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.

Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did not contain an apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-mRNA contamination during library construction, or alternatively represent nonspecific "transcriptional noise." Here we show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts of unknown function whose expression appears to be regulated. The ncRNA transcripts have larger exons and fewer introns than protein-coding transcripts. Analysis of the genomic landscape around these sequences indicates that some cDNA clones were produced not from terminal poly(A) tracts but internal priming sites within longer transcripts, only a minority of which is encompassed by known genes. A significant proportion of these transcripts exhibit tissue-specific expression patterns, as well as dynamic changes in their expression in macrophages following lipopolysaccharide stimulation. Taken together, the data provide strong support for the conclusion that ncRNAs are an important, regulated component of the mammalian transcriptome.[Supplemental material is available online at www.genome.org. The microarray data from this study have been submitted to the Gene Expression Omnibus under accession nos. GSD275 and GSE3098.] In recent years there have been increasing reports of functional non-protein-coding RNAs (ncRNAs) that are involved or implicated in developmental, tissue-specific, and disease processes, including X-chromosome dosage compensation, germ cell development and embryogenesis, neural and immune cell development, kidney and testis development, B-cell neoplasia, lung cancer, prostate cancer, cartilage-hair hypoplasia, spinocerebellar ataxia type 8, DiGeorge syndrome, autism, and schizophrenia (see Pang et al. 2005). Many putative ncRNAs are alternatively spliced and/or polyadenylated (Sutherland et al. 1996;Tam et al. 1997;Bussemakers et al. 1999;Raho et al. 2000;Charlier et al. 2001;Wolf et al. 2001). Smaller ncRNAs, termed microRNAs, have also been shown to be involved in developmental processes in both plants and animals, as well as implicated in disease (Carrington and Ambros 2003;Mattick and Makunin 2005). Recent evidence suggests that these microRNAs are derived from the introns of capped and polyadenylated protein-coding transcripts as well as the exons and introns of non-protein-coding transcripts, many of which are derived from "intergenic" regions (Cai et al. 2004;Rodriguez et al. 2004;Seitz et al. 2004;Mattick and Makunin 2005;Ying and Lin 2005). In addition, many complex genetic phenomena, including cosuppression, imprinting, methylation, and gene silencing (see Mattick...

Long noncoding RNAs (lncRNAs) are increasingly recognized to play major regulatory roles in development and disease. To identify novel regulators in breast biology, we identified differentially regulated lncRNAs during mouse mammary development. Among the highest and most differentially expressed was a transcript (Zfas1) antisense to the 59 end of the protein-coding gene Znfx1. In vivo, Zfas1 RNA is localized within the ducts and alveoli of the mammary gland. Zfas1 intronically hosts three previously undescribed C/D box snoRNAs (SNORDs): Snord12, Snord12b, and Snord12c. In contrast to the general assumption that noncoding SNORD-host transcripts function only as vehicles to generate snoRNAs, knockdown of Zfas1 in a mammary epithelial cell line resulted in increased cellular proliferation and differentiation, while not substantially altering the levels of the SNORDs. In support of an independent function, we also found that Zfas1 is extremely stable, with a half-life >16 h. Expression analysis of the SNORDs revealed these were expressed at different levels, likely a result of distinct structures conferring differential stability. While there is relatively low primary sequence conservation between Zfas1 and its syntenic human ortholog ZFAS1, their predicted secondary structures have similar features. Like Zfas1, ZFAS1 is highly expressed in the mammary gland and is down-regulated in breast tumors compared to normal tissue. We propose a functional role for Zfas1/ ZFAS1 in the regulation of alveolar development and epithelial cell differentiation in the mammary gland, which, together with its dysregulation in human breast cancer, suggests ZFAS1 as a putative tumor suppressor gene.

Inherited white-matter disorders are a broad class of diseases for which treatment and classification are both challenging. Indeed, nearly half of the children presenting with a leukoencephalopathy remain without a specific diagnosis. Here, we report on the application of high-throughput genome and exome sequencing to a cohort of ten individuals with a leukoencephalopathy of unknown etiology and clinically characterized by hypomyelination with brain stem and spinal cord involvement and leg spasticity (HBSL), as well as the identification of compound-heterozygous and homozygous mutations in cytoplasmic aspartyl-tRNA synthetase (DARS). These mutations cause nonsynonymous changes to seven highly conserved amino acids, five of which are unchanged between yeast and man, in the DARS C-terminal lobe adjacent to, or within, the active-site pocket. Intriguingly, HBSL bears a striking resemblance to leukoencephalopathy with brain stem and spinal cord involvement and elevated lactate (LBSL), which is caused by mutations in the mitochondria-specific DARS2, suggesting that these two diseases might share a common underlying molecular pathology. These findings add to the growing body of evidence that mutations in tRNA synthetases can cause a broad range of neurologic disorders.

Temple-Baraitser syndrome (TBS) is a multisystem developmental disorder characterized by intellectual disability, epilepsy, and hypoplasia or aplasia of the nails of the thumb and great toe. Here we report damaging de novo mutations in KCNH1 (encoding a protein called ether à go-go, EAG1 or KV10.1), a voltage-gated potassium channel that is predominantly expressed in the central nervous system (CNS), in six individuals with TBS. Characterization of the mutant channels in both Xenopus laevis oocytes and human HEK293T cells showed a decreased threshold of activation and delayed deactivation, demonstrating that TBS-associated KCNH1 mutations lead to deleterious gain of function. Consistent with this result, we find that two mothers of children with TBS, who have epilepsy but are otherwise healthy, are low-level (10% and 27%) mosaic carriers of pathogenic KCNH1 mutations. Consistent with recent reports, this finding demonstrates that the etiology of many unresolved CNS disorders, including epilepsies, might be explained by pathogenic mosaic mutations.