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RNA. Nov 2009; 15(11): 2013–2027.
PMCID: PMC2764477

Complex architecture and regulated expression of the Sox2ot locus during vertebrate development

Abstract

The Sox2 gene is a key regulator of pluripotency embedded within an intron of a long noncoding RNA (ncRNA), termed Sox2 overlapping transcript (Sox2ot), which is transcribed in the same orientation. However, this ncRNA remains uncharacterized. Here we show that Sox2ot has multiple transcription start sites associated with genomic features that indicate regulated expression, including highly conserved elements (HCEs) and chromatin marks characteristic of gene promoters. To identify biological processes in which Sox2ot may be involved, we analyzed its expression in several developmental systems, compared to expression of Sox2. We show that Sox2ot is a stable transcript expressed in mouse embryonic stem cells, which, like Sox2, is down-regulated upon induction of embryoid body (EB) differentiation. However, in contrast to Sox2, Sox2ot is up-regulated during EB mesoderm-lineage differentiation. In adult mouse, Sox2ot isoforms were detected in tissues where Sox2 is expressed, as well as in different tissues, supporting independent regulation of expression of the ncRNA. Sox2dot, an isoform of Sox2ot transcribed from a distal HCE located >500 kb upstream of Sox2, was detected exclusively in the mouse brain, with enrichment in regions of adult neurogenesis. In addition, Sox2ot isoforms are transcribed from HCEs upstream of Sox2 in other vertebrates, including in several regions of the human brain. We also show that Sox2ot is dynamically regulated during chicken and zebrafish embryogenesis, consistently associated with central nervous system structures. These observations provide insight into the structure and regulation of the Sox2ot gene, and suggest conserved roles for Sox2ot orthologs during vertebrate development.

Keywords: noncoding RNA, Sox2, embryonic stem cells, zebrafish, central nervous system

INTRODUCTION

Over the last decade, transcriptomic studies have revealed that most of the genome of higher eukaryotes is transcribed in developmentally regulated patterns, to produce a complex set of overlapping and interlacing coding and noncoding transcripts from both strands (Mattick and Makunin 2006; Willingham and Gingeras 2006). Over 30,000 putative full-length long (>200 nucleotides [nt]) non-protein-coding RNAs (ncRNAs) have been catalogued in mouse (Carninci et al. 2005), many of which are subjected to splicing, but the biological roles of only a small number have been investigated. Nevertheless, their specific expression patterns, conservation of promoters, splice site sequences and predicted structures, association with particular chromatin signatures, regulation by morphogens and transcription factors, alternative splicing and altered expression in disease states, as well as evidence of evolutionary selection, all suggest that many of these transcripts are functional (Cawley et al. 2004; Carninci et al. 2005; Washietl et al. 2005; Pang et al. 2006, 2007; Pollard et al. 2006; Ravasi et al. 2006; Torarinsson et al. 2006; Ponjavic et al. 2007; Rinn et al. 2007; Dinger et al. 2008a; Mercer et al. 2008; Guttman et al. 2009; Zhang et al. 2009). Indeed, ncRNAs have been shown to regulate gene expression through a large variety of mechanisms, including the control of chromatin structure, transcription, mRNA processing, and translation (Amaral et al. 2008) and are emerging as important players in gene regulation during eukaryotic development (Amaral and Mattick 2008).

Given the lack of currently known intrinsic features that might provide insight into specific long ncRNA functions, a common strategy to study ncRNAs has been the analysis of their expression and genomic relationship with adjacent protein-coding loci. This approach has successfully revealed cis-regulatory roles for several ncRNAs (Sleutels et al. 2002; Feng et al. 2006; Martianov et al. 2007; Beltran et al. 2008; Dinger et al. 2008a; Ebralidze et al. 2008; Pandey et al. 2008; Wang et al. 2008; Yu et al. 2008). Recently, we identified 174 long, spliced ncRNAs that are dynamically regulated during mouse embryonic stem (ES) cell differentiation, many of which are associated and coordinately expressed with genes that have important roles in the differentiation process, including several homeobox-containing genes (Dinger et al. 2008a). In fact, long ncRNA genes are commonly associated with transcription factor loci that control development and are proposed to be a general component in their regulation (Engstrom et al. 2006; Amaral and Mattick 2008). Moreover, a number of ncRNAs exhibit expression profiles similar to genes that have crucial roles in ES pluripotency, such as Sox2, Nanog, and Pou5f1, indicating that long ncRNAs may also participate in the regulatory networks that define the pluripotent state (Dinger et al. 2008a).

Sox2 (SRY-box containing gene 2) is a key regulatory factor involved in establishing and maintaining pluripotency in vertebrate stem cells. It is a highly conserved single-exon gene that encodes a high-mobility group transcription factor expressed very early in development, including in totipotent cells from the morula and inner cell mass of blastocysts, and its ablation causes early embryonic lethality in mice (Avilion et al. 2003; Masui et al. 2007). Conditional knockouts of Sox2 have further demonstrated important roles in conserved ontogenetic processes, especially in neural development (Mizuseki et al. 1998; Bylund et al. 2003; Okuda et al. 2006). Sox2 is required for both neural induction of the ectoderm and maintenance of progenitor and neural stem cells during embryonic and adult neurogenesis (Cai et al. 2002; Wegner and Stolt 2005; Cavallaro et al. 2008; Kim et al. 2008). Sox2 is also expressed in glial precursors and proliferating astrocytes, but it is down-regulated in neurons and in quiescent astrocytes (Ferri et al. 2004; Komitova and Eriksson 2004; Miyagi et al. 2004, 2006; Bani-Yaghoub et al. 2006). However, the mechanisms determining its differential regulation in these neural cell lineages are unclear. In addition, Sox2 is required for mouse retina and lens development (Kamachi et al. 2001; Taranova et al. 2006), and heterozygous deficiency in humans is associated with eye defects, such as microphthalmia and anophtalmia, as well as other abnormalities, including forebrain and hippocampal malformation, and epilepsy (Fantes et al. 2003; Kelberman et al. 2006, 2008; Sisodiya et al. 2006).

