The long non-coding RNA Dali is an epigenetic regulatorof neural differentiation
Abstract
Many intergenic long noncoding RNA (lncRNA) loci regulate the expression of adjacentprotein coding genes. Less clear is whether intergenic lncRNAs commonly regulatetranscription by modulating chromatin at genomically distant loci. Here, we reportboth genomically local and distal RNA-dependent roles of Dali, aconserved central nervous system expressed intergenic lncRNA. Daliis transcribed downstream of the Pou3f3 transcription factor geneand its depletion disrupts the differentiation of neuroblastoma cells. Locally,Dali transcript regulates transcription of thePou3f3 locus. Distally, it preferentially targets activepromoters and regulates expression of neural differentiation genes, in part throughphysical association with the POU3F3 protein. Dali interacts withthe DNMT1 DNA methyltransferase in mouse and human and regulates DNA methylationstatus of CpG island-associated promoters in trans. These resultsdemonstrate, for the first time, that a single intergenic lncRNA controls theactivity and methylation of genomically distal regulatory elements to modulatelarge-scale transcriptional programmes.
eLife digest
Traditionally genes are considered to contain all the instructions necessary to buildproteins. For these instructions to be followed they need to be‘transcribed’ into molecules called messenger RNA, which are then‘translated’ to form the protein. Messenger RNAs are not the only typeof RNA molecule made in a cell; long non-coding RNAs (or lncRNAs), for example, aretranscribed but never translated into proteins. Instead, some lncRNAs control theexpression of nearby genes and some alter how the DNA is packaged within thecell.
Several lncRNAs have been found to control their neighbouring genes, but it isunclear how many of these molecules can also regulate genes that are much furtheraway, even on other chromosomes. One lncRNA called Dali is made incells of the nervous system of mammals. In the genome, the gene forDali is situated next to a gene called Pou3f3,which encodes a protein that contributes to the growth and development of nerves andthe kidneys.
Chalei et al. have now shown that artificially reducing the amount of theDali lncRNA restricts the development of mouse cells called N2Acells, which are commonly used to study the development of nerve cells. ReducingDali lncRNA levels in these cells caused Pou3f3messenger RNA levels to also decrease, which demonstrates that Daliis a lncRNA that controls its neighbouring gene. The levels of many other genes werealso changed when Dali levels were reduced, including many genesthat are needed to grow working nerve cells.
Chalei et al. also showed that the Dali lncRNA binds to 1427different regions of the genome of N2A cells, most often near to the start of activegenes; Dali could be carried to these sites by the POU3F3 protein.The DNA sequences with which the Dali lncRNA binds were alldifferent. Chalei et al. found that Dali also binds to an enzyme,called DNMT1, that chemically modifies DNA to change how it is packaged into a cell,and they predict that this enzyme helps Dali to find its bindingsites. Furthermore, when Dali lncRNA levels were artificiallyreduced, the chemical modifications that affect the packaging of DNA in thecell—and hence the expression of genes encoded by this DNA—were changedfor several genes. Some of these genes were located far away from the gene thatencodes Dali, indicating that this lncRNA can regulate the packagingand expression of distant genes.
Many genes that are regulated by Dali are also regulated by thePOU3F3 protein; this suggests that the lncRNA might work together with this proteinto affect the expression of some genes. Further work is now needed to uncover howmany other lncRNAs act away from their sites of synthesis, and how many also formcomplexes with DNA-binding and DNA-modifying proteins.
Introduction
A growing number of nuclear localised long noncoding RNAs (lncRNA, ≥ 200 nt) areknown to regulate gene transcription and chromatin organisation (reviewed in (Vance and Ponting, 2014)). Many of thesetranscripts appear to act near to their site of synthesis to regulate the expression ofgenes locally on the same chromosome (cis-acting).Cis-acting lncRNA regulatory mechanisms have been described in detailfor a number of enhancer associated nuclear lncRNAs, as well as lncRNAs involved in theprocesses of genomic imprinting and X chromosome inactivation (Tian et al., 2010; Melo et al.,2013; Monnier et al., 2013; Mousavi et al., 2013; Santoro et al., 2013; Vallot etal., 2013). Some cis-acting lncRNAs bind to DNAmethyltransferase (DNMT) proteins and regulate genomic DNA methylation levelsspecifically at their sites of transcription (Mohammadet al., 2010; Di Ruscio et al.,2013).
Trans-acting lncRNAs that regulate gene expression across multiplechromosomes and on either allele have been documented less frequently. The ability ofsuch lncRNAs to exert widespread effects on gene expression in trans ispoorly understood, in large part because direct transcriptional targets for only veryfew of these transcripts have thus far been identified (Chu et al., 2011; Ng et al., 2013;Simon et al., 2011; Vance et al., 2014). Moreover, it is not clear whether thesetranscripts commonly act directly, or within ribonucleoprotein complexes, and how theymight modify their target genes’ regulatory landscape such as by regulating theirDNA methylation profiles.
Many thousand mammalian intergenic lncRNAs have now been identified. Not all lncRNAtranscript models will be functional, however. Single exon models, in particular, can beartefacts arising from genomic DNA contaminating sequencing libraries, and transcriptsthat are expressed at average levels lower than one copy per cell are less likely toconfer function. Highly and broadly expressed, and bona fide monoexonic intergeniclncRNAs, such as Neat1 andMalat1/Neat2, however, appear not to have essentialroles because their knockout mouse models are viable and fertile (Eissmann et al., 2012; Zhang etal., 2012). Transcript sequences and levels are thus not reliable predictorsof mechanism. Instead, the significant temporal and spatial co-expression of genomicallyadjacent intergenic lncRNA and transcription factor genes might suggest that suchlncRNAs commonly modulate transcriptional programmes that are initiated by thesetranscription factors (Ponjavic et al., 2009).Indeed, several intergenic lncRNAs have well-documented cis-actingregulatory roles (Wang et al., 2011; Zhang et al., 2012; Berghoff et al., 2013).
