It is likely that other nuclear enriched lncRNAs operate in a simi-
lar manner to Paupar. The CTBP1-AS lncRNA has recently been
demonstrated to possess cis- as well as trans-acting functions
(Takayama et al, 2013) while Prncr1 and Pcgem1 have been shown
to bind the AR and associate with androgen responsive enhancers
genome-wide (Yang et al, 2013). Hotair and Terc occupy hundreds
of short genomic regions of up to 1 kb in length across multiple
chromosomes while Drosophila roX2 interacts with Chromosome
Entry Sites on the X-chromosome (Chu et al, 2011; Simon et al,
2011), regions that can recruit the dosage compensation machinery
when inserted into autosomes (Fagegaltier & Baker, 2004). Human
Alu RNA, transcribed from short interspersed elements, binds poly-
merase II and therefore has the potential to function as a general
transcriptional repressor (Mariner et al, 2008) while the lateral
mesoderm specific lncRNA Fendrr interacts in vitro with Foxf1
promoter fragments in trans (Grote et al, 2013). We therefore
propose that Paupar is a member of a class of nuclear enriched
lncRNAs that can interact with multiple transcriptional enhancers
and silencers to regulate gene expression in trans in a transcript-
dependent manner.
LncRNAs that play important roles in the development and func-
tion of the nervous system may be dysregulated in neurological dis-
orders. We have shown that Paupar loss of function disrupts the
normal cell cycle profile of N2A neuroblastoma cells and induces
neuronal differentiation. Furthermore, its transcript binds and mod-
ulates the activity of Sox2,Hes1 and E2f2 gene transcriptional regu-
latory elements in trans, in addition to regulating the expression of
Paupar’s adjacent Pax6 gene. Although the roles of Sox2 and Hes1
in neural progenitor cell maintenance and neurogenesis are well
characterised (Pevny & Nicolis, 2010; Kageyama et al, 2008), the
Keith W Vance et al Paupar,atrans-acting lncRNA The EMBO Journal
ª2014 The Authors The EMBO Journal 11

function of the E2f2 gene in neural lineages has been less well
studied. We found that E2f2, and the related E2f7 and E2f8 genes,
are up-regulated upon Paupar knockdown in our profiling experi-
ments and that the Paupar transcript operates to restrict the activity
of an E2f2 enhancer element. E2f2 functions as a pro-survival factor
during retinal development (Chen et al, 2009) and is involved in
maintaining the differentiated state of post-mitotic neurons in cells
in culture (Persengiev et al, 2001). Furthermore, E2f2 over-expres-
sion promotes cell cycle arrest and inhibits S-phase progression in
PC12 neurons (Persengiev et al, 2001). Up-regulation of E2f family
members in Paupar knockdown N2A cells may thus contribute to
the accumulation of cells in S and G2 phases of the cell cycle and
the observed neural differentiation phenotype.
The mechanisms of action of a growing number of cis-acting
lncRNAs have been studied and have revealed roles for both the
RNA molecule and lncRNA transcription in regulating the expres-
sion of genomically neighbouring genes (Lai et al, 2013; Melo
et al, 2013; Wang et al, 2011). By way of contrast, we have
uncovered the mode of action of a lncRNA molecule that is able
to regulate the expression of genomically local as well as distal
genes and suggest that lncRNAs which modulate cellular functions
via genome-wide targets may be more widespread than previously
anticipated.
