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Single cell lncRNAs in hematopoiesis
Coordinated activation and suppression of signature messenger RNAs and long noncoding RNAs during hematopoiesis
To systematically assess expression of lncRNAs that might be activated or suppressed during hematopoiesis, we focused on dynamic changes of the mRNA and lncRNA transcriptomes along differentiation trajectories defined by pseudotime ordering of HSC/MLP into MEP and GM/L (granulocyte/monocyte/lymphocyte progeni- tors) (Figure 5 and Online Supplementary Tables S7 and S8). Sequentially upregulated/downregulated mRNAs and lncRNAs along the two trajectories were analyzed and gene expression was visualized in heatmaps (MEP trajec- tory in Figure 5A and GM/L trajectory in Figure 5B). Common downregulated mRNAs in MEP and GM/L tra- jectories (Figure 5C) were involved in signaling pathways related to stemness, including NRF2, AP-1, ATF-2, C- MYB, HIF-1, and IL-6 signaling. Downregulated genes specifically in the MEP differentiation pathway were mostly enriched in T cells and for broad immune response; enrichment in the EPO signaling pathway was observed only among GM/L downregulated genes. Frequently upregulated genes were involved in DNA replication, cell cycle, and cell proliferation; genes specifically upregulated in GM/L were enriched in B- and T-cell signaling and immune response (Figure 5D, right); hemoglobin synthesis and androgen receptors were enriched only among MEP upregulated genes (Figure 5D, left). lncRNA expression along the two differentiation trajectories was synchro- nously coordinated with lineage-specific coding genes and interrelated in functional pathways of stemness, megakaryocyte/erythrocyte development, and granulo- cyte/monocyte/lymphocyte development. Collectively, these data suggest the ordered expression of lncRNAs in hematopoietic differentiation and involvement in the reg- ulation of hematopoiesis.
Long noncoding RNAs are bound by lineage-specific transcription factors and might be regulated by epige- netic mechanisms
Transcription factors are critical in cell fate decisions and thus in the regulation of lineage-specific gene expression. Given the observation of highly ordered expression pat- terns of lncRNAs during hematopoiesis and co-expression with lineage-specific transcription factors, we investigated roles of lineage-specific transcription factors in regulating lncRNA expression during hematopoiesis. The transcrip- tion factor GATA1 regulates erythrocyte and megakary- ocyte differentiation,45,46 and indeed its expression was sequentially increased as HSC differentiate into MEP (Figure 5A). Using data obtained by chromatin immuno- precipitation sequencing (ChIP-seq) for GATA1 binding (Encode Ref# ENCSR000EFT), we found that GATA1 binding to promoters was higher in lncRNA-encoding (Figure 6A, top) as well as protein-coding genes (Figure 6A, bottom) preferentially expressed in MEP than for other cell types. lncRNA-encoding genes preferentially expressed in MEP, such as SNHG3 and RP11-620J15.3 (Figure 6B), bound to GATA1 and had high read coverage of active histone marks (H3K27Ac, H3K79me2, and H3K4me2) and low coverage of repressive histone marks (H3K27me3) in erythroid cells. Our analysis, together with published data,8,13,14,16,18,39,47 indicated that cell fate deci- sions were controlled by critical lineage-specific transcrip- tion factors, as evidenced by expression of both lineage-
specific mRNAs and lncRNAs bound and regulated by cor- responding transcription factors, probably involving epi- genetic modification.
Long noncoding RNAs exhibit aberrant expression
in aneuploid cells from patients with myelodysplastic syndromes
Gene expression of 588 single CD34+ cells from five MDS patients was compared with that of cells from four healthy donors. lncRNAs were differentially expressed in MDS cells compared with those from healthy donors (P<0.05): 372 and 590 lncRNAs were upregulated and downregulated, respectively (Figure 7A and Online Supplementary Table S10). By guilt-by-association, down- regulated lncRNAs were associated with gene sets involved in immune response, cellular response, and gene expression and DNA damage response; upregulated lncRNAs were involved in cell metabolism and cell signal- ing (Figure 7B,C).
We adopted three bioinformatics methods to distin- guish cells with abnormal karyotypes from diploid cells.31 We observed that 200 and 56 lncRNAs were downregulat- ed and upregulated, respectively, in monosomy 7 cells, compared to diploid cells (P<0.05) (Figure 7D and Online Supplementary Table S11). By guilt-by-association, down- regulated lncRNAs were associated with genes involved in immune response, cell apoptosis and cell death, and DNA modification; upregulated lncRNAs displayed involve- ment in Ras signaling, Wnt signaling, and interleukin-8 production (Figure 7E,F).
Discussion
In the current study, we profiled the repertoire of lncRNAs in human bone marrow-derived CD34+ cells, with the goal of understanding lncRNA biology in early human hematopoiesis. The majority of the human genome is transcribed but only a small proportion of tran- scripts encode proteins,4,48,49 and thus the number of lncRNA genes is predicted to be very large. Deep RNA sequencing followed by de novo transcriptome reconstruc- tion was adopted for genome-wide annotation and func- tional characterization of novel lncRNAs.12-14,16-18 Moreover, by scRNA-seq, we and others observed higher cell-to-cell variation of lncRNA expression compared to mRNA expression.26,28,30,50 The validation of defined lncRNAs, including potential novel ones, with quantitative RT-PCR in single cells and a new set of sorted bulk samples proved the validity of scRNA-seq and bioinformatic analysis in defining lncRNAs in the current study. Our strategy of sin- gle cell deep sequencing in combination with de novo tran- script assembly could be adopted to further facilitate annotation of the complete lncRNA repertoire.
The very large number of both annotated and novel lncRNAs presents a challenge to functional validation. Based on earlier studies,4,15,39-41 we adopted a systematic, computational guilt-by-association method, from which we could confirm defined lncRNAs in human HSPCs to be likely involved in hematopoietic differentiation and antic- ipated cell functions. Conventional functional validation of the many hundreds of known and new lncRNAs would not only be prohibitively costly and time-consuming, but the choice of assays and conditions of testing is not obvi- ous, nor is there an established statistic by which to judge
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