Long non-coding RNAs control hematopoietic stem cell function

Abstract

Hematopoietic stem cells (HSCs) possess unique gene expression programs that enforce their identity and regulate lineage commitment. Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression and cell fate decisions, although their functions in HSCs are unclear. Here we profiled the transcriptome of purified HSCs by deep sequencing and identified 323 unannotated lncRNAs. Comparing their expression in differentiated lineages revealed 159 lncRNAs enriched in HSCs, some of which are likely HSC specific (LncHSCs). These lncRNA genes share epigenetic features with protein-coding genes, including regulated expression via DNA methylation, and knocking down two LncHSCs revealed distinct effects on HSC self-renewal and lineage commitment. We mapped the genomic binding sites of one of these candidates and found enrichment for key hematopoietic transcription factor binding sites, especially E2A. Together, these results demonstrate that lncRNAs play important roles in regulating HSCs, providing an additional layer to the genetic circuitry controlling HSC function.

Figure 1. Identification of HSC-specific LncRNAs

(A) Flowchart for identification of LncHSCs. Filters indicate exclusion criteria. (B) Heatmap to compare gene expression between HSCs, B cells and Granulocytes, including protein-coding genes, previously annotated lncRNAs and unannotated transcripts. (C) Expression of the transcripts identified in HSCs, B cells, Granulocytes and 20 other tissues, including Cerebellum, Cortex, ESC, Heart, Kidney, Lung, Embryonic fibroblasts (MEFs), Spleen, Colon, Duodenum, Mammary gland, Ovary, SubcFatPad (Subcutaneous Adipose tissue, Sfat), GenitalFatPad (Gfat), Stomach, Testis and Thymus (GSE36025 and GSE36026). (D) Coding potential prediction by CAPT for 503 unannotated transcripts identified in HSC. (E) UCSC browser track showing two LncHSCs, with expression (green), H3K4me3 signal (pink) and H3K36me3 (blue). (F) UCSC browser track showing one LncHSC is located in the UMR, with expression (green), H3K4me3 signal (pink) and DNA methylation (red), and UMR (blue bar). See also Figure S1 and Table S1.

Figure 2. Transcriptional regulation of LncHSCs

(A) Heatmap depicting expression of 159 LncHSCs: 4mo HSC vs 12mo HSC and 24mo HSC (Left panel); WT vs Dnmt3a KO HSC (Right panel). (B) UCSC browser track showing expression (green), H3K4me3 signal (pink) and DNA methylation (red) for LncHSC-3 and LncHSC-4 in WT and Dnmt3a KO (3a KO) HSCs. Grey bars show differential DNA methylation between WT and 3a KO HSCs. (C) Quantitative RT-PCR analysis of selected LncHSCs. 50,000 KSL cells (c-Kit⁺Sca-1⁺Lin), CD4 T cells (CD4⁺), CD8 T cells (CD8⁺), B cells (B220⁺), Macrophages (Mac1⁺), Granulocytes (Gr1⁺) and Red blood cells (TER119⁺) were sorted for RT-PCR. The expression levels were plotted relative to that in KSL cells (set as 100, n=3, Mean ± SD). (D) UCSC browser track showing DNA methylation (red), expression (green), H3K4me3 (pink), H3K27me3 (orange), H3K27ac (dark green) and H3K4me1 (blue). (E) Representative images of LncHSC-1 expression in HSCs analyzed by RNA-FISH. Green is 18srRNA signal and Red is LncHSC-1 signal. (F) Representative images of LncHSC-2 and LncGr-1 expression in HSC and Granulocytes analyzed by RNA-FISH. Green is LncHSC-2 signal and red is LncGr-1 signal. See also Figure S2 and Figure S3.

Figure 3. LncHSCs regulate HSC differentiation in vitro

(A) Flow chart depicts knockdown of LncHSC for in vitro and in vivo functional studies. (B) Q-RT-PCR to show LncHSCs knockdown. Sca-1⁺ cells were transduced with knockdown constructs, cultured in vitro for two days, then 20,000 GFP⁺ cells were sorted for RT-PCR (n=3, Mean ± SD). (C) Methylcellulose CFU assay using 200 KSL-GFP⁺ cells (transduced by LncHSC-1 KD-1 and LncHSC-2 KD-1). Sorted cells were put into a well of 6-well plate containing Methocult3434 and average colony number counted after 14 days. For the second plating, 2,000 live cells from the colonies obtained in the first plating were plated as before and cultured for 14 days. ** P< 0.01. (n=3, Mean ± SD). Representative of 3 experiments. (D) Morphology of cells from the colonies at the second plating by cytospin. See also Figure S3.