Spatiotemporal co-expression of intergenic lncRNA and transcription factor genes is mostpronounced during the development of the mouse central nervous system (CNS) (Ponjavic et al., 2009). To investigate themechanistic basis of this physical linkage we chose to study a 3.5-kb, CNS-expressed,monoexonic, intergenic lncRNA termed Dali (DNMT1-Associated LongIntergenic), owing to its conservation of sequence and transcription across therianmammals and its genomic proximity to a transcription factor gene,Pou3f3 (also known as Brn1 orOct8), which encodes a class III POU family transcription factor.Dali is transcribed in the sense orientation, relative toPou3f3, from a locus 50 kb downstream of Pou3f3within the flank of an extended genomic region (Figure1A) that is characterised by near pervasive transcription in neuronal lineages(Ramos et al., 2013). Sauvageau et al.recently generated mouse knockout models for two of these intergenic lncRNA loci,linc-Brn1a, and linc-Brn1b (Figure 1A). Genomic deletion of the linc-Brn1blocus resulted in significant (∼50%) down-regulation of the upstreamPou3f3 gene, andlinc-Brn1b-/- mice exhibitedabnormalities of cortical lamination and barrel cortex organization (Sauvageau et al., 2013). These abnormalities mayderive from loss of the linc-Brn1b RNA transcript, or from the deletionof DNA functional elements (Bassett et al.,2014). The Dali locus is more distally located and does notoverlap previously described lncRNA loci or regulatory elements (Figure 1A).
Pou3f3 is a single exon gene whose protein binds to DNA in asequence-specific manner. Pou3f3 contributes to both neuronal andkidney development by regulating the proliferation and differentiation of progenitorcells (Nakai et al., 2003). Mouse mutants withhomozygous loss of Pou3f3 die of renal failure within 36 hrpost partum (Nakai et al.,2003), with severe defects of the hippocampus and forebrain among others(McEvilly et al., 2002). In the developingneocortex, Pou3f3 is expressed in late neuronal precursors and inmigrating neurons and, together with its closely related paraloguePou3f2, is required in ventricular zone progenitors fordeep-to-upper layer fate transition, sustained neurogenesis and cell migration (Dominguez et al., 2013).
Our experiments show that Dali is required for the normaldifferentiation of neural cells in culture. Furthermore, our results indicate thatDali functions by modulating the expression of its neighbouringPou3f3 gene, as well as by interacting with the POU3F3 protein, andby directly binding and regulating the expression of genes involved in the neuronaldifferentiation programme in trans. Unexpectedly, Daliassociates with the DNMT1 DNA methyltransferase and reduction of Dalilevels increases DNA methylation at a subset of Dali-bound and-regulated promoters in trans. Our data therefore provide the firstevidence that a lncRNA transcript can regulate multiple genes situated away from itssite of synthesis by binding to promoter-proximal regulatory elements and altering theirDNA methylation status in trans.
Results
Conserved Dali genomic organisation and transcription
Full-length mouse Dali is approximately 500 nt (2.6 kb) longer thana previously identified AK034039 cDNA cloned from the telencephalon(Figure 1—figure supplement 1A).Its locus, downstream of the Pou3f3 gene, contains mammalianconserved sequence both just upstream of its transcriptional start site, whichpresumably contributes to this locus’ promoter, and within its transcribedsequence. A positionally equivalent and sequence-similar human DALI(∼3.7 kb) transcript was identified by RT-PCR and RACE in human foetal brain(Figure 1B; Figure 1—figure supplement 1B,C). Transcriptionalevidence also exists for the orthologous locus in rat embryonic, as well as heart andkidney, samples (data not shown).
Dali is a chromatin-associated transcript that is co-expressedwith Pou3f3 in neural cell lineages
ENCODE data indicate that both mouse and human Dali loci have theproperties of a weak (or poised) enhancer in both brain and kidney tissues (Figure 1—figure supplement 1D,E).Consistent with this, Dali was most highly expressed in the adultbrain and kidney, two of the three tissues displaying highest Pou3f3expression, when profiled across a panel of adult mouse organs (Figure 1—figure supplement 1F,G). In adult mouse (P56),Dali and Pou3f3 were expressed in all threeregions of adult neurogenesis, the sub-ventricular zone (SVZ), olfactory bulb (OB),and dentate gyrus (DG) (Figure 1—figuresupplement 1I) (Reviewed in Ming andSong, 2011). Dali was also co-expressed withPou3f3 temporally and spatially in the developing mouse embryonicbrain (Figure 1C,D). Both transcripts wereup-regulated with the first appearance of cortical neurons (E10.5), and increased inexpression further as the ratio between neurons and progenitors grew (Figure 1C,D). Furthermore, bothDali and Pou3f3 transcripts were undetectable inself-renewing mouse E14 embryonic stem (ES) cells, but after 3 days of retinoic acid(RA)-induced differentiation, a stage corresponding to the cell cycle exit ofneuronal progenitors and their differentiation into neurons, these transcripts wererapidly up-regulated, their levels subsequently peaking at days 7(Pou3f3) and 8 (Dali) (Figure 1E,F).
Mouse neuroblastoma N2A cells, which are frequently used as a neuronalprogenitor-like cell type and an in vitro model of neuronal differentiation (Tremblay et al., 2010), express bothDali (at a population-average level of 2 copies per cell (Figure 1—figure supplement 1K)) andPou3f3. When first detected in neuronal-progenitor-dominatedareas of the developing brain (E10.5), Dali is expressed at a levelat least two orders of magnitude higher than in N2A cells (Figure 1—figure supplement 1H). However, in N2A cellstreated with RA for 72 hr, Dali is up-regulated approximately4.5-fold, similar to the up-regulation observed in embryonic cortical plate (bothdorsal and lateral) between days E10.5 to E18.5 (Figure 1—figure supplement 1H). Therefore, despiteDali expression level differences in N2A cells and the in vivosystem, N2A cells represent an appropriate model system in which to studyDali function. Furthermore, Dali, but not acontrol mRNA (Gapdh), was highly enriched in the nucleus of N2Acells, most abundantly in the chromatin fraction (Figure 1G,H). Taken together, the data suggest that Dalimay be involved in regulating nuclear function during neuronal development,potentially in coordination with Pou3f3.
Dali regulates neural differentiation of N2A cells
We next investigated whether Dali regulates neural differentiationby generating three independent stable Dali knockdown N2A cell lineseach showing approximately 50–70% reduction of Dalitranscript levels and inducing neural differentiation using RA (Figure 2A). Compared to a stable non-targeting control line,fewer differentiated cells of Dali knockdown lines developedneurites. Those that did exhibited shorter neurites, often with multiple shortoutgrowths emanating from the same cell, compared to one or two long neuritesdeveloped by differentiated control cells (Figure2B,C) indicating that Dali is required for normaldifferentiation of N2A cells.
