Role of Long Coding RNAs in Epigenetic Modulation of Hematopoiesis

Long noncoding (Lnc) RNAs are RNA transcripts greater than 200 nucleotides (nt) that regulate gene expression independent of protein coding potential (1-3). It is estimated that thousands of lncRNAs play vital roles in diverse cellular processes. LncRNAs modulate many stages of gene expression by regulating transcription, epigenetics, splicing, translation, and protein localization. We hypothesize that multiple lncRNAs are expressed specifically during erythrocyte and megakaryocyte differentiation, and are likely to have important roles. To identify lncRNAs in erythro-megakaryopoiesis, we performed strand-specific, paired-end deep sequencing (RNA-Seq) to a depth of 200 million reads per sample on two replicates each of murine Ter119⁺erythroblasts, CD41⁺ megakaryocytes and bipotential megakaryocyte-erythroid progenitors (MEPs) [lin^- Kit⁺ Sca1^- CD16/32^- CD34^-], and used bioinformatic filtering tools to identify approximately 1,100 candidate lncRNAs. Over 60 percent of these lncRNAs are novel unannotated transcripts with exquisite lineage-specific expression. Using erythroid and megakaryocytic primary cell ChIP-Seq for key transcription factors (TFs) GATA1, TAL1, GATA2,and FLI1, we found that the loci of lncRNAs show similar degree of TF binding as coding genes. We used the erythroid line G1E-ER4 (which expresses estrogen-activated GATA1) to confirm that lncRNAs bound by GATA1 are also directly regulated by it. Furthermore, we used histone methylation ChIP-Seq to show that most lncRNAs arise from classical “promoters” with high H3K4me3 levels and low H3K4me1 levels. Thus, we find that lncRNAs show epigenetic features similar to the promoters of coding genes and are directly regulated by similar TF networks. Comparison of the transcriptomes of mouse fetal liver and human cord blood erythroblasts demonstrated that lncRNAs are expressed in a highly species-specific fashion, i.e., most lncRNAs identifiable in one species are not transcribed in the other, even though the corresponding genomic region is present in both species. Numerous non-conserved but functional lncRNAs are reported in the literature, and the significance of conservation in lncRNA biology is greatly debated. In order to identify functional lncRNAs, we are currently performing RNAi knockdown on numerous candidates to assess how loss of function affects erythroid maturation. We are also performing HITS-CLIP of key chromatin modifying complexes and erythroid transcription factors to identify lncRNAs bound to them. Our studies are beginning to define new layers of gene regulation in normal erythro-megakaryopoiesis, which may be relevant to the pathophysiology of related disorders including various anemias, myeloproliferative and myelodysplastic syndromes and leukemias.

1. Wang K.C., Chang H.Y. Molecular mechanisms of long noncoding RNAs. Molecular Cell. 2011;43(6):904-914. Prepublished on 2011/09/20 as DOI 10.1016/j.molcel.2011.08.018.

2. Hu W., Alvarez-Dominguez J.R., Lodish H.F. Regulation of mammalian cell differentiation by long non-coding RNAs. EMBO reports. 2012;13(11):971-983. Prepublished on 2012/10/17 as DOI 10.1038/embor.2012.145.

3. Paralkar V.R., Weiss M.J. Long noncoding RNAs in biology and hematopoiesis. Blood. 2013;121(24):4842-4846. Prepublished on 2013/05/07 as DOI 10.1182/blood-2013-03-456111.