Materials and Methods
Plasmid construction
To generate shRNA expression plasmids we first used the White-
head Institute siRNA selection program to design shRNAs targeting
multiple regions of Paupar and Pax6. Selected sequences were
filtered to eliminate off-target effects by performing a BLAST search
of the NCBI RefSeq database and removing hits with >15 matched
bases of the anti-sense strand. Double stranded DNA oligonucleo-
tides containing sense-loop-antisense targeting sequences were then
cloned downstream of the U6 promoter in pBS-U6-CMVeGFP
(Sarker et al, 2005) by linker ligation. To generate the Paupar
expression plasmid the full length Paupar transcript was PCR ampli-
fied as a XhoI fragment from mouse N2A cell cDNA and inserted
into pCAGGS. Paupar CHART-Seq peak regions were PCR cloned
from N2A genomic DNA and inserted upstream of the SV40 pro-
moter in pGL3-Pro (Promega) to generate a panel of luciferase
reporters to test for transcriptional regulatory activity. The
sequences of all oligonucleotides used for cloning are shown in
Supplementary Table 1.
Cell culture
N2A mouse neuroblastoma cells were grown in DMEM supple-
mented with 10% fetal bovine serum. E14 mouse ES cells were
grown on 0.1% gelatin-coated dishes in DMEM supplemented with
15% fetal calf serum, Leukemia-Inhibitor factor, 1×non-essential
amino acids, 2 mM L-glutamine, 50 mg/ml penicillin/streptomycin
and 50 lM 2-mercaptoethanol. To induce neuronal differentiation
ES cells were seeded onto gelatinised plates and grown in differ-
entiation medium (DMEM supplemented with 10% fetal calf
serum, 1×non-essential amino acids, 2 mM L-glutamine, 50 mg/ml
penicillin/streptomycin, 50 lM 2-mercaptoethanol and 10
7
M all-
trans retinoic acid).
Transcriptomic analysis
Total RNA was isolated using the Qiagen Mini RNeasy kit following
the manufacturer’s instructions and RNA integrity assessed on a
BioAnalyzer (Agilent Technologies). 200 ng RNA was used to gener-
ate labelled sense single stranded DNA (ssDNA) for hybridization
with the Ambion WT Expression Kit, the Affymetrix WT Terminal
Labelling and Controls Kit and the Affymetrix Hybridization, Wash,
and Stain Kit as described by the manufacturer. Sense ssDNA was
fragmented and the distribution of fragment lengths was measured
on a BioAnalyzer. Fragmented ssDNA was then labelled and hybrid-
ized to the Affymetrix GeneChip Mouse Gene 1.0 ST Array (Affyme-
trix). Chips were processed on an Affymetrix GeneChip Fluidics
Station 450 and Scanner 3000. Affymetrix CEL files were analysed
using the Limma, oligo, and genefilter R Bioconductor packages
(Carvalho & Irizarry, 2010; Smyth, 2004). Arrays were RMA back-
ground corrected, quantile normalised and summary expression
values calculated for Refseq and full length mRNAs. Genes changing
upon Paupar knockdown were filtered to remove genes showing
little variation in expression (variance cut off of 0.5), while for the
Pax6 knockdown analysis, genes with consistently low expression
were removed before the identification of significant changes. In
each case, differential expression between three knockdown and
three control samples (biological replicates) was tested using the
Limma Ebayes algorithm. Gene Ontology analyses were performed
using GOToolBox, and representative significantly enriched catego-
ries selected from a hypergeometric test with a Benjamini-Hochberg
corrected P-value threshold of 0.05 (http://genome.crg.es/GOTool-
Box/).
CHART-seq and analysis
CHART Enrichment and RNase H Mapping experiments were per-
formed as previously described (Simon, 2013). CHART extract
was prepared from approximately 8 ×10
7
N2A cells per pull down
and hybridized with 810 pmol biotinylated oligonucleotide cocktail
(Supplementary Table 1) overnight with rotation at room tempera-
ture. Complexes were captured using 250 ll MyOneC1 streptavidin
beads (Invitrogen) overnight at room temperature with rotation.
Bound material was extensively washed and eluted using RNase H
(New England Biolabs) for 30 min at room temperature. Samples
were treated with Proteinase K and cross-links were reversed.
RNA was purified from 1/5 total sample volume using the Qiagen
miRNeasy kit while DNA was purified from the remaining sample
by Phenol:CHCl
3
:isoamyl extraction and ethanol precipitation.