Dali regulates neural gene expression
To investigate the molecular function of Dali, we performedmicroarray analysis to profile the transcriptome of N2A cells in whichDali transcript levels had been depleted by ∼70% usingtransient transfection of a specific Dali targeting shRNA expressionvector (Figure 2D; shRNA and RT-qPCR oligosequences and positions can be found in Supplementary file 1). Dali knockdownresulted in statistically significant changes in expression levels for 270 genes(False Discovery Rate [FDR] < 10%) compared to a non-targeting control (Supplementary file 2;Figure 2E). 14 of 15 of these genes werealso determined as being differentially expressed, with similar fold changes, usingRT-qPCR and two additional independent shRNA expression constructs targetingDali (Figure 2—figuresupplement 1C). Gene expression changes we observed using microarrays werethus unlikely to be dominated by off-target effects of the shRNA used. Gene Ontology(GO) analysis revealed that Dali-regulated genes were significantlyenriched in cell cycle, DNA repair, cellular response to stimulus, and cellprojection assembly functions (Figure 2F andSupplementary file2; Benjamini-Hochberg corrected p ≤ 0.05). Taken together, theseexpression and loss of function studies suggest that Dali acts as apro-differentiation factor in neural development.
Dali and Pou3f3 share transcriptionaltargets
To investigate whether Dali knockdown affects expression of theadjacent Pou3f3 gene, we reduced its levels by transienttransfection of three different shRNA constructs in N2A cells. After 72 hr, reductionof Dali levels by an average of 60–70% was found to reducePou3f3 transcript levels by approximately 40% (Figure 2G). Three independent stableDali knockdown clones in which Dali levels werereduced by 50–60% (Figure 2A) alsoshowed ∼15–40% lower Pou3f3 levels (Figure 2H). This suggests that theDali transcript positively regulates Pou3f3expression in an RNA-dependent manner. The genome-wide transcriptional response toDali knockdown thus could be explained, in part, by its effect onPou3f3.
Levels of another transcript, AK011913, expressed downstream ofPou3f3 (Figure 1A) werereduced by approximately 55% upon Dali knockdown (Figure 2—figure supplement 1A).Reduction of AK011913 levels by approximately 60% using shRNAsresulted in Dali and Pou3f3 levels decreasing by73% and 82%, respectively (Figure 2—figuresupplement 1B). Linc-Brn1a, a lncRNA upstream of andsharing a bi-directional promoter with Pou3f3, was up-regulated byapproximately 90% upon AK011913 depletion (Figure 2—figure supplement 1B). This is reminiscent ofthe down-regulation of Pou3f3 and up-regulation oflincBrn-1a following knockdown of another lncRNA downstream ofPou3f3, lincBrn-1b (Figure 1A) (Sauvageau et al.,2013). Together with previous reports, our data show the opposingregulatory influences of lncRNAs transcribed up- and downstream ofPou3f3 on its expression. Non-coding transcripts expressed fromthe extended Pou3f3 locus thus contribute to a complex network ofregulatory interactions.
Furthermore, Chromatin Conformation Capture (3C) showed that theDali promoter contacted three regions across thePou3f3 locus (Figure 1A)in ES derived neuronal precursors (Figure1—figure supplement 2) : 1) an enhancer element sequence lyingupstream of Pou3f3 within the linc-Brn1a locus, 2)a region overlapping the 3′ UTR of Pou3f3 and full-lengthAK53590 (which are both regulated by Dali), aswell as parts of TCONS_00000039 and linc-Brn1b,including a differentially methylated region reported to be important in regulatingPou3f3 expression (Mutai etal., 2009), and 3) a region lying within another non-coding locus(TCONS_00000040) (Ramos et al.,2013). Neither Dali nor Pou3f3 appears toplay a role in initiating these DNA looping interactions because these contacts werepresent in E14 ES cells where neither is expressed (Figure 1—figure supplement 2B). Nevertheless, theDali locus appears to contribute to an extended structurally andtranscriptionally complex region centred on the Pou3f3 gene.
To examine to what extent the transcriptional response to Daliknockdown can be explained by its effect on Pou3f3, we reduced thelevel of Pou3f3 transcript in N2A cells by 35% using transienttransfection of a Pou3f3 targeting shRNA vector (Figure 3A) and using microarrays observedstatistically significant expression changes in 1041 genes (FDR <10%; Figure 3B). Dali transcriptlevels do not change upon Pou3f3 depletion (Figure 3A). Genes differentially expressed afterPou3f3 knockdown were enriched in categories related to celldivision and cell cycle (Figure 3C). Theintersection between the sets of genes differentially expressed inDali or in Pou3f3 knockdown cells was 6.2-foldgreater than expected by chance (p-value < 2.2 × 10−16),and represented 31% of all genes differentially expressed in Daliknockdown cells (Figure 3D). Approximatelyequal numbers of genes shared between the two datasets were down- (43 genes) orup-regulated (41 genes) in both experiments (Supplementary file 3). A strong correlation was observedbetween the fold-change values of differentially expressed genes inDali and Pou3f3 knockdown experiments (R =0.74; Figure 3E). Genes that weresignificantly differentially expressed only when Dali was depletedwere enriched in chromatin assembly and MAPKKK signalling functions, whilst genesthat were differentially expressed only when Pou3f3 transcripts weredepleted were preferentially involved in dendrite development and axon guidance(Figure 3F). Cell cycle, DNA repair, andcellular response to stimulus genes were regulated by Dali in bothPou3f3-dependent and -independent manners. We conclude thatDali and Pou3f3 interact, either genetically ormolecularly, to regulate a subset of common targets involved in neuraldifferentiation, and that Dali also likely possessesPou3f3-independent transcriptional regulatory functions.
Dali regulates gene expression programmes during neuraldifferentiation of N2A cells
To further investigate the role of Dali in neuronal differentiationwe profiled the transcriptomes of proliferating or RA differentiated control andDali stable knockdown N2A cell lines. In proliferating cells, 733genes were differentially expressed between Dali knockdown andcontrol cells (Figure 4A), including manygenes with functions related to neuronal differentiation, apoptosis, neuronalfunction (Figure 4B). RA-mediated neuronaldifferentiation induced expression changes in 958 genes in control cells and 1016genes in Dali knockdown cells (Figure 4—figure supplement 1A,B). Based on GO category annotations,differentiation of control or Dali knockdown cells was broadlysimilar, and was associated with altered expression of cell cycle, celldifferentiation, energy metabolism, and neuron projection (Figure 4—figure supplement 1C,D). However, 804 geneswere differentially expressed between terminally differentiated control andDali knockdown cells (Figure4C), of which 376 genes (46.8%) also differed in expression betweenDali knockdown and control cells prior to their differentiation(Figure 4E). The 428 genes that weresignificantly altered in expression only between stable Dali andcontrol differentiated cells were enriched in functional categories relating tosterol biosynthesis, energy metabolism, cell cycle, response to chemical stimulus,cell cycle, adhesion and small GTPase signalling (Figure 4D). All 11 (of 34 known) sterol biosynthesis genes weredown-regulated in Dali knockdown cells. This observation isconsistent with the impaired neurite outgrowth of stable Daliknockdown cells because neuritogenesis and neurite outgrowth critically rely onmembrane biosynthesis, and therefore, on expression of sterol biosynthesis genes(Paoletti et al., 2011).
