Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells

Abstract

Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes.development | expression T he non-protein-coding portion of the mammalian genome is transcribed into a vast array of RNA species (1), some of which play important roles in cellular regulation, development, and disease (2). The long noncoding RNAs (lncRNAs) are of particular interest because they are known to contribute to gene silencing (3), X inactivation (4), imprinting (5, 6), and development (7-9), but there is limited understanding of the genomic origin, regulation, and function of lncRNA molecules in individual cell types.Embryonic stem cells (ESCs) are widely used as a model system to study transcriptional control of cell state during early development (10-13), yet there is no catalog of lncRNAs in human (h) ESCs, and it is not clear how lncRNAs are regulated in these cells. Catalogs of lncRNAs have been recently described in various murine (14, 15) and human cell types (16)(17)(18)(19), but the majority were limited to spliced lncRNA species (14-16, 18) and those distant from protein-coding genes (14-17). Because lncRNAs tend to be cell-type-specific (16, 18), these catalogs likely contain only a very small fraction of lncRNAs expressed in hESCs.We describe here catalogs of human and murine ESC lncRNAs and the genomic regions from which these RNA species arise. We find that the majority of these lncRNAs originate from divergent transcription of lncRNA/mRNA gene pairs and that many such gene pairs are coordinately regulated when ESCs differentiate.Results lncRNAs Expressed in Human ESCs. We compiled a catalog of lncRNA species expressed in hESCs as summarized in Fig. 1A. An initial pool of RNA candidates was generated by sequencing polyadenylated RNA species from hESCs and supplementing these with EST data from the full-length long Japan (FLJ) collection of sequenced human cDNAs, which contains transcripts expressed in >60 human tissues, including embryonal tissue (20). An initial pool of 170,162 ncRNA candidates (Dataset S1) was obtained after removing protein-coding transcripts based on the National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq). This pool was further filtered by using multiple criteria to identify lncRNAs. The RNA species were required to have a 5′ end that originates from a genomic site where there is corroborating evidence of active transcript...

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“…33 Within a region of 2 kb around the TSSs of PCG promoters, a promoter size that has previously been used to define PCG-associated long ncRNAs, we found 3891 long ncRNA/PCG pairs. 16 This set of long ncRNA/PCG pairs exhibits a major enrichment of promoters between 100 and 200 bp in length separating the TSSs (Supplementary Figure 1), which is in accordance with previous studies and supports the idea that most long ncRNAs in our data set share a promoter with their associated PCG rather than both genes being expressed from distinct promoters. 34 When filtering for bidirectional promoters of up to 500 bp in length, the set was narrowed down to nearly half (~45%) of the initial PCG-associated long ncRNAs.…”

“…5,6,10,11 Although many genome-wide studies in a wide range of organisms established that most PCG promoters are characterized by divergent non-coding transcription, the functional role of those promoter-associated long ncRNAs remains elusive. [12][13][14][15][16] In many cases, antisense transcripts upstream of promoters are unstable and their emergence might be explained by aberrant transcription initiation of RNA polymerase II due to the depletion of nucleosomes on active promoters. 14,[17][18][19][20] In evolutionary terms, divergent transcription initiation could fuel the emergence of new functional genes, by exposing the transcribed DNA strand to mutagenic alterations as has been reported for the acquisition of splice sites.…”

“…14,[17][18][19][20] In evolutionary terms, divergent transcription initiation could fuel the emergence of new functional genes, by exposing the transcribed DNA strand to mutagenic alterations as has been reported for the acquisition of splice sites. 21 Several studies have addressed the regulation of bidirectional transcription, 13,16,18,19,[22][23][24][25][26][27] with three of these shedding light on the functionality of ncRNAs that are bidirectionally expressed from human PCG promoters: Hung et al 28 investigated human cell cycle-regulated genes and identified 58 functional long ncRNAs that are co-expressed with PCGs from bidirectional promoters. One of these transcripts, PANDA (P21 associated ncRNA DNA damage activated), is induced by DNA damage at the CDKN1A promoter and mediates regulation of pro-apoptotic genes.…”

“…28 In another study, human embryonic stem cells have been shown to robustly express more than 2000 long ncRNAs bidirectionally from within 2 kb of the transcription start sites (TSS) of PCGs. 16 Transcriptional activation of specific PCGs via the induction of cellular differentiation is coordinated with expression of paired long ncRNAs, suggesting similar functional roles for bidirectionally expressed genes. 16 Furthermore, the long ncRNA Fendrr that seems to be bidirectionally expressed from the Foxf1 promoter in mouse and partly co-expressed with the Foxf1 transcript has been shown to contribute to heart and body wall formation.…”

“…16 Transcriptional activation of specific PCGs via the induction of cellular differentiation is coordinated with expression of paired long ncRNAs, suggesting similar functional roles for bidirectionally expressed genes. 16 Furthermore, the long ncRNA Fendrr that seems to be bidirectionally expressed from the Foxf1 promoter in mouse and partly co-expressed with the Foxf1 transcript has been shown to contribute to heart and body wall formation. 29 The coexpression of two genes from a bidirectional promoter suggests a mutual regulation mediated by a shared upstream transcriptional network, and implies that their functions might contribute to the same cellular response independently.…”