Human SOX2 is required for ES self-renewal and pluripotency (Fong et al. 2008). It has recently generated great therapeutic interest as a reprogramming factor (along with Oct4/Klf4/c-Myc or Oct4/Nanog/Lin28) capable of inducing pluripotency when ectopically expressed in a variety of differentiated murine and human cells, the so-called induced pluripotent stem (iPS) cells (Takahashi and Yamanaka 2006; Okita et al. 2008; Park et al. 2008). Neural progenitor cells, which express Sox2, can be reprogrammed into iPS cells when transduced with only Oct4/Klf4, in which case endogenous Sox2 is thought to facilitate reprogramming (Eminli et al. 2008). Therefore, elucidating the mechanisms that regulate Sox2 expression is important to better understand and manipulate its roles in the control of pluripotency.

Multiple mechanisms are implicated in the developmental and tissue-specific regulation of Sox2, notably transcriptional control via an array of highly conserved enhancers located upstream and downstream from the gene, which have stage- and tissue-specific activities (Zappone et al. 2000; Uchikawa et al. 2003, 2004; Catena et al. 2004; Miyagi et al. 2004).

Recently, a study reporting the involvement of mutations in SOX2 in anophtalmia made the intriguing observation that the SOX2 gene lies in an intron of a long multiexon ncRNA that is transcribed in the same orientation (Fantes et al. 2003). The described transcript in humans, named SOX2OT or “Sox2 overlapping transcript” (approved by the HUGO Gene Nomenclature Committee; also “non-protein-coding RNA 43,” HGNC ID:20209), is 3.4 kb long, and shares 88% identity with orthologous sequences in mouse (Fantes et al. 2003). SOX2OT has been postulated to participate in SOX2 regulation (Fantes et al. 2003), but its expression patterns and potential functions have not been studied.

Our group previously observed that at least one isoform of Sox2ot is expressed in the mouse brain in regions of adult neurogenesis (Mercer et al. 2008), indicating that this ncRNA may have roles in processes related to the Sox2 gene. In this work, to obtain insights into its function and regulation, we examined the expression of Sox2ot transcripts in different developmental systems and its relationship to Sox2 expression. We found that Sox2ot is dynamically expressed in different developmental processes in which Sox2 plays important roles, including mouse ES cell and neural cell differentiation, but it is also present in tissues where Sox2 is not detected. Sox2ot orthologs are expressed in different vertebrates, and are regulated during development of mouse, chicken, and zebrafish embryos, with a conserved association with the central nervous system. The expression profiles of Sox2ot, as well as its evolutionarily preserved genomic association with Sox2, suggest that it has conserved functions in vertebrate development, and that it may participate in the regulation of Sox2 or related processes.

RESULTS

Sox2ot transcripts and locus structure

SOX2OT was originally reported as a spliced ncRNA mapping to human chromosome 3q26.3-q27, with an intron overlapping the SOX2 gene in the same transcriptional orientation (Fantes et al. 2003). We analyzed the orthologous region in mouse and observed expressed sequence tags (ESTs) derived from several alternative isoforms of Sox2ot, with multiple alternative promoters, splicing and polyadenylation sites (Fig. 1; Supplemental Fig. 1). AceView annotation (Thierry-Mieg and Thierry-Mieg 2006) supports 16 different canonical GT-AG introns in at least 13 splicing variants and six alternative transcription start sites (TSSs). Four Sox2ot full-length sequences cloned from mouse tissues (Strausberg et al. 2002; Numata et al. 2003; Carninci et al. 2005) range in size from 638 nt (GenBank accession no. BY721402) to ~3.5 kb (accession no. AK031919).

FIGURE 1.
Genomic organization of Sox2ot locus in mouse. (A) Genome Browser (Hinrichs et al. 2006) view of Sox2 overlapping transcripts showing “Full-length clones” (mouse mRNAs from GenBank) and distal “spliced ESTs” tracks (accession ...

Analysis of protein-coding potential of mouse and human Sox2ot/SOX2OT full-length cloned sequences with the software CRITICA, which uses a combination of statistical and comparative parameters, such as open reading frame (ORF) length, synonymous versus nonsynonymous base substitution rates, and similarity to known proteins (Badger and Olsen 1999; Frith et al. 2006), indicates no significant coding potential, although it remains possible that small proteins or peptides are encoded within the transcript (Dinger et al. 2008b).

The existence of poly(A) and 5′ cap structures, inferred from full-length cloning strategies, suggests that Sox2ot is transcribed by RNA polymerase II (RNAP II), and therefore represents a putative “mRNA-like” ncRNA (Numata et al. 2003). Mapping of TSSs of Sox2ot determined from EST clones that include the 5′end of transcripts (Carninci et al. 2005; Kimura et al. 2006) showed several novel isoforms with alternative TSSs (Fig. 1A; Supplemental Fig. 1). We also analyzed published chromatin modification maps of active (tri-methylated histone 3 lysine 4, H3K4me3) or repressed (trimethylated histone 3 lysine 27, H3K4me27) promoters obtained by chromatin immunoprecipitation-sequencing (ChIP-Seq) (Mikkelsen et al. 2007), and found support for multiple TSSs of Sox2ot (Fig. 1A). These promoter regions are associated with noncoding sequence blocks of high conservation in vertebrates (Woolfe et al. 2004, 2007), here termed highly conserved elements (HCEs) 1–7 encompassed in the Sox2ot locus. These blocks also largely correspond to “transposon free regions” (TFRs) (sequences over 5 kb long, which have remained resistant to transposon fixation throughout vertebrate evolution and appear to comprise extended regulatory sequences controlling the expression of genes that are central to early development) (Fig. 1A; Supplemental Fig. 1; Simons et al. 2006). The novel transcripts include a distal isoform associated with HCE 1, whose TSS is located more than 0.5 Mb upstream of Sox2 in mouse, and is hereafter referred to as Sox2dot (“Sox2 distal overlapping transcript”). HCE 1 has been shown to have enhancer activity specific to the forebrain of developing mouse embryos (Supplemental Fig. 2; Pennacchio et al. 2006).