DNA was sheared to an average fragment size of 150–300 bp
using a Bioruptor (Diagenode) and sequenced on an Illumina
HiSeq.
CHART-seq was performed in replicate with two independent
pull down samples and matched controls using non-targeting LacZ
oligos. A single sample of input DNA from N2A cells was prepared
and sequenced separately. 50 bp, paired-end reads were mapped to
the mouse genome (mm9) using bowtie with the options ‘–m1 –v2 –
best –strata –a’. For each Paupar sample, peaks were called against
the matched LacZ control and against the N2A input sample. Peak
The EMBO Journal Paupar,atrans-acting lncRNA Keith W Vance et al
The EMBO Journal ª2014 The Authors
12

calls were made using the MACS2 algorithm (Zhang et al, 2008;
https://github.com/taoliu/MACS/blob/master/README) with the
options ‘–mfold 10 30 –gsize=2.39e9 –qvalue=0.01’ using the CGAT
pipeline ‘pipeline_mapping.py’ (https://github.com/CGATOxford/
cgat). Peak calls were then filtered such that only peak calls with a
log10 qvalue >5 were retained (FDR 0.001%).
Characterisation of Paupar binding sites
The chromosomal distribution of Paupar peaks was visualised
using the R Bioconductor package ‘ggbio’ (Yin et al, 2012). Gen-
ome territory enrichments were identified using the Genome Asso-
ciation Tester (GAT; Heger et al, 2013), using a mappability
filtered workspace, an isochore file partitioning the genome into
8 bins based on regional GC content and 10,000 simulations.
Chromosomal enrichments were analysed by proportionally
assigning chromosomal territories to a single virtual meta-chromo-
some before using GAT to test for GC and mappability corrected
enrichments as before. Peak shapes were visualised using read
count normalised (MACS2–SPMR), background subtracted
(MACS2–bdgcmp) coverage tracks from which regions covering
peaks were extracted and centred based on the location of the
peak maximum. Gene ontology categories enriched for Paupar
binding were identified by intersecting regulatory regions for
known coding genes with Paupar binding sites. Regulatory regions
for genes were defined as a basal domain surrounding the TSS of
5kb to+1 kb plus an extended domain of upstream and down-
stream to the nearest gene’s basal domain or to a maximum dis-
tance of 1 Mb, following the GREAT definition (McLean et al,
2010). Enrichments were identified with GAT using the regulatory
regions of all genes as the workspace, and 10,000 simulations.
Because we noted some correspondence between Paupar binding
and gene expression level, we supplied GAT with a file stratifying
the workspace into six bins based on gene expression level in
N2A cells under the ‘–isochore’ option to conservatively avoid
associations solely due to expression level.
Paupar peaks were characterised using DNase I hypersensitiv-
ity sites identified by the Stamatoyannopoulos lab at the Uni-
versity of Washington and regulatory elements identified by the
Ren lab at the Ludwig Institute for Cancer Research (ENCODE
Project Consortium, 2011). Enrichments of DNase I HS and regu-
latory elements overlapping Paupar peaks were assessed using
GAT to control for mappability and regional GC content as
before.
Complementarity of Paupar sequence and binding locations was
assessed using the EMBOSS Water algorithm (Rice et al, 2000) to
perform Smith-Waterman alignment with a range of gap opening
and extension penalties. De novo motif discovery was performed
using the MEME-ChIP (Machanick & Bailey, 2011) algorithm to
examine the unmasked DNA sequence of the central regions of top
scoring (MACS2 peak score) peak locations. MEME-ChIP was run
with the options ‘-meme-mod zoops -meme-minw 5 -meme-maxw
30—meme-nmotifs 50’ using a custom background file prepared
from regions flanking the peak locations using the command ‘fasta-
get-markov -m 2’. Enrichment of known vertebrate transcription
factor binding sites from the TRANSFAC Professional database
(Matys et al, 2006) was assessed using the AME algorithm
(McLeay & Bailey, 2010) with the options ‘–method fisher –length-
correct’ using the sequence and background file prepared for
MEME-ChIP analysis.
