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Isolation and molecular signature of stress-BFU-Es
files of steady-state progenitors from BM as recently defined by single-cell transcriptomics and fate assays,15 demonstrated that splenic stress-progenitors map closely with their steady-state BM counterparts (Online Supplementary Figure S6). The most widely used protocol for delineating myelo-erythroid progenitors in steady-state BM nicely demonstrates that BFU-E potential resides in the Lin–cKit+CD150+CD105+ “Pre-CFU-E” fraction,9 also con- firmed in our hands (Figure 4A-B). However, since the “Pre- CFU-E” population contains relatively few BFU-E progeni- tors (Figure 4B and Pronk et al.),9 a specific cell population possessing the BFU-E potential remains poorly defined. We therefore asked if CD9 could be used to also enrich for steady-state BFU-E. Progenitor populations were FACS-sorted from steady-state BM using the same marker- combinations as in stressed spleen (CD150+CD9+Sca1–), and plated for erythroid colony formation. The FACS profile of steady-state BM was very similar to that of stressed spleen (Figure 1C and Figure 4C), and both Sca1– and Sca1+ CD150+CD9+ BM cells efficiently gave rise to BFU-E colonies (BFU-E/CFU-E colony formed from CD150+CD9+Sca1–: 30.0, frequency: 7.5±0.9%, Figure 4C- D), representing a 45-fold improved BFU-E/CFU-E ratio compared to previously used marker combination. In con- trast, CD150+CD9+ cells were hardly present in spleens from steady-state mice, and of these only a few comprised BFU-E-forming potential (BFU-E/CFU-E colony formed from CD150+CD9+Sca1–: 10.8, frequency: 1.0±0.4%; Online Supplementary Figure S7). Furthermore, steady-state BM pro- genitors gave rise to highly proliferative BFU-E colonies, whereas steady-state spleen progenitors resulted in much smaller BFU-E colonies (data not shown). In conclusion, CD150 and CD9 expression mark BFU-E potential, both during steady-state in the BM and in the spleen during acute anemia.
Compared to steady-state BFU-E, stress-BFU-E have enhanced expression of genes associated with BMP sig- naling, erythropoiesis and proliferation
To investigate prospective mechanisms giving stress- BFU-E their unique capacity to rapidly produce large num- bers of erythroid cells in response to anemia, BFU-E (CD150+CD9+Sca1–) were sorted from steady-state BM and day 8 stressed spleens and analysed for transcriptional dif- ferences using RNA-seq. Analysis demonstrated a large overlap between stress- and steady-state BFU-E, with only 277 genes being differentially expressed (Figure 5A, full list of differentially expressed genes in the Online Supplemental Table S2). Gene set enrichment analysis demonstrated that sBFU-E expressed higher levels of genes associated with BMP and glucocorticoid signaling, proliferation, maturation block, and erythropoiesis (Figure 5B and Online Supplementary Figure S8) with several erythropoiesis-rela- ted genes among the most highly up-regulated genes in stress- compared to steady-state BFU-E (Table 1). Steady- state BFU-E on the other hand retained gene signatures associated with myeloid cell development and immune response (Figure 5C), which was also reflected by the genes most down-regulated in stress- compared to steady-state BFU-E (Table 2).
Strong prevalence for the binