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.