In addition, we identified a putative promoter between HCEs 2 and 3 that is marked by H3K4me3 and H3K27me3 modifications, as well as a TFR, but which is not associated with any mapped alternative TSS of Sox2ot or any EST sequence (Fig. 1A). However, we found a novel ncRNA associated with this element from a published collection of recently identified long intergenic ncRNAs (lincRNAs) (Guttman et al. 2009; data not shown). This, and additional ESTs located in the introns of Sox2ot, further highlight the transcriptional complexity associated with the Sox2ot locus.

Expression of Sox2ot in mouse tissues and ES cell differentiation

Inspection of the distribution of spliced ESTs in mouse tissues indicated a predominance of Sox2ot transcription in the central nervous system (CNS) (Supplemental Table 1). Moreover, analysis of RNA deep sequencing (RNA-Seq) data (Cloonan et al. 2008; Mortazavi et al. 2008) showed the presence of alternative splicing isoforms of Sox2ot in mouse brain, as well as in pluripotent and differentiating embryonic stem cells (Fig. 1B).

Because of the importance of Sox2 in these systems, we first examined the regulation of Sox2ot, along with Sox2, in undifferentiated mouse ES cells and during differentiation as embryoid bodies (EBs) over a 16-d time course (see Materials and Methods) using quantitative real-time PCR (qRT-PCR). Similarly to Sox2, Sox2ot is expressed in pluripotent ES cells and is initially down-regulated upon EB differentiation (Fig. 2A). However, unlike Sox2, the ncRNA is reactivated after 6 d of differentiation (Fig. 2A), which coincides with induction of mesoderm differentiation programs (Bruce et al. 2007; Dinger et al. 2008a). We did not detect expression of the Sox2dot isoform in these samples at any time point (data not shown).

FIGURE 2.
Expression profile of Sox2 and Sox2ot in mouse ES cells and tissues. (A) qRT-PCR analysis of Sox2 and Sox2ot levels in EB over a 16-d differentiation time course (expressed relative to day 0), and (B) in differentiating EBs treated (solid lines) or nontreated ...

In addition, to confirm the transcription of Sox2ot by RNAPII and to assess transcript stability, we treated mouse ES cells with RNAPII inhibitor α-amanitin for 12, 24, and 48 h, and quantified the levels of Sox2 and Sox2ot in treated and nontreated cells during EB differentiation. While Sox2 mRNA levels are dramatically reduced after 12 h of treatment, Sox2ot levels were not significantly affected in the same period and significant reduction was observed only after 24 h of treatment (Fig. 2B). Similar results were observed using nondifferentiating ES cells treated with α-amanitin (data not shown), indicating that Sox2ot is more stable than Sox2 mRNA in ES cells.

By examination of Sox2ot expression in mouse embryos (E10–E12 days post-fertilization [dpf]) and in adult brain using Northern blot, we detected a major band of ~3 kb (Fig. 2C). The expression was stronger in the brain, where multiple additional bands of ~1, 4, 6, and >10 kb, presumably consisting of alternative isoforms, can be detected. To further evaluate the regulation of the expression of Sox2ot RNAs and the relationship with Sox2 in mouse, we analyzed their expression in mouse embryo, adult tissues, and cell lines by RT-PCR. By using a primer pair that detected Sox2ot last exon (“Sox2ot a”) (Fig. 1B), the ncRNA was detected in mouse embryo (E10–E12 dpf) and in different adult mouse tissues, both in tissues where Sox2 was also present, such as brain, lung, and heart (Fig. 2D, right panels), and in tissues where the mRNA was not detected, such as testis (Fig. 2D, left panels). Using primers spanning an exon–exon junction (“Sox2ot b”) (Fig. 1B), a cassette exon was detected, which is differentially regulated in mouse tissues (Fig. 2D, cf. lower band of “Sox2ot b” in brain and ES cells). The bands corresponding to the different isoforms were sequenced to confirm their identity, and the regulated expression of isoforms was also evidenced by detection of different splicing junctions in the RNA-Seq data from different sources (Fig. 1B).

Sox2dot isoform was present in whole embryo and newborn mouse, but was detected only in the brain among the adult tissues tested (Fig. 2D). The expression of Sox2dot and isoforms of Sox2ot with alternative TSSs in the mouse brain is supported by the presence of H3K4me3 histone modifications associated with their promoter regions (Fig. 1A). In addition, the observation that H3K27me3 modifications can also be associated with alternative TSSs indicates that initiation of Sox2ot transcription from different promoters can be regulated at the chromatin level (Fig. 1A).

Sox2ot and Sox2dot are dynamically regulated during mouse neurogenesis

Sox2ot expression was previously observed in the mouse brain in zones of adult neurogenesis (Mercer et al. 2008). This pattern was detected by in situ hybridization (ISH) with a probe targeting a long (~2.8 kb) last exon (Fig. 1B, GenBank accession no. AK031919). In addition, Sox2ot ESTs are found in mouse differentiating neural stem cells libraries (Supplemental Table 1), and its expression can be detected in a variety of mouse primary neuronal cell cultures (data not shown).