In addition, several key neuronal differentiation genes, for example Nrcam,Dscam, Dlx1 and Pax3, were differentially expressedbetween Dali knockdown and control cells both prior to and afterdifferentiation. Furthermore, multifactorial analysis of RA-induced gene expressionchanges in control and stable Dali knockdown cells showed that 174genes responded to RA differently depending on the presence or knockdown ofDali (FDR 5%; Supplementary file 4). These were significantly enriched incategories relating to neuronal development (Figure4F), including pro-differentiation factors such as the inhibitor of Wntsignaling Dkk1 (Cajanek et al.,2009) and Wnt receptor Fzd5 (Kemp et al., 2007).
In summary, compared to control cells, stable Dali knockdown cellsexhibit contrasting alterations in gene expression programmes before and afterRA-induced differentiation. In both cases, these programmes are enriched infunctional categories related to neural differentiation and function, consistent withthe proposed role for Dali in neural development.
Dali preferentially binds to active promoters intrans
We next sought to identify and characterise genes that are both bound and regulatedby Dali. To do so, we determined the genome-wide binding profile ofDali in N2A cells using Capture Hybridisation Analysis of RNATargets (CHART)-Seq (Simon et al., 2011;Simon, 2013) (Figure 5—figure supplement 1A–C). We discovered1427 focal Dali-associated regions genome-wide (Figure 5A,B; Supplementary file 5), of which all nine selected loci werevalidated by CHART-qPCR in an independent experiment (Figure 5—figure supplement 1D).
Dali binding sites were typically limited to less than 1 kb inlength (Figure 5—figure supplement1E) and were distributed across the genome with no apparent chromosomal biasesother than a depletion on the X chromosome which may reflect the inactivation of oneX chromosome copy in these female N2A cells (Figure5C). These sites were preferentially located at the 5′ end ofprotein coding genes (Figure 5D): 30.5% ofpeaks were within 5 kb of a transcriptional start site (TSS) (Figure 5E). Dali bound sequences weresignificantly enriched for H3K4me3, H3K4me1 and H3K27ac modified histones and PolIIoccupancy, and were depleted for repressive histone marks (Figure 5F). This suggests that Dalipreferentially associates with regions of active chromatin. GO category enrichmentanalysis showed that genes associated with Dali peaks contribute toprocesses related to neuronal differentiation (cell cycle), neuronal projectiondevelopment (cytoskeleton organization and small GTPase mediated signaltransduction), neuronal function (synaptic transmission), and more general cellularprocesses, such as gene expression, intracellular signalling, and cellularhomeostasis (Figure 5G). 150 genes (8.6% ofall Dali bound genes) regulated by Dali containedDali binding sites within their regulatory regions (Figure 5H) and presumably represent directtranscriptional targets.
Dali interacts with chromatin modifying proteins
To investigate the mechanisms of its genomic targeting, we next performedcomputational analysis of Dali bound sequences. We discovered thatDali binding sites do not exhibit significant sequencecomplementarity with the Dali transcript (Figure 5—figure supplement 1F, see Methods), and arenot likely to form RNA-DNA:DNA triplex structures (Figure 5—figure supplement 1G), suggesting thatDali does not bind DNA directly. We therefore speculated thatDali may be targeted to the genome indirectly thoroughRNA-protein interactions. To identify proteins that interact directly withDali, we performed a pull down assay in which in vitrotranscribed and 5′ end-biotinylated Dali was incubated withnuclear extract prepared from day 4 RA-differentiated ES cells. We identified, usingmass spectrometry, 50 proteins that associated with Dali, but notwith antisense Dali or a size-matched unrelated control transcript(Supplementary File7). Direct interactions between the endogenous Dalitranscript and four of these candidate binding proteins, the DNA methyltransferaseDNMT1, the BRG1 core component of the SWI/SNF family chromatin remodelling BAFcomplex, and the P66beta, and SIN3A transcriptional co-factors, were subsequentlyvalidated using UV-crosslinked RNA Immunoprecipitation (UV-RIP) in N2A cells (Figure 6A,B). Human DALI wasalso found, using UV-RIP, to interact with human DNMT1, yet not with BRG1, in humanneuroblastoma SH-SY5Y cells (Figure 6B).Consequently, in further experiments, we focused on the evolutionarily conservedDNMT1 interaction.
Interestingly, 9 of 58 human transcription factors reported by Hervouet et al. asinteracting with the DNMT1 protein (Hervouet etal., 2010), including CTCF, but also AP-2, C-ets-1, LRH1, PARP, PAX6,STAT1, YY1, and Sp1, were found to have binding site motifs that were significantlyenriched within our stringent Dali bound CHART-seq peaks (Supplementary File 6).Motifs for none of 42 transcription factors that do not interact with DNMT1 butinteract with DNMT3a and/or DNMT3b (Hervouet etal., 2010) were enriched in these peaks (Supplementary File 6). Inparticular, using a de novo motif discovery approach, we found a highly-enrichedCTCF-binding site-like motif in 125 out of 1427 Dali peaks (9%; MEMEE-value = 3.1 × 10−62; Figure 6C) (Supplementary File 7). This result was concordant with the greater thanexpected overlap between Dali-associated regions and known CTCFbinding sites in neuronal tissues (Figure 5F)(Shen et al., 2012). Using ChromatinImmunoprecipitation and qPCR (ChIP-qPCR) in N2A cells, we confirmed theCTCF-enrichment of previously-known CTCF-binding sites within 7Dali-bound and regulated promoters, but not at four control regions(Figure 6D). However, despite CTCF andDali thus occupying a subset of shared genomic binding sites,UV-RIP provided no evidence of a direct physical interaction (Figure 6E). Consequently, Dali and CTCF may benon-interacting molecular subunits of a larger ribonucleoprotein complex, oralternatively they might independently bind adjacent sequence, or compete for bindingto the same region. Taken together, the data suggest that Dali isrecruited to chromatin via indirect interactions with several DNA-binding proteinsthrough its direct association with DNMT1.
