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.

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 numbers on the left). *The depicted ESTs represent some of Sox2ot splicing isoforms (for complete list of spliced ESTs see Supplemental Table 1), including “Sox2 distal overlapping transcript” (Sox2dot). Mouse highly conserved elements (HCEs 1–7) associated with Sox2ot start sites and exons in mouse or human (guide gray boxes, see Supplemental Fig. 1) are depicted by a Condor identifier (http://condor.fugu.biology.qmul.ac.uk/) (Woolfe et al. 2007). Also indicated are the Multiz 17-way vertebrate conservation (phastCons score histogram), TFRs, H3K4me3 (green), and H3K27me3 (red) chromatin marks from mouse brain obtained from UCSC Genome Browser tracks. (B) Detailed view of Sox2ot region proximal to Sox2, indicating positions of primer pairs used in RT-PCR analysis of Sox2 (blue arrows) and Sox2ot expression (opposite arrows below and above transcript representing “Sox2ot a” and “Sox2ot b” primer pairs, respectively); splicing isoforms identified by available RNA-Seq data in mouse brain (blue), undifferentiated ES cells (green), and differentiating EBs (orange). The positions of an RNAz predicted secondary structure (P > 0.9) (Supplemental Fig. 3) and the Allen brain probe (ABA probe) used to determine Sox2ot expression (Mercer et al. 2008) in mouse brain sections are 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 (dashed lines) with α-amanitin (expressed relative to 0 h of treatment). Error bars show standard deviation (SD) for at least three replicates. (C) Northern blot detection of Sox2ot expression in mouse embryos and adult brain. (D) RT-PCR performed with different primer pairs (see Materials and Methods for primer sequences) detecting different regions and isoforms of Sox2ot (“Sox2ot a,” “Sox2ot b,” and Sox2dot). *Embryo: RNA pool from E10–E12 dpf embryos. RT-: No reverse transcriptase control with embryonic RNA.

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), and Glial fibrillary acidic protein (Gfap) were also measured, and error bars showing SD were determined from at least three replicates. (C) In situ hybridization of sagittal adult mouse brain section showed expression of Sox2dot in zones of adult neurogenesis. Detailed anterior view indicating the olfactory bulb (OB), rostral migratory stream (RMS), and subventricular zone (SVZ).

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 by qRT-PCR (B,D) (expressed relative to eF1α levels), and whole mount in situ hybridization with zebrafish embryos at tailbud stage and 28 h post-fertilization (hpf) embryos (C; anterior to the top with lateral view of embryos in upper and middle panels, and dorsal view in lower panel). alpm: Anterior lateral plate mesoderm, ba: branchial arches, Fb: forebrain, Hb: hindbrain, Mb: midbrain, Ne: neurectoderm, nt: neural tube, olp: olfactory placode, otp: otic placode, picm: posterior intermediate cell mass, and Re: retina. (E) Expression of SOX2OT and SOX2 in human tissues determined by qRT-PCR (relative to 18S rRNA levels). (F) Normalized EST distribution of SOX2OT ESTs in human tissue libraries (%), where (x:n) indicates the number of SOX2OT spliced ESTs (x) over the total number of ESTs (n) in each tissue (see Materials and Methods). n/a: Not available. Primer sequences are provided in Supplemental Table 2.