To further explore the possible involvement of Sox2ot in neural differentiation processes, we next examined the expression of the RNAs using the “neurosphere assay,” an in vitro system of neurogenesis with cultures of neurospheres originated from neural stem cells and undifferentiated precursors present in the adult mouse subventricular zone (Reynolds and Rietze 2005, 2006). Neurospheres were cultured for up to 7 d in differentiation media, which progressively enriches for neurons and glial cells (Rietze and Reynolds 2006). Analysis of Beta-III tubulin (Tubb3) and Glial fibrillary acidic protein (Gfap) mRNA expression by qRT-PCR indicates an increasing enrichment for neurons and astrocytes, respectively, after 2 and 7 d of culture (Fig. 3A). Sox2 and Sox2ot were present in the cultured neurospheres (passage 2) (Fig. 3A), observed at similar levels after eight passages (data not shown). In addition, both transcripts were concordantly up-regulated in neurospheres after 2 d of culture in differentiation medium, and slightly down-regulated after 7 d, a pattern similar to that observed for Nestin (Fig. 3A). Sox2ot absolute expression levels were significantly higher in these neural cell types than in ES cells (data not shown).

FIGURE 3.
Sox2ot dynamic expression in mouse adult neurogenesis. Detection of (A) Sox2 and Sox2ot, and (B) Sox2dot, in mouse differentiating neurospheres by qRT-PCR (expressed relative to Hprt levels). Levels of neural markers Nestin, Beta-III tubulin (Tubb3), ...

Sox2dot was expressed at low levels in neurospheres, but showed a marked increase after 7 d of culture, indicating specific expression in the more differentiated population of neurons and glial cells (Fig. 3B). We then performed ISH to examine the expression of Sox2dot in adult mouse brain sections. Like Sox2ot, Sox2dot was also detected in zones of adult neurogenesis (Zhao et al. 2008) in the subventricular zone, rostral migratory stream and olfactory bulb, with a higher intensity in the latter (Fig. 3C), which contains a higher number of differentiated neurons, consistent with the expression observed in the neurospheres enriched in differentiated cells.

Although the in vitro neurosphere differentiation system originates a heterogeneous population of cells, including progenitors and differentiating neuronal and glial cells (Jensen and Parmar 2006), these results suggest that Sox2ot isoforms are present in neural cells and are dynamically regulated during differentiation. Interestingly, highly enriched binding of neuronal transcriptional repressor NRSF/REST has been previously validated in conserved sites upstream of Sox2ot (Mortazavi et al. 2006). NRSF/REST has been proposed to target several long ncRNAs in mouse and human neural cell differentiation models, and may represent a general mechanism of regulation of ncRNA expression in neuronal development (Johnson et al. 2009).

Sox2ot is transcribed from highly conserved elements in vertebrates

We observed that multiple SOX2OT TSSs in humans, determined by 5′-end sequencing of “oligo-cap” cDNA libraries (Kimura et al. 2006), are associated with orthologous HCEs (Supplemental Fig. 1; Supplemental Table 1). Analysis of ultraconserved elements (UCEs), blocks with at least 100 base pairs (bp) that have 100% conservation in placental mammals (Stephen et al. 2008), showed that the ~850 kb locus spanned by SOX2OT/DOT transcripts in humans contains 46 UCEs that occupy 1.09% of this region (average block size of 204 bp) (Supplemental Fig. 1A). This represents 15.6-fold enrichment relative to the entire genome (UCE coverage of 0.07%, average block size of 155 bp), underlying a high density of functional elements spanned by SOX2OT transcription. The longest UCE in the region in humans (646 bp long) corresponds to HCE 1, which in mouse comprises a sequence of 554 bp that presents 95% identity with human and 83% with zebrafish genomic sequences.

As observed in mouse, these HCEs associated with SOX2OT TSSs closely correspond to TFRs in humans; which further suggests that these sequences have been subjected to functional constraints across mammals (Supplemental Fig. 1). Moreover, HCEs upstream of Sox2 are also identified in chicken (Uchikawa et al. 2004) and zebrafish genomes (Supplemental Fig. 1), and associated Sox2ot ESTs are not only found in these species, but also in other vertebrates with low EST coverage, such as Xenopus, cow, macaque, and orangutan (data not shown). Not all HCEs associated with Sox2ot TSSs in human and mouse are associated with TSSs in zebrafish (Supplemental Fig. 1), but this may be due to the small EST coverage and lack of 5′ end-cloned EST information in the latter.

At the primary sequence level, although Fantes et al. (2003) reported an identity of 88% of human SOX2OT transcribed from HCE 4 and 5 with mouse ESTs, the sequence similarity is restricted to specific regions of the transcript, and, because different exons have different conservation levels, the overall sequence conservation varies among alternative isoforms (Supplemental Fig. 1A). For example, the sequence of 2,971 bases [excluding the poly(A) tail] from Sox2ot isoform transcribed from HCE 4 in mouse (GenBank accession no. BC057611) is highly conserved in the rat genome, presenting ~90% identity in a continuous stretch of 2967 bases and all GT-AG splice sites conserved. However, the same sequence restricts to 384 nt with 91.2% identity in the human genome (concentrating in the exons immediately flanking Sox2, whose splice sites are also conserved). These stretches can overlap highly conserved elements, such as HCE 7 that falls inside the last exons of mouse and human Sox2ot/SOX2OT (Supplemental Fig. 4). This element is part of an evolutionarily constrained region that encompasses a sequence of 106 nt predicted to form a conserved secondary structure (RNAz, P > 0.9) (Fig. 1B; Supplemental Fig. 3), which may represent a conserved functional element within Sox2ot. Finally, some exons of SOX2OT may be species- or clade-specific, as evidenced by the exon between HCEs 1 and 2, which is present in all SOX2DOT ESTs in humans and has splice site conserved only in primates, but is not detected in mouse (not present in ESTs nor detected by RT-PCR) (Supplemental Fig. 1A,B, data not shown).