Paupar knockdown and flow cytometry
Approximately 2 ×10
5
cells were plated per well in a six well
plate. 16–24 h later cells were transfected with 1.5 lg shRNA
expression construct using FuGENE 6 (Promega) according to the
manufacturer’s instructions. Total RNA was extracted from the
cells 2–3 days later using the Qiagen Mini RNeasy kit according to
the manufacturer’s instructions. For stable transfections, N2A cells
were co-transfected with a 5:1 ratio of pBSU6-sh408 expression
vector and pTK-Hyg (Clontech). Three days after transfection
200 lg/ml Hygromycin B was added to the cells and individual
drug resistant clones were isolated and expanded under selection
conditions. Individual clones were characterized for Paupar
expression using qRT-PCR. For flow cytometry, cells were har-
vested by trypsinization, washed twice with PBS and fixed as a
single cell suspension in 20°C filtered 70% ethanol. After
incubation at 4°C for 10 min cells were pelleted, treated with
40 lg/ml RNase A and propidium iodide (40 lg/ml) for 30 min at
room temperature and then analysed using a FACSCalibur (BD
Biosciences) flow cytometer.
qRT-PCR and RACE
The QuantiTect Reverse Transcription Kit (Qiagen) was used for
reverse transcription and followed by SYBR Green quantitative
PCR using a Step One Plus Real-Time PCR System (Applied
Biosystems). RACE was performed using the GeneRacer Kit
(Invitrogen) following the manufacturer’s instructions. Human
foetal brain RNA was obtained from Promega. Primers are shown
in Supplementary Table 1.
Cell fractionation
Approximately 2.5 ×10
6
cells were pelleted, washed with PBS,
resuspended in 250 ll Lysis Buffer (15 mM HEPES pH7.5, 10 mM
KCl, 5 mM MgCl
2
, 0.1 mM EDTA, 0.5 mM EGTA, 250 mM
Sucrose, 0.4% Igepal, 1 mM DTT, 40 U/ml RNaseOUT (Invitro-
gen), protease inhibitor cocktail [Roche]) and incubated on ice for
20 min. Nuclei were centrifuged at 2,000 gfor 10 min at 4°C and
the supernatant was collected as the cytoplasmic fraction. Nuclei
were then resuspended in 50 ll Nuclei Lysis Buffer (10 mM HE-
PES pH7.5, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 40 U/ml
RNaseOUT (Invitrogen), protease inhibitor cocktail [Roche]) and
incubated on ice for 5 min. Nuclei were pelleted at 17,000 gfor
5 min at 4°C and the supernatant was removed as the nucleo-
plasm fraction. The pellet was then resuspended in 50 ll Salt
Extraction Buffer (25 mM HEPES pH7.5, 10% glycerol, 420 mM
NaCL, 5 mM MgCl
2
, 0.1 mM EDTA, 1 mM DTT, 40 U/ml
RNaseOUT (Invitrogen), protease inhibitor cocktail [Roche]) and
incubated for 30 min at 4°C with rotation. The sample was then
centrifuged at 17,000 gfor 20 min at 4°C. The supernatant was
collected as the salt extracted fraction and the pellet resuspended
in 50 ll Salt Extraction Buffer to generate the chromatin fraction.
RNA was isolated from each fraction using the Qiagen Mini
RNeasy kit following the manufacturer’s instructions.
