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. 2008 Sep;18(9):1433-45.
doi: 10.1101/gr.078378.108. Epub 2008 Jun 18.

Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation

Marcel E Dinger  1 , Paulo P Amaral, Tim R Mercer, Ken C Pang, Stephen J Bruce, Brooke B Gardiner, Marjan E Askarian-Amiri, Kelin Ru, Giulia Soldà, Cas Simons, Susan M Sunkin, Mark L Crowe, Sean M Grimmond, Andrew C Perkins, John S Mattick
Affiliations

Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation

Marcel E Dinger et al. Genome Res. 2008 Sep.

Abstract

The transcriptional networks that regulate embryonic stem (ES) cell pluripotency and lineage specification are the subject of considerable attention. To date such studies have focused almost exclusively on protein-coding transcripts. However, recent transcriptome analyses show that the mammalian genome contains thousands of long noncoding RNAs (ncRNAs), many of which appear to be expressed in a developmentally regulated manner. The functions of these remain untested. To identify ncRNAs involved in ES cell biology, we used a custom-designed microarray to examine the expression profiles of mouse ES cells differentiating as embryoid bodies (EBs) over a 16-d time course. We identified 945 ncRNAs expressed during EB differentiation, of which 174 were differentially expressed, many correlating with pluripotency or specific differentiation events. Candidate ncRNAs were identified for further characterization by an integrated examination of expression profiles, genomic context, chromatin state, and promoter analysis. Many ncRNAs showed coordinated expression with genomically associated developmental genes, such as Dlx1, Dlx4, Gata6, and Ecsit. We examined two novel developmentally regulated ncRNAs, Evx1as and Hoxb5/6as, which are derived from homeotic loci and share similar expression patterns and localization in mouse embryos with their associated protein-coding genes. Using chromatin immunoprecipitation, we provide evidence that both ncRNAs are associated with trimethylated H3K4 histones and histone methyltransferase MLL1, suggesting a role in epigenetic regulation of homeotic loci during ES cell differentiation. Taken together, our data indicate that long ncRNAs are likely to be important in processes directing pluripotency and alternative differentiation programs, in some cases through engagement of the epigenetic machinery.

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Figures

Figure 1.
Figure 1.
Correlation of expression profiles of ncRNAs with protein-coding gene markers during EB differentiation. Genes with well-characterized roles in EB differentiation (A) were used to identify ncRNAs with correlated expression profiles (Pearson’s coefficient > 0.9) in pluripotency (B; red lines; Sox2, Pou5f1), primitive streak formation (C; green lines; Evx1, T), and mesoderm differentiation along the hematopoietic lineage (D; Hba-a1, Hba-x). Expression was detected by microarray from 11 RNA samples isolated from differentiating EBs over a 16-d period.
Figure 2.
Figure 2.
Correlation of expression between ncRNAs and associated protein-coding genes. (A–C) Density plots of correlation coefficients between the expression of ncRNAs and their associated intronic (A), bidirectional (B), or cis-antisense (C) protein-coding gene (purple line) and randomized pairs (black line). (D–F) Examples of correlated (positive or negative) expression between ncRNAs (red) and the associated protein-coding genes (blue). The upper panel shows the genomic region of the ncRNA (GenBank accession nos. indicated) and protein-coding gene; the lower panel shows the corresponding expression profiles with Pearson’s correlation coefficients (R2) as indicated. Arrows indicate the direction of transcription.
Figure 3.
Figure 3.
ncRNAs associated with Dlx1/Dlx2 and Dlx5/Dlx6 loci. (A,B) Genomic context of the Dlx1/Dlx2 (A) and Dlx5/Dlx6 (B) loci showing the position of the Dlx genes (blue), ncRNAs (Dlx1as and Evf1, light red; Evf2, dark red), and the highly conserved enhancers (I12a [Park et al. 2004]; VISTA ID290 [Pennacchio et al. 2006]; m1561, [Zerucha et al. 2000]; green). Arrows indicate the direction of transcription. (C,D) Relative expression profiles of Dlx1 and Dlx1as (C) and Evf1, Evf2, and Dlx6 (D) during EB differentiation as determined by qRT-PCR (relative to day 0 or 1; primer positions indicated in A,B). Error bars show standard deviation (SD) determined from at least three replicates. (E–G) ISH of sagittal adult mouse brain sections for Dlx1 (E), Dlx1as (F), and Dlx2 (G). Whole brain is shown in the left panels; subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB) in the middle panels; and the hippocampus (HP) in the right panels. Dlx1, Dlx1as, and Dlx2 show similar expression in the OB, RMS, and SVZ in the brain and in addition Dlx1as is strongly expressed in cells dispersed throughout the cortex (CX) and HP.
Figure 4.
Figure 4.
Characterization of ncRNAs associated with Hoxb5/Hoxb6 and Evx1 loci. (A) Genomic context of Hoxb5/Hoxb6 (top) and Evx1 (bottom) and their associated ncRNAs, Hoxb5/6as, and Evx1as. (B) Relative expression profiles of Hoxb5, Hoxb6, and Hoxb5/6as (left) and Evx1 and Evx1as (right) during EB differentiation as determined by qRT-PCR (relative to day 0; primer positions indicated in A). Error bars show standard deviation (SD) determined from three replicates. (C) Whole-mount ISH showing expression of Hoxb6 and Hoxb5/6as (upper panels) and Evx1 and Evx1as (lower panels) in the tail bud of E9.5 mouse embryos. (D) Association of Hoxb5/6as and Evx1as RNAs with H3K4me3 chromatin and MLL1 fractions, as detected by ChIP followed by RT-PCR detection (see Methods). Normal IgG was used as a negative control antibody, and input corresponds to RNA present in the samples before ChIP.
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