Developmental regulation of Sox2ot in vertebrates

In light of the association of Sox2ot with highly conserved sequences and the identification of ESTs in different vertebrates, we evaluated whether Sox2ot transcripts are present and regulated during vertebrate embryonic development by examining the expression of Sox2 and Sox2ot/dot orthologs in chicken and zebrafish embryos (Fig. 4). Using qRT-PCR, Sox2ot was detected during chicken development, with increasing levels observed after 2 d of embryonic development, similar to the pattern observed for Sox2 (Fig. 4A), although with lower absolute expression levels. Expression of both Sox2 and Sox2ot was enriched to the head of 6.5-d chicken embryos, compared to the limbs and whole body (Fig. 4A).

FIGURE 4.
Expression of Sox2ot orthologs in different vertebrate species. (A) Relative expression of Sox2 and Sox2ot in chicken embryos as determined by qRT-PCR (relative to 2.5-d embryos). (B–D) Expression of sox2 and sox2ot/dot in zebrafish embryos determined ...

In zebrafish embryos, Northern blot analysis of sox2ot expression at 10, 30, and 78 h post-fertilization (hpf) stages showed a major band of ~2.5 kb at 30 hpf, as well as less intense bands of ~1.5 and ~6 kb, which are also present in the other stages (Supplemental Fig. 4A), indicating developmental regulation of alternative isoforms. Moreover, PCR using cDNA from zebrafish embryos and primers positioned in the first and last exons of sox2ot EST (accession no. EB947627), followed by sequencing, demonstrated the presence of different splicing isoforms (Supplemental Fig. 1C), including an isoform that skips the exon associated with HCE 5 and an isoform that contains a novel exon of 207 bases positioned between HCE 4 and 5 (deposited as GenBank accession nos. GO599877, GO599878, and GO599879).

In qRT-PCR analysis of a zebrafish developmental series from two-cell stage embryos to 5 dpf, sox2 and sox2ot presented concordant up-regulation after sphere stage (Fig. 4B). This corresponds to the activation of zygotic transcription at midblastula transition, suggesting that sox2 and sox2ot are both early zygotic genes in zebrafish. However, there is an apparent divergence in their expression patterns after tailbud stage when sox2ot total levels are reduced, although later in development at 5 dpf, both sox2 and sox2ot are highly expressed (Fig. 4B). Interestingly, after zygotic activation, sox2dot is expressed at very low levels, but is dramatically induced at 5 dpf (Fig. 4D). By this developmental stage, the zebrafish has completed most aspects of organogenesis and already contains well-developed neuroanatomical features (Kimmel et al. 1995), a result that may parallel the expression of Sox2dot in mouse brain and in differentiated neurosphere cells.

To explore the temporospatial expression pattern of the transcripts in zebrafish embryos, we used whole-mount ISH with riboprobes for sox2 and sox2ot at tailbud and 28 hpf stages. We detected presumptive neurectodermal expression of sox2 at tailbud stage, as previously described (Okuda et al. 2006). At the same stage, sox2ot was also expressed in neurectodermal tissue, but with a more diffused pattern (Fig. 4C). Later at 28 hpf, both transcripts were highly expressed throughout the developing brain and eyes, and also at lower levels posteriorly in the neural tube (Fig. 4C). Specific expression of sox2ot in the retina and CNS was also observed by section ISH using 48 hpf embryos (Supplemental Fig. 4), and this was maintained in the brain throughout embryonic development until at least 6 dpf (data not shown). However, sox2ot expression at 28 hpf was broader, observed at high levels in the pharyngeal arches, but lower in some regions of sox2 expression, such as the otic placodes. Moreover, at this stage, unlike sox2, sox2ot was also detected at posterior mesoderm, with distinct expression in the gut, lateral mesoderm, and posterior blood island (Fig. 4C), a pattern that was supported by section ISH with 48 hpf embryos (Supplemental Fig. 4). These results point to a complex expression pattern for sox2ot, which overlaps with sox2 particularly in the CNS, but presents a broader distribution, especially in mesoderm tissues during development.

Finally, to gain insight into the expression of SOX2OT in humans, we used EST and published expression data information to examine transcript distribution. Available microarray expression data for several normal human tissues show the highest expression of SOX2OT in the brain and spinal cord (GEO Data Set Records GDS423 and GDS1085) (Shyamsundar et al. 2005; Yanai et al. 2005). qRT-PCR analysis of several human tissues confirmed that the levels of SOX2OT are orders of magnitude higher in the brain, where SOX2 is also predominantly expressed, but is also detected in other tissues such as testis (Fig. 4E). In addition, we analyzed the presence of 202 spliced ESTs originating from the SOX2OT-associated HCEs in different EST libraries (Supplemental Table 1). The majority of SOX2OT spliced ESTs in humans (85% or 172/202 ESTs) originate from HCE 5, which is also the HCE with the highest number of associated Sox2ot ESTs in mouse (Supplemental Table 1). Consistent with the microarray and qRT-PCR data, the relative EST frequency of SOX2OT in human tissues (see Materials and Methods) indicates a predominance in CNS libraries, including fetal brain and eyes, with only 16 out of 202 spliced ESTs not assigned to brain or eye tissues (of which two were obtained from testis and 10 from kidney tumor) (Fig. 4F), with similar results in mouse (Supplemental Table 1). The tissue with greatest enrichment in human SOX2OT ESTs is “lens,” with a proportion of 17.5% of SOX2OT expression relative to all libraries (although this library has a smaller coverage, with ~1300 ESTs), followed by subthalamic nucleus (13.5%) and Alzheimer cortex (13%), as well as an enrichment in kidney tumor libraries (7.4%), while it is found in lowest relative abundance in cerebellum (0.3%). It is important to notice that we only considered spliced ESTs, for which there is information about the transcript orientation and connectivity to SOX2OT. Nonetheless, this distribution shows that SOX2OT is highly enriched in the human brain and suggests that its expression is spatially regulated.

