Long Non-Coding RNAs Control Hematopoietic Stem Cells (HSC) Function

The mammalian genome encodes a significant number of long non-coding RNAs (lncRNAs). The functions of some lncRNAs have been determined in biological processes, such as cancer progression, cell-cycle regulation and embryonic stem cell (ESC) pluripotency. However, our understanding of the basic function of lncRNAs in hematopoietic stem cell (HSC) is still limited. Here, we aim to identify the full complement of lncRNAs expressed in mouse HSCs and to determine whether they control HSC function.

To uncover lncRNAs expressed in HSC across different ages, we performed RNA-seq on highly purified HSCs (SP-KSL-CD150⁺) from 4 month (m04), 12 month (m12) and 24 month (m24) old mice. With two biological replicates for each age, deep sequencing generated 368, 311 and 293 million mapped reads for m04, m12 and m24 HSC, respectively. After combining these datasets, assembly of over 1 billion mapped reads for the HSC transcriptome reconstructed 3,104 novel transcripts, which do not correspond to any UCSC, RefSeq or Ensemble known genes. Among them, 2,853 transcripts have multiple assembled exons and a total length >200 bp, representing potential lncRNAs. It has been shown that lncRNAs usually exhibit stage- or cell type-specific expression. To identify lncRNAs specifically expressed in HSC, we further performed RNA-seq on differentiated lymphoid lineage B cells (B220⁺) and myeloid lineage Granulocytes (Gr1⁺). Comparison of the expression of these 2,853 transcripts in the three cell types revealed that 173 transcripts are specifically expressed in HSC.

As epigenetic mechanisms play critical roles to regulate gene transcription, we checked the chromatin map associated with those novel transcripts by ChIP-seq for H3K4me3, H3K27me3 and H3K36me3 in purified HSCs. Like protein-coding genes, these HSC specific novel transcripts typically contain the H3K4me3 mark at their transcriptional start site (TSS) and H3K36me3 along the gene body. Remarkably, one fifth of those 173 transcripts showed altered expression with HSC aging. Given that HSC function declines with aging, we hypothesize that those transcripts contribute to control HSC homeostasis.

We selected three of these transcripts for further validation: LncHSC-1, LncHSC-2 and LncHSC-3. RT-PCR confirmed that they were highly expressed in stem and progenitor populations (KSL), but not in differentiated lineages (B220, CD4, CD8, Mac1, Gr1 and Ter119). Next, we generated retrovirally expressed-miRNA constructs to knockdown these transcripts. In vitro methocult colony forming assay showed that knockdown of LncHSC-1 in HSC significantly increased the colony number after second plating. Lineage analysis revealed that the majority of those cells are c-Kit⁺, and exhibit similar morphology, possibly representing expanded myeloid progenitors.

To confirm our in vitro findings, we further examined their functions in vivo by HSC transplantation. Progenitors in which LncHSC-3 was knocked down failed to contribute to long-term hematopoietic reconstitution, as revealed by loss of retrovirally transduced population in the peripheral blood and bone marrow. In contrast, progenitors in which LncHSC-1 was knocked down resulted in augmented myeloid differentiation, consistent with in vitro CFU results that knockdown increased myeloid colony number.

To understand the molecular mechanism through which lncRNAs influence hematopoiesis, we checked gene expression changes upon knockdown of specific transcripts in KSL cells. Overall, 80-100 genes were significantly changed after knockdown of specific transcripts, including cell cycle regulators and chromatin modification enzymes. For example, after LncHSC-3 knockdown, cell cycle regulator Cdkn1a (p21) expression increased, possibly contributing to the inhibition of hematopoietic reconstitution.

In summary, here we carried out a comprehensive lncRNAs analysis in HSC and determined HSC specific novel transcripts. Loss-of-function experiments demonstrated that these transcripts may play important roles for HSC self-renewal and differentiation. These findings provide a useful resource to study lncRNA functions in normal hematopoiesis and disease progression.