Figure 4. LncHSCs control HSC function in vivo

(A) Contribution of retrovirally-transduced donor HSCs (CD45.2⁺GFP⁺) to recipient mouse PB after primary transplantation. *** P <0.001, ** P< 0.01, * P<0.05. Error bars represent Mean ± SEM. (n=10 for Control KD, n=5–8 for LncHSCs KD). (B) Analysis of HSC differentiation in peripheral blood at 16 weeks post-primary transplant. The % of the indicated lineages within CD45.2⁺GFP⁻ or CD45.2⁺GFP⁺ cell compartment are shown. Myeloid cells (Mye) were defined as Gr1⁺ and Mac1⁺, B-cells (B) are B220⁺, T-cells (T) are CD4⁺ and CD8^+.. *** P <0.001, ** P< 0.01, * P<0.05. Error bars represent Mean ± SEM. (n=10 for Control KD, n=5–8 for LncHSCs KD). (C) Contribution of donor HSCs (CD45.2⁺GFP⁻ or CD45.2⁺GFP⁺) to recipient mouse PB after secondary transplantation. Mean ± SEM. (n=5–6). For secondary transplantation, 500 CD45.2+GFP⁺ KSL cells from primary recipients were re-sorted 20 weeks after transplantation and mixed with 250,000 CD45.1 WBM cells, and injected into new lethally irradiated CD45.1 recipients. (D) Analysis of peripheral blood cells at 16 weeks post-secondary transplant. The % of the indicated lineages within CD45.2⁺GFP⁻ and CD45.2⁺GFP⁺ cell compartment are shown. Myeloid cells (Mye) were defined as Gr1⁺ and Mac1⁺, B-cells (B) are B220⁺, T-cells (T) are CD4⁺ and CD8^+.. *** P <0.001, ** P< 0.01, * P<0.05. Error bars represent Mean ± SEM. (n=5–6). (E) Bone marrow FACS analysis showing frequencies of side population, LK and LSK cells 16 weeks after secondary transplantation in mice. Error bars represent Mean ± SEM. **P < 0.01. (n=3–6) See also Figure S4 and Table S2.

Figure 5. ChIRP-seq reveals LncHSC-2 binding sites in genome

(A) LncHSC-2 binding sites are enriched in promoter-proximal regions. Left, pie chart shows the distribution of LncHSC-2 binding sites across the indicated intergenic or intragenic regions. Right, enrichment of LncHSC-2 sites (versus genomic background) among transcript features. (B) Enriched sequence motif associated with lncHSC-2 binding sites (bottom) strongly resembling the mouse Tcfe2a secondary motif (top). (C) Co-enrichment analysis of lncHSC-2 binding sites with sequence features, ChIP-seq profiles of hematopoietic transcriptional regulators and epigenetic marks in HSC, multipotent progenitors (HPC-7 cells), bone marrow, thymus, and spleen. Enrichment for LncHSC-2 binding compared to LacZ negative control was assessed by Fisher’s exact test with multiple testing correction. Colors subdivide the results into three classes: epigenetic mark (red), sequence feature (green), and TF binding site (blue). Dot sizes are proportionate to the Odds Ratio. The x-axis values represent the – log Benjamini-Hochberg corrected p-value. (D) Hierarchical clustering of genomic regions bound by lncHSC-2 and published hematopoietic lineage TF or epigenetic marks. The major partition of columns separates LncHSC–2 occupancy into two main branches, with unmethylated promoter proximal regions associated with transcriptional activation marks (H3K4me3/H3K27ac/PolII) and Erg/Fli/Meis1/Pu.1 TFs to the left and promoter distal intronic or intergenic regions associated with bone marrow tissue enhancer or insulator elements (CTCF), E2A sequence motifs to the right. Each line corresponds to a LncHSC-2 peak where blue/white coloring indicates the presence/absence of the additional given factors. (E) LncHSC-2 occupancy at the genes Pml (top) and Itpkb (bottom). LncHSC-2 and LacZ control ChIRP-seq signal density tracks generated by MACS2 representing the fragment pileup signal per million reads. Additional overlaid tracks are HSC H3K4me3, RNA-seq, undermethylated regions (Jeong et al., 2014) and hematopoietic lineage TF binding sites (Wilson et al., 2010). (F) ChIP-qPCR to show E2A binding to three LncHSC-2 binding peaks after LncHSC-2 KD in primary Sca-1⁺ cells. Y-axis represents the % of immunoprecipitated DNA compared to input. Mean ± SEM values are shown (n=4). See also Figure S5 and Table S3–S6.