Depletion of Dali leads to DNA methylation changes at bound andregulated promoters
Increasing numbers of lncRNAs have been shown to direct DNA methylation changes attheir sites of synthesis (Mohammad et al.,2010; Di Ruscio et al., 2013). Thedirect interaction of Dali with DNMT1, however, suggests that it maybe able to regulate DNMT1-mediated CpG methylation at CpG island-associated promotersof Dali-bound and -regulated genes in trans. Toinvestigate this, we performed Combined Bisulfite Restriction Analysis (COBRA) (Xiong and Laird, 1997) in parallel at 10different CpG islands. Selection of these regions was on the basis that they eachcontained several COBRA-compatible restriction enzyme sites and could be efficientlyamplified from bisulfite-converted template. COBRA demonstrated that five of theseregions (corresponding to four genes) exhibited altered restriction profilesindicative of altered DNA methylation status after Dali depletiondepletion (Figure 7—figure supplement1). The inability of COBRA to detect changes at all sites may indicate thatthe DNA methylation status of the remaining regions did not change uponDali depletion or that changes that occurred were undetected dueto technical limitations of the assay.
Bisulfite sequencing demonstrating that the Dlgap5,Hmgb2, and Nos1 promoters each display increasedCpG methylation in two independent stable Dali knockdown linescompared to control further confirmed these results (Figure 7A). Importantly, these data show that methylation changes occurwithin the core of these CpG islands and are not limited to their shores. Althoughother unidentified factors are also likely to play a role, our results are consistentwith Dali (or a Dali:POU3F3 complex) acting intrans, as part of a multi-subunit ribonucleoprotein complex, toreduce DNMT1-mediated CpG methylation at a subset of bound and regulated genepromoters away from its site of transcription.
One of these genes, Nos1, has multiple alternative promoters fallinginto two distinct regions (for simplicity referred to here as Exon 1 and Exon 2)whose differentiated use is proposed to fine-tune its expression in response tovarious physiological and developmental stimuli (Bros et al., 2006). Only the 5′-most region contains a CpG islandand is bound by Dali (Figure7B). By measuring expression levels of the three 5′-mostNos1 exons in stable Dali knockdown and controllines we observed that the expression level of the 5′ mostDali-bound Exon 1 was reduced, relative to that for Exon 3, whenDali was depleted, whereas the expression ratio between Exons 2and 3 was unaffected (Figure 7C). Thepreferential use of the 5′ most CpG site could reflect a secondary effect ofDali knockdown. Nevertheless, the observation that this site isbound by Dali transcript suggests that Dali mayfunction by promoting the preferential use of a distantly located (and more rarelyused) alternative promoter potentially through its effect on promoter-associated CpGisland methylation.
Dali and POU3F3 protein form a trans-actingtranscriptional regulatory complex
A recognisable binding motif for POU III family transcription factors, such asPOU3F3, was present in 115 out of 1427 Dali CHART-Seq peaks (8.0%;E-value = 3.8 × 10−5; Figure 6F). This finding, together withDali and Pou3f3 regulating a set of common genes(Figure 3D) and Dalioccupying regulatory regions within 135 (13%) of Pou3f3 targets(Figure 5H), suggested thatDali and POU3F3 protein may interact physically. Indeed, weobserved direct RNA-protein interactions between over-expressed FLAG-tagged POU3F3and co-transfected Dali, using UV-RIP in N2A cells (Figure 6G). Using ChIP-qPCR, we then determinedthat at least five genes that were regulated by both Dali andPou3f3 contained regions that were bound both byDali and by POU3F3 protein (Figure 6H). These results provide further mechanistic insights intoDali's mode of action and indicate that Dali andPOU3F3 form a complex that binds to and regulates a subset of genes intrans in N2A cells.
Induction of the endogenous Dali transcript in mouse ES cellsregulates Pou3f3 locally and E2f2 distally
Finally, we tested whether de novo expressed Dali transcript can actas a transcriptional regulator in order to further substantiate the observation thatDali functions as a novel regulator of both local and distal geneexpression. To achieve this, we induced Dali expression from itsendogenous locus in E14 mouse ES cells, which do not express Dali orPou3f3 to detectable levels, using transient transfection of anartificial Transcription Activator-Like effector (TALE) transcription factor. After72 hr, up-regulation of Dali transcript was shown to significantlyincrease Pou3f3 expression (Figure7D). Dali expression from its own locus is thus sufficientto induce the expression of its genomically neighbouring Pou3f3 gene(Figure 7D). We next investigated theexpression levels of E2f2, a gene that we found to be negativelyregulated by Dali using shRNA mediated knockdown (Supplementary file 2), andfound that Dali up-regulation reduced E2f2transcript levels by approximately 40% (Figure7D). Taken together, these results indicate that Dali canregulate both local and distal target genes when its expression is induced from itsendogenous locus.
Discussion
The ability of nuclear localised lncRNAs to act in trans at distalgenomic locations to regulate gene expression programs has been poorly understood. Thisis in large part because the direct transcriptional targets of only a small number ofsuch transcripts (for example, Paupar (mouse), HOTAIR,NEAT1, TERC, RMST (all human), androx2 (Drosophila)) have been identified thus far(Chu et al., 2011; Simon et al., 2011; Ng et al.,2013; Vance et al., 2014).Consequently, it has been unclear how these transcripts are targeted to distalfunctional elements and whether thereafter they alter chromatin structure in situ.
In this study we found evidence that the intergenic lncRNA Dali actsboth locally to regulate the expression of its nearest protein-coding gene,Pou3f3, and distally to regulate bothPou3f3-dependent and -independent target genes in an RNA-dependentmanner. 8.8% (150) of all genes whose expression altered following Dalidepletion were associated with Dali binding sites within 1 Mb (although30% of peaks reside within 5 kb of a TSS, see Figure5E) and, therefore, are likely to represent direct regulatory targets. Thisproportion lies within the range of functional sites observed for transcription factors(Cusanovich et al., 2014). Our results areconsistent with a model in which mouse or human Dali is recruited tochromatin indirectly via RNA-protein interactions with both sequence-specifictranscription factor proteins, such as POU3F3 which is encoded by its neighbouring gene,or non-sequence specific DNA binding cofactors including DNMT1, which in turn mayinteract with sequence-specific DNA-binding proteins. In this model,Pou3f3-dependent target genes are regulated by Daliboth indirectly, via its transcriptional regulatory effect on thePou3f3 gene, and directly via its physical interaction with thePOU3F3 protein and their co-occupancy at regulatory regions of target genes.