DISCUSSION

Most RNAs transcribed from eukaryotic genomes have little or no protein-coding potential, and the vast majority is currently defined as “transcripts of unknown function” (TUF) (Willingham and Gingeras 2006). A first necessary step toward their characterization is to determine the tissues and physiological processes in which such RNAs are expressed and may play specific roles. Several studies have shown specific expression of large numbers of ncRNAs (Ravasi et al. 2006; Louro et al. 2007; Nakaya et al. 2007; Dinger et al. 2008a; Mercer et al. 2008; Sunwoo et al. 2008). Moreover, long ncRNAs are dynamically regulated during animal development, supporting the idea that they are involved in differentiation and developmental processes (Amaral and Mattick 2008).

An emerging common feature of the set of conserved genes for regulatory proteins that direct multicellular development is their genomic association with long ncRNAs, with accumulating examples of ncRNAs that regulate their associated genes at different levels (Amaral and Mattick 2008). This set includes the transcription factor SOX2, embedded within the intron of a putative long ncRNA, SOX2OT, which was proposed to play a role in the transcriptional regulation of SOX2 expression (Fantes et al. 2003). In mouse, Sox2 regulates a large number of genes (Boyer et al. 2005; Chen et al. 2008), and precise regulation of its dosage is critical for the temporal and spatial regulation of differentiation processes, with small changes dramatically altering the self-renewal and pluripotency capacity of ES cells (Taranova et al. 2006; Kopp et al. 2008). Indeed, Sox2 is thought to function as a molecular “rheostat” controlling key transcriptional regulatory networks in stem cells (Boer et al. 2007; Kopp et al. 2008). However, the mechanisms regulating Sox2 expression are still being elucidated, and include regulation at the chromatin structure level (Sikorska et al. 2008), multiple transcription factors (Boyer et al. 2005), and post-transcriptional regulation by microRNAs (Tay et al. 2008).

In this work, we examined the expression patterns of Sox2ot and Sox2 in developmental systems, especially those where Sox2 has well-characterized roles. We found that Sox2ot is a stable RNA transcribed in mouse ES cells, and its expression profile was similar to Sox2 in mouse early differentiating EBs and neurospheres, as well as in some adult tissues. However, Sox2ot was also differentially regulated and present in tissues where Sox2 was not detected, indicating that expression of Sox2ot does not simply reflect the transcriptional status of Sox2. Indeed, sox2ot expression in zebrafish embryos was broader than for sox2, including in mesodermal tissues, a result that may parallel the up-regulation of Sox2ot in mouse ES differentiation into mesoderm lineages.

These expression patterns indicate that Sox2 and Sox2ot may have complex functional relationships. Because Sox2ot expression overlapped with Sox2 expression in all of the different systems examined, Sox2ot could be involved in the activation of Sox2 in some tissues. If this is the case, the fact that Sox2ot is also present in tissues where Sox2 is not detected indicates that it is not sufficient for the activation, but must act in conjunction with the different regulatory factors that control the expression of Sox2 (see above). Nevertheless, although the conserved association between Sox2ot and Sox2 in vertebrates supports a functional relationship, Sox2ot may have additional roles that are unrelated or tangential to the regulation of Sox2. The evolutionarily preserved association and the overlap in the expression patterns could be a consequence of the sharing of regulatory elements that control the expression of both Sox2ot and Sox2. Finally, regulation of Sox2ot by Sox2 is also possible, but we did not find evidence of Sox2 binding in Sox2ot proximal promoter regions in published ChIP-Seq data for Sox2 in mouse ES cells (Chen et al. 2008; data not shown).

Supporting a role for Sox2ot in vertebrate development, we observed that the expression of the ortholog transcripts is differentially regulated in human tissues and dynamically modulated in zebrafish and chicken embryos. The observed expression was consistently associated with CNS tissues, which is a pattern observed for a large number of ncRNAs that may play roles in animal brain development and function (Amaral and Mattick 2008). Nevertheless, while the inferred promoter regions of Sox2ot are highly conserved, the primary sequence of the transcript has conservation in limited regions and contains repeat elements, features that are also present in other characterized ncRNAs (Amaral and Mattick 2008). In addition, these observations are consistent with previous findings that ncRNA sequences have more plasticity to evolve (Pang et al. 2006), but have also, on average, higher promoter conservation compared to protein-coding genes (Carninci et al. 2005; Ponjavic et al. 2007).

We observed that several Sox2ot isoforms originating from alternative TSSs are also associated with chromatin modifications characteristic of bona fide promoters and with HCEs (Fig. 1; Supplemental Fig. 1). Indeed, it is known that when HCEs (which often have enhancer activity) are localized in introns, they are generally associated with alternative downstream TSSs from the host gene (Vavouri et al. 2007). Moreover, the upstream TSSs observed for Sox2ot isoforms are common in the organization of the metazoan transcriptome, in which many transcripts have distal 5′ exons, sometimes located several hundreds of kilobases upstream, and are expressed from alternate promoters that are tissue- or cell-line-specific (Kimura et al. 2006; Manak et al. 2006; Denoeud et al. 2007). We found evidence that Sox2ot alternative promoters can be differentially regulated, as indicated by Sox2dot, a novel isoform of Sox2ot that has a unique tissue expression pattern (detection in adult mouse restricted to brain) and different profiles in the ES and neurosphere differentiation assays. The differential regulation of Sox2ot isoforms, including the extensive occurrence of alternative splicing, may indicate specific roles for these different transcripts.