Keith W Vance et al Paupar,atrans-acting lncRNA The EMBO Journal
ª2014 The Authors The EMBO Journal 13

UV-RIP
UV-RIP was performed as described in (Zhao et al, 2010) with
minor modifications. Approximately 1 ×10
7
N2A cells per UV-RIP
were UV crosslinked in ice-cold PBS at 254 nm, 120 mjoules/cm
2
using a Stratalinker (Stratagene). Nuclei were isolated, disrupted by
sonication (three cycles, 30 sec ON/OFF) using a Bioruptor (Diagen-
ode) and treated with 20 ll Turbo DNase (Ambion) before over-
night incubation with either anti-rabbit PAX6 (AB2237) or rabbit
IgG (both Millipore) polyclonal antibodies. Complexes were
captured using Protein-A magnetic beads (Pierce), washed using
low- and high-stringency buffers and then treated with RNA grade
Proteinase K (Invitrogen). RNA was extracted using Trizol (Invitrogen)
and analysed by qRT-PCR.
ChIP
ChIP was performed using approximately 1 ×10
7
N2A cells per
assay. Cells were trypsinized, resuspended in 10 ml PBS containing
1% final concentration formaldehyde and incubated for 10 min at
room temperature with rotation. Cross-linking reactions were
quenched with 0.125 M glycine for 5 min at room temperature and
washed twice with ice-cold PBS. Nuclei were then isolated and chro-
matin was sheared to approximately 500 bp using a Bioruptor
(Diagenode). Cross-linked chromatin was immunoprecipitated using
5lg anti-rabbit PAX6 or anti-rabbit IgG control antibodies (both
Millipore) overnight at 4°C. Complexes were collected using
Protein-A magnetic beads (Pierce) pre-blocked with BSA (New Eng-
land Biolabs) and transfer RNA (Roche), then washed and eluted.
Cross-links were reversed at 65°C overnight and DNA was precipi-
tated, treated with Proteinase K (Roche) and then purified using a
PCR Purification Kit (Qiagen).
Luciferase assays
Approximately 5 ×10
4
N2A cells were seeded per well in a 12-well
plate. On the next day, cells were transfected with the indicated
ratios of reporter constructs and expression vectors using FuGENE 6
(Promega) according to the manufacturer’s instructions. The pRL-tk
plasmid (Promega) was co-transfected into each well to normalize
for transfection efficiency. The total amount of DNA was made up
to 1 lg for each transfection by the addition of empty expression
vector. Forty-eight hours after transfection lysates were prepared
and assayed for firefly and renilla luciferase activity.
Data deposition
Microarray and CHART-Seq data have been deposited in the
GEO database under accession number GSE52571 (http://www.
ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52571).
Supplementary information for this article is available online:
http://emboj.embopress.org.
Acknowledgements
We thank Drs Andrew Bassett, Ana Marques and Wilfried Haerty for critical
reading of the manuscript and Dr Matthew Simon (Yale University) for his
invaluable help and advice in establishing the CHART protocol. We thank
the High-Throughput Genomics Group at the Wellcome Trust Centre for
Human Genetics for the generation of the sequencing data and OXION for
use of their microarray facility. We also thank Dr Ana Marques for the non-
targeting control shRNA expression vector, Dr Shirin Bonni (University of
Calgary) for the pBS-U6-CMVeGFP plasmid and Prof Veronica van Heynin-
gen, Dr Dirk-Jan Kleinjan and Shipra Bhatia (University of Edinburgh) for
helpful discussions. This project has been funded by the European Research
Council (Project Reference 249869, DARCGENs; KWV, VC, LK), the Medical
Research Council (CPP, SNS; and MRC Hub Grant G0900747 91070 for
Sequencing) and the Wellcome Trust (Grant Reference 090532/Z/09/Z for
Sequencing).
Author contributions
KWV and CPP conceived the study. KWV designed experiments with input from
SNS. KWV, SNS and CPP interpreted data and wrote the manuscript. KWV, SL,
VC and PLO performed experiments. SNS performed computational analysis of
expression and CHART-seq data with input from KWV. LK conducted EST
profiling. SEC provided reagents.
Conflict of interest
The authors declare that they have no conflict of interest.
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