Our data show that both human and mouse Dali associate with DNMT1 andthat depletion of Dali levels increases CpG methylation atDali bound and regulated promoters in trans. Whilsta growing body of literature has implicated lncRNAs, such as Kcnq1ot1and ecCEBPA (Mohammad et al.,2010; Di Ruscio et al., 2013), inmodulating CpG methylation in a DNMT1-dependent manner at their sites of synthesis, ourfindings represent the first evidence that an intergenic lncRNA can regulate DNAmethylation in trans at distal genomic locations away from its site oftranscription.
Our findings suggest that Dali inhibits DNA methylation at a subset ofbound and regulated regions, presumably deposited by the DNMT1 DNA methyltransferase, towhich it binds. DNMT1 binds structured RNA with higher affinity than its DNA substrate(Di Ruscio et al., 2013). It is thuspossible that Dali competes for binding to DNMT1 with either proteinco-factors such as UHRF1, which loads and orients the enzyme on the DNA substrate (Inomata et al., 2008), or its DNA substrate.Targeting of DNMT1 to specific loci is believed to be mediated by DNMT1-interactingtranscription factors. 58 transcriptional factors have been reported as DNMT1interactors (Hervouet et al., 2010), of which 9have enriched sequence motifs in Dali CHART-Seq peaks. We thus proposea model in which such transcription factors promote the sequence-specificity ofDali-modulated DNA methylation changes. The genomic co-localisationof DNMT1 and transcription factors using ChIP remains unknown owing to the poorperformance of the available anti-DNMT1 antibodies in this application.
We have shown that Dali regulates genes involved in neural developmentand function and its depletion disrupts terminal stages of neuronal differentiation,more particularly neurite outgrowth development. Dali RNA binds to andup-regulates the promoters or promoter-proximal regions of key pro-differentiationfactors, such as E2f2 (Persengiev etal., 2001), Fam5b (Terashima et al., 2010), Sparc (Bhoopathi et al., 2011) and Dkk1 (Cajanek et al., 2009) (Watanabe et al., 2005), as well as binding and negativelyregulating genes such as Kif2c and Kif11 which areknown to block neurite outgrowth (Laketa et al.,2007; Myers and Baas, 2007; Nadar et al., 2012). Therefore,Dali works as a pro-differentiation factor in neural development byregulating the balance between proliferation and differentiation, as well as processesassociated with terminal neuronal differentiation.
Cis- or trans-acting modes of action have beenproposed for a growing number of lncRNAs (Fatica andBozzoni, 2014). Dali is unusual in acting in atranscript-dependent manner to perform both local and distal gene regulatory roles likeanother such lncRNA, Paupar (Vance etal., 2014). Dali is transcribed in the vicinity of a neuronaltranscription factor Pou3f3. Both Dali andPaupar lncRNAs are CNS-expressed and evolutionarily constrainedtranscripts that are co-expressed with their neighbouring transcription factor genesboth spatially and temporally. Moreover, both lncRNAs interact directly with the proteinproduct of their neighbouring genes, POU3F3 and PAX6, respectively, to regulate a largeset of targets in trans. These observations, together with thepreferential genomic location of intergenic lncRNA loci adjacent to transcription factorgenes (Ponjavic et al., 2009) imply thatlncRNAs may commonly interact with the product of genomically adjacent transcriptionfactor genes to act in trans on distal genes.
Materials and methods
Plasmid construction
We used the Whitehead Institute siRNA selection program to design shRNAs that targetmultiple regions of Dali or Pou3f3. To minimise thepossibility of off-target effects, we compared candidate sequences against the NCBIRefSeq database and removed those with ≥15 bases in the anti-sense strand thatmatched a database entry. We then cloned the double stranded DNA oligonucleotidescontaining sense-loop-antisense targeting sequences downstream of the U6 promoter inpBS-U6-CMVeGFP (Sarker et al., 2005) bylinker ligation. The Dali expression plasmid was constructed by PCRamplifying the full length Dali sequence as anEcoRI-XhoI fragment from mouse N2A cell genomicDNA and inserting it into pcDNA3. The FLAG-tagged Pou3f3 expressionplasmid was constructed by excising the full length Pou3f3 ORF fromPou3f3 (NM_008900) mouse cDNA clone in pCMV Entry vector(Cambridge Biosciences, UK) and inserting it into the multiple cloning site (MCS) ofthe N-terminal pFLAG-CMV-6a vector (Sigma–Aldrich, UK) betweenEcoRI and EcoRV sites. The sequences of alloligonucleotides used for cloning are shown in Supplementary file 1.
Dali and Pou3f3 knockdown
Cells were plated at a density of approximately 2 × 105 cells perwell in a six well plate. 16–24 hr later cells were transfected with 1.5μg shRNA expression construct using FuGENE 6 (Promega, UK) following themanufacturer's instructions. Total RNA was extracted from the cells 48–72 hrlater using TRIzol-chloroform extraction method. For stable transfections, N2A cellswere co-transfected with the pBSU6-shRNA expression vector and pTK-Hyg (Clontech,Mountain View, CA) at a 5:1 ratio. 72 hr post-transfection 200 μg/ml HygromycinB was added to the cells to select individual drug resistant clones that were laterisolated and expanded under selective conditions. Dali expression inindividual clones was measured by qRT-PCR.
qRT-PCR and RACE
Reverse transcription was performed using the QuantiTect Reverse Transcription Kit(Qiagen, Netherlands). SYBR Green quantitative PCR was performed using a Step OnePlus Real-Time PCR System (Applied Biosystems, UK). For RACE, GeneRacer Kit(Invitrogen, UK) was used according to the manufacturer's instructions. Human foetalbrain RNA was purchased from Promega. Primers are listed in Supplementary file 1.
Cell culture
Mouse N2A neuroblastoma and E14 ES cells were cultured as described in (Vance et al., 2014). The N2A cell line waschosen because it has been used extensively as a model to study neuraldifferentiation in vitro (Shea et al.,1985). Human neuroblastoma (SH-SY5Y) cells were grown in DMEM/F12 mediumsupplemented with 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine at37°C in a humidified atmosphere with 5% CO2. Biochemicalfractionation, ChIP and UV-RIP experiments was performed exactly as described inVance et al. (2014). The followingantibodies were used: anti-DNMT1 (ab87656; Abcam, UK), anti-BRG1 (ab4081; Abcam),anti-P66beta (ab76924; Abcam), anti-SIN3A (Active Motif, Belgium, 39,865), anti-CTCF(Abcam, 70,303), anti-rabbit IgG control antibodies (Millipore, Billerica, MA) andmouse monoclonal anti-FLAG M2 beads (Sigma–Aldrich) for FLAG-tagged POU3F3experiments.