Similar to Sox2ot, the long spliced ncRNAs Evf-1 and Evf-2 are transcribed from alternative TSSs that correspond to ultraconserved enhancers associated with the Dlx5/6 genes, which have important roles in development (Kohtz and Fishell 2004; Feng et al. 2006). Interestingly, in mouse neural cells, the Evf-2 isoform was shown to mediate the transcriptional activity of the associated ultraconserved element by interacting and recruiting the Dlx2 transcriptional activator to the enhancer (Feng et al. 2006). Most recently, the function of Evf-2 was analyzed in mouse embryos by insertion of premature termination sites that truncate Evf-2, which caused a specific neural phenotype and demonstrated regulation of the enhancer by the ncRNA in vivo (Bond et al. 2009). A similar mechanism has been suggested for ncRNAs transcribed from highly conserved elements associated with other Dlx genes (Dinger et al. 2008a). Given the high density of conserved elements in the locus and the association of Sox2ot transcripts with different HCEs, including Sox2dot with the HCE 1 neural enhancer, one possibility is that Sox2ot transcripts or transcription are also involved in regulation of enhancer activity.

Sikorska et al. (2008) showed that epigenetic modifications of the SOX2 enhancers SRR1 and SRR2A correlate with high level of SOX2 expression during cell differentiation (Sikorska et al. 2008), and here we observed that Sox2ot-associated HCEs are marked by chromatin activation and/or repression marks (Fig. 1A). In addition, the region surrounding Sox2 flanked by Sox2ot exons corresponds to a very large TFR (>30 kb in mouse and human), and comprises a broad region (>20 kb) marked by histone modifications characteristic of transcriptionally active promoters (H3K4me3) in the mouse brain (Fig. 1A; Supplemental Fig. 1). This unusual chromatin structure has been reported in mouse ES cells, in which transcriptional elongation marks (H3K36me3) also extend far downstream (>15 kb) from the Sox2 annotated 3′ end (Mikkelsen et al. 2007). Considering these features associated with the chromatin at Sox2 enhancers and flanking region, it is also possible that Sox2ot plays a role in regulating chromatin structure, a rapidly emerging theme for regulatory RNAs (Rinn et al. 2007; Ebisuya et al. 2008; Pandey et al. 2008; Yu et al. 2008; for review, see Mattick et al. 2009). Indeed, recent evidence indicate that a large proportion of lincRNAs, which are also conserved long, spliced ncRNAs, directly associate with different chromatin modifying complexes and can affect the expression of genes that are targeted by these complexes (Khalil et al. 2009). Finally, a regulatory role for Sox2ot may depend on the act of transcription itself, as observed for specific ncRNA loci involved in chromatin and transcriptional remodeling in yeast and mammalian cells (Ebisuya et al. 2008; Hirota et al. 2008). However, the stability, regulated splicing patterns, predicted secondary structure, and conservation of Sox2ot transcripts (Fig. 1) suggest that the transcript has intrinsic functions.

Gain- and loss-of-function strategies to determine Sox2ot mechanism of action and relationship with Sox2 are warranted, for example, using morpholinos to block sox2ot splicing in zebrafish embryos and siRNA knockdown in ES cells, an approach that has proved successful in identifying functional roles for an increasing number of ncRNAs (Mattick 2009). The elucidation of these roles may help to understand the different developmental processes, and potentially the related disorders, in which Sox2ot is expressed. Linkage disequilibrium analysis has already implicated the upstream regions of SOX2OT as a myopia common susceptibility locus, and the ncRNA was suggested to be a plausible candidate (Andrew et al. 2008), with perturbations in its expression patterns possibly involved, as has also been proposed for ncRNAs present in other disease susceptibility loci (Shirasawa et al. 2004; He et al. 2009).

In conclusion, this study provides insight into the range of structural isoforms, epigenetic modifications, and expression patterns of the Sox2ot gene and its transcripts. It also highlights the hidden complexity of such loci and of the biological processes in which they are likely to participate, consistent with the emerging view of the eukaryotic genome as an RNA machine (Amaral et al. 2008). Indeed, another ncRNA located ~100 kb downstream from Sox2 was recently shown to be expressed specifically in mouse ES cells and to be a direct target of Sox2 and Oct4, as part of a collection of over a thousand novel spliced ncRNAs identified (Guttman et al. 2009). Similar to the Sox2ot locus, identification and characterization of unnanotated transcripts with orthologs in amenable model organisms may represent a general approach to explore the roles of ncRNAs in developmental processes.

MATERIALS AND METHODS

Mouse tissue samples and cell culture

Mouse W9.5 ES cells at low passage number were maintained in 15% fetal calf serum (FCS) on mitotically inactive MEFs with 1000 U/mL LIF, as previously described (Bruce et al. 2007; Dinger et al. 2008a). Embryoid bodies were differentiated in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS in 1% methylcellulose (GIBCO 10912-012), which efficiently induces mesoderm-differentiation programs (Bruce et al. 2007; Dinger et al. 2008a). For RNA Polymerase II inhibition experiments, ES cells were seeded at 1 × 105 cells/mL in 6 cm plates in 10% FCS DMEM with or without 20 μg/mL α-amanitin (Sigma A2263) (and with or without LIF) and harvested after 0, 12, 24, and 48 h of culture. Whole neurospheres were generated from adult neural stem cells and proliferating precursor cells harvested from the subventricular zone of 12 mo old mice, passaged and differentiated as described by Rietze and Reynolds (2006), and were kindly provided by Beatrice Large and Rod Rietze. Mouse mammary epithelial HC11 cells (Ball et al. 1988) were cultured in RPMI 1640 medium supplemented with 10% FCS, 5 μg/mL insulin, and 10 ng/mL EGF. Mouse testis-derived cell lines TM3 (ATCC no. CRL-1714) and TM4 (ATCC no. CRL-1715) were cultured in high-glucose DMEM medium containing 10% FCS, 2.5 mM L-glutamine and 0.5 mM sodium pyruvate.