Animal work
All animal experiments were conducted in accordance to schedule one UK Home Officeguidelines (Scientific Procedures Act, 1986). C57BL/6J, postnatal day P56 male andpregnant females were killed by cervical dislocation; whole brains were dissected inice-cold phosphate-buffered saline (PBS) from adult (n = 2), and intrauterinestages E9 (n = 6), E10.5 (n = 6), E13.5 (n = 6), E15.5 (n = 6)and E18.5 (n = 6) mice. Brains were embedded in 5% agarose (low melting,Bioline) and sectioned using a vibrating microtome (Leica, VT1000S) into 200 μmcoronal sections using a chilled solution of 1:1 mixture of RNAlater (Ambion) andPBS. Regions of interest (adult: dentate gyrus, subventricular zone and olfactorybulb; embryos: preplate, proliferative compartmenst combining ventricular andsubventricular zones, and cortical plate from lateral and dorsal tiers) weredissected from individual sections using 27 gauge needles under visual guidance,using transillumination on a dissecting microscope (MZFLIII, Leica, Switzerland).Dissected samples were rinsed in RNAse free PBS/RNAlater 1:1, submerged in ice-coldRNAlater kept for 24 hr at 4°C and stored at −80°C in RNAlater untilprocessing.
Transcriptomic analysis
Total RNA was isolated using the Qiagen Mini RNeasy kit according to themanufacturers' instructions. RNA integrity was assessed on a BioAnalyzer (AgilentTechnologies, UK). 200 ng RNA was used to produce labelled sense single stranded DNA(ssDNA) for hybridization with the Ambion WT Expression Kit, the Affymetrix WTTerminal Labelling and Controls Kit and the Affymetrix Hybridization, Wash, and StainKit following the manufacturer’s instructions. Sense ssDNA was fragmented andthe distribution of fragment lengths was assessed on a BioAnalyzer. Next, fragmentedssDNA was labelled and hybridized to the Affymetrix GeneChip Mouse Gene 1.0 ST Array(Affymetrix, UK). Arrays were processed on an Affymetrix GeneChip Fluidics Station450 and Scanner 3000.
CEL files were analysed using the Limma, oligo, and genefilter R Bioconductorpackages (Smyth, 2004; Carvalho and Irizarry, 2010). Arrays were RMA backgroundcorrected and quantile normalised. Summary expression values were calculated at thegene level. Genes whose expression changed upon Dali andPou3f3 knockdown, as well as upon retinoic acid induceddifferentiation of control and stable Dali knockdown cells, werefiltered to remove genes showing little variation in expression (variance cut off of0.5) before the identification of significant changes. In every case, the LimmaEbayes algorithm was used to identify differential expression between three knockdownand three control samples (biological replicates). 1.3-fold change cutoff was appliedin every case. GOToolbox was used to perform Gene Ontology analyses ((Martin et al., 2004); http://genome.crg.es/GOToolBox/). Representative significantlyenriched categories were selected from a hypergeometric test with aBenjamini-Hochberg corrected p-value threshold of 0.05.
CHART
CHART Enrichment and RNase H Mapping experiments were performed as described in(Simon, 2013). We designed 10biotinylated DNA capture (C)-oligos: 5 oligos complementary to the most accessibleregions of Dali, as determined by RNase H mapping, and 5 oligostargeting the most evolutionarily conserved regions of the transcript (Figure 5A). These oligos were used as twococktails of 5 oligos, and as a pool of all 10. As controls, we used an oligodesigned to target the antisense Dali sequence (absent from the N2Atranscriptome). Additionally we require peaks to not overlap with those identified inan analogous CHART-sequence experiment using the E. coli lacZsequence (GSE52571) (Vance et al.,2014). Compared to controls, all three cocktails of Dalioligos showed significant enrichment of the Dali transcript (10-foldcompared to lacZ), but no enrichment of the abundant mRNAGapdh (Figure 5B). Withoutany prior information about Dali genomic binding, we considered itsendogenous site of synthesis to assess the enrichment of transcript-associated DNAloci. Specific enrichment of Dali at its locus was observed asexpected (Figure 5—figure supplement1).
CHART extract was prepared from approximately 3 × 108 N2A cells perpull down and hybridized overnight with 810 pmol biotinylated oligonucleotidecocktail (Supplementary File1) at room temperature with rotation. 250 μl MyOneC1 streptavidinbeads (Invitrogen) were used to capture the complexes overnight at room temperaturewith rotation. After extensive washes, bound material was eluted using RNase H (NewEngland Biolabs (NEB), UK) for 30 min at room temperature. Samples were treated withProteinase K and cross-links were reversed. RNA was purified from 1/5 total samplevolume using the QIAGEN miRNeasy kit. DNA was prepared from the remaining sampleusing the phenol:chloroform:isoamyl alcohol extraction and ethanol precipitationmethod. DNA was further sheared to an average fragment size of 150–300 bpusing a Bioruptor (Diagenode, Belgium) and sequenced on an Illumina HiSeq (50 bppaired end).
Computational analysis of CHART-seq data
CHART-seq was performed with three independent pull down samples (using twoindependent cocktails of 5 C-oligos, and one cocktail containing all 10 C-oligos) andsequenced simultaneously with a matched input sample. 50 bp, paired-end reads weremapped to the mouse genome (mm9) using bowtie with the options ‘–m1–v2 –best–strata–a’. For eachDali sample, peaks were called against the matched N2A inputsample (4208 peaks) and CHART-seq peaks previously analogously identified in N2Acells using two lacZ controls (1928 peaks) (Vance et al., 2014). Peak calls were made using the MACS2algorithm ((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 filteredsuch that only peak calls with a −log10 q value >5 were retained (FDR0.001%).
We discovered 1427 Dali-associated regions genome-wide calledagainst both input and lacZ control samples (Figure 5A; Supplementary file 5).
Characterisation of Dali binding sites
The chromosomal distribution of Dali peaks was visualised using theR Bioconductor package ‘ggbio’ (Yinet al., 2012). Genome territory enrichments analysis was performed usingthe Genome Association Tester (GAT; (Heger et al.,2013)). 10,000 simulations were performed using a mappability filteredworkspace and an isochore file partitioning the genome into eight bins based onregional GC content. For the chromosomal enrichment analyses, chromosomal territorieswere proportionally assigned to a single virtual meta-chromosome before using GAT totest for GC and mappability corrected enrichments as above. Gene Ontology categoriesenriched for Dali binding were identified by intersecting regulatoryregions for known coding genes with Dali binding sites. Regulatoryregions for genes were defined following the GREAT definition (McLean et al., 2010) as a basal domain surrounding the TSS(from −5 kb to +1 kb) and extending domains upstream and downstream tothe nearest gene's basal domain or to a maximum distance of 1 Mb. Enrichments wereidentified using GOToolbox.