Mouse tissues dissected from adult CD1 or C57BL mice, and embryos dissected from pregnant C57BL mice at 10, 11, and 12 dpc (E10–E12) were frozen in liquid nitrogen and stored at −80°C until later used for RNA extraction.

RNA isolation and RT-PCR expression analysis

RNA from zebrafish embryos, mouse tissues, and cell cultures was purified using Trizol (Invitrogen) or RNeasy Mini Kit (Qiagen) and treated with DNase I (Invitrogen), according to instructions of the manufacturers. RNA samples from chicken embryos were kindly provided by Evgeny Glazov, and total RNA from human tissues was purchased from Ambion (AM6000). cDNA preparation and quantitative real-time PCR (qRT-PCR) analysis were performed as described (Bruce et al. 2007). Primers were designed based on Sox2ot EST sequences available for different vertebrates, spanning splice sites in most cases (Supplemental Table 2), and PCR products were sequenced to confirm the identity of the fragments. For normalization of transcript expression levels, Hprt and eF1α were used as internal controls in mouse and zebrafish samples, respectively. For qRT-PCR analysis of α-amanitin inhibition experiments, as well as for human and chicken expression analysis, cDNA was produced using random primers and expression values normalized relative to 18S RNA expression. For mouse tissue expression analysis, cDNA was used in PCR for 35 cycles and amplification products were visualized after electrophoresis in 2%–3% agarose gels. For in situ hybridization (ISH) probe preparation, cDNA from ES cells, and mouse or zebrafish embryos was amplified (see primers in Supplemental Table 2) and PCR products were cloned into pGEM-T Easy Vectors (Promega) (sequenced to confirm the identity of the insert), which were then used in PCR with T7 and SP6 primers to generate DNA templates for in vitro transcription reactions. ISH probe sequences are listed in Supplemental Table 2.

Northern blot analysis

For Northern blot analysis, 10 μg of total RNA were electrophoresed at 90 V for 3 h and 20 min through 1.2% agarose gels containing 1.1% formaldehyde in buffer 1X MOPS, 0.2 M sodium acetate (pH 7.0), and 0.01 M Na2EDTA (1 M stock solution to pH 8.0). After electrophoresis, RNA gels were rinsed twice in distilled water and then washed in 10X SSC (3 M NaCl, 0.3 M sodium citrate at pH 7.0) at room temperature for 45 min. RNA was transferred overnight to Hybond-N+ membranes (Amersham Biosciences) by capillary blotting and fixed to the membranes by baking at 80°C for 2 h. The blots were probed with 32P-labeled probes (Supplemental Table 2), which were prepared by PCR and purified amplification products labeled using Megaprime DNA Labeling Systems (Amersham Biosciences), following the manufacturer's instructions. Band size was estimated using 0.5–10 kb RNA Ladder (Invitrogen).

Zebrafish material and in situ hybridization

AB strain zebrafish stocks were reared and embryos obtained as described (Westerfield 1994). Embryos were incubated at 28.5°C and staged as previously described (Kimmel et al. 1995). All experiments were performed subject to animal ethics committee approval (AEC no. IMB/167/06/ARC).

For whole-mount in situ hybridization with zebrafish embryos, riboprobe templates were generated by PCR using primers that included T7/SP6 promoter sequences as described above. Riboprobe synthesis and in situ hybridizations were performed as previously described (Wilkins et al. 2008), and section ISH performed with 7 μm sections, as described by Wilhelm et al. (2007).

Electronic expression and secondary structure analysis

Available microarray expression data for SOX2OT in several normal human tissues were obtained from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/), deposited under GEO Data set Record GDS423, which contains expression data for 12 normal human tissues interrogated with Affymetrix GeneChip HG-U95B (ProbeSet 55950) (Yanai et al. 2005), and Data set Record GDS1085, which analyzed the expression profile of 35 different types of normal tissues (ProbeSet 34804) (Shyamsundar et al. 2005).

For electronic expression analysis, SOX2OT spliced ESTs in human tissue libraries present in GenBank were catalogued, and the number of SOX2OT EST clones was computed for each tissue library (Supplemental Table 1). SOX2OT EST frequency (f) in each tissue was calculated by dividing the number of SOX2OT EST clones (x) by the total number of clones per tissue (n). Finally, the “relative SOX2OT expression” in human tissues was obtained by normalizing the frequency in each library (f) by the total number of SOX2OT ESTs in all libraries (expressed as a percentile) (Fig. 4F; Supplemental Table 1).

In the secondary structure analysis, a 106 nt region associated to the conserved sequence of Sox2ot was identified by RNAz (Washietl et al. 2005) with P > 0.9, as described by Dinger et al. (2008a). Additional secondary structure analysis was performed using the most recent RNAalifold algorithm with RIBOSUM scoring (http://rna.tbi.univie.ac.at/cgi-bin/RNAalifold.cgi). RNAalifold predicts the consensus structure from a set of aligned sequences taking into account both thermodynamic stability and sequence covariation (Bernhart et al. 2008). The 17-Way species multiple genome alignment from the UCSC genome browser was used as input.

SUPPLEMENTAL MATERIAL

Supplemental material can be found at http://www.rnajournal.org.

ACKNOWLEDGMENTS

We thank Ke-lin Ru for laboratory assistance, Martin Smith for assistance with secondary structure analysis, Beatrice Large and Rod Rietze for providing cultured neurospheres, Evgeny Glazov for providing chicken embryonic RNA, and Marcel Dinger for critical reading of the manuscript. This work was supported by the Australian Research Council (Grant nos. FF0561986 and S00001543) and The University of Queensland, and the experiments with zebrafish were supported by the Australian Zebrafish Phenomics Facility (NHMRC Grant no. 455871).

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1705309.

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