Dali peaks were characterised using DNase I hypersensitivity (HS)data generated by the Stamatoyannopoulos lab at the University of Washington andchromatin features identified by the Ren lab at the Ludwig Institute for CancerResearch ((Shen et al., 2012); ENCODE Project Consortium, 2012). Enrichmentsof DNase I HS and chromatin features overlapping Dali peaks were assessed using GATto control for mappability and regional GC content as above.
Complementarity between Dali sequence and binding locations wasassessed using the EMBOSS Water algorithm (Rice etal., 2000) which performs Smith-Waterman alignment with a range of gapopening and extension penalties. RNA-DNA:DNA triplex formation was assessed using theTriplexator search software suit (Buske et al.,2012). The MEME-ChIP (Machanick andBailey, 2011) algorithm was used to perform de novo motif discoveryanalysis by examining the unmasked DNA sequence of the central regions of peaklocations. MEME-ChIP was run with the options ‘-meme-mod zoops -meme-minw 5-meme-maxw 30–meme-nmotifs 50’ using a custom background file preparedfrom regions flanking the peak locations using the command ‘fasta-get-markov-m 2’. Enrichment of known vertebrate transcription factor binding sites fromthe TRANSFAC Professional database (Matys et al.,2006) was assessed using the AME algorithm (McLeay and Bailey, 2010) with the options‘–method fisher–length-correct’ using the sequence andbackground file prepared for MEME-ChIP analysis.
3C
E14 ES cells or day 4 ES-derived neuronal were cross-linked with 2% formaldehyde.Nuclei were prepared and permeabilized with 0.3% SDS in 1.2× restriction buffer(NEB3 for BglII) for 1 hr at 37°C. Then, SDS was sequestered byadding 1.8% Triton X-100. 1 × 106 nuclei (∼15 μg ofchromatin) were digested with 400 units of BglII restriction enzymeovernight, and the enzyme was inactivated. Nuclei were diluted in 1.15× T4 DNAligation buffer (NEB), and SDS sequestered by adding 1% Triton X-100. The digestedchromatin was ligated using 100 Weiss units of T4 DNA ligase for 4 hr at 16°Cand treated with Proteinase K to reverse cross-links. Samples were further treatedwith RNase A, and DNA was phenol-chloroform extracted and ethanol precipitated.
A RP23-92N4 (CHORI; BACPAC) Bacterial Artificial Chromosome (BAC) clone covering thePou3f3-Dali locus was treated as above and used as a controltemplate for the 3C assay. Ligation products of 3C and BAC samples were quantified byqPCR. PCR reactions consisted of 300 ng 3C sample, 0.2 μM test primers and aprimer corresponding to Dali promoter and 1× SYBR Green PCRMastermix (Life Technologies, UK). All reactions were performed in triplicate. Themean threshold cycle (Ct) value was calculated and used to calculate relative amountsof PCR products. To normalise for different primer efficiencies, interactionfrequencies were calculated by dividing the amount of PCR product obtained from the3C sample by the amount of DNA obtained from control BAC DNA. Interaction frequencieswere also normalised to Gapdh internal controls prepared fromgenomic DNA in the same manner as the BAC clone sample. All primers used are listedin Supplementary file1.
COBRA
We used COBRA to study 9 out of 44 CpG island-containing promoters bound byDali and associated with genes differentially expressed betweenstable Dali knockdown and control cell lines prior to or subsequentto the RA-induced differentiation. 80–350 ng of genomic DNA wasbisulfite-treated using EZ DNA Methylation Gold kit according to the manufacturer'sinstruction and used for PCR amplification. Primers for amplifying bisulfiteconverted template DNA were designed using MethPrimer software accessible athttp://www.urogene.org/methprimer/ (Li and Dahiya, 2002). PCR products were on-column purified with QIAquickPCR Purification Kit. 250 ng to 1 μg of purified products were incubated withappropriate COBRA-compatible (BstUI (NEB), MspI(NEB), TaqI (Thermo Scientific), HpyCH4IV (NEB)) orcontrol (Hsp92II (Promega), BfaI (NEB)) restrictionenzymes overnight. Restriction products were analysed on 3% low melting point agarosegels.
TALE-mediated up-regulation
Target regions were selected and TAL effector constructs were designed usingsoftware, tools, and information found on the TAL Effector Nucleotide Targeter2.0 website accessible from https://tale-nt.cac.cornell.edu/. Construction of custom TALE-TFsdesigned to target promoter-proximal region of Dali to up-regulatetranscription from the locus was performed as described by Sanjana et al. (2012). The TALE-TF was designed to target thefollowing region lying upstream of the TSS of Dali: chr1 (mm9):42807019-42807038 ("TGTCCCTTGTCCACATATCT"). The TAL domain sequence used was asfollows: NH NG HD HD HD NG NG NH NG HD HD NI HD NI NG NI NG.
Data deposition
Microarray and CHART-Seq data have been deposited in the GEO database under accessionnumber GSE62035 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE62035).
Acknowledgements
We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for HumanGenetics for the generation of the sequencing data and OXION for use of their microarrayfacility. This project has been funded by the European Research Council (ProjectReference 249869, DARCGENs; KWV, VC, LK), the Medical Research Council (CPP, SNS; andMRC Hub Grant G0900747 91070 for Sequencing) and the Wellcome Trust (Grant Reference090532/Z/09/Z for Sequencing). VC is a recipient of The Darwin Trust of EdinburghPostgraduate research Scholarship.
Funding Information
This paper was supported by the following grants:
http://dx.doi.org/10.13039/501100000781 European Research Council (ERC) 249869, DARCGENs to Vladislava Chalei, Lesheng Kong, Keith W Vance. to Stephen N Sansom, Chris P Ponting.http://dx.doi.org/10.13039/501100000265 Medical Research Council (MRC) The Darwin Trust of Edinburgh Postgraduate Scholarship to Vladislava Chalei.http://dx.doi.org/10.13039/501100000780 European Commission (EC) Human Brain Project to Juan F Montiel.
Additional information
Competing interests
CPP: Senior editor, eLife.
Author contributions
Ethics
Additional files
Major dataset
The following dataset was generated:
The following previously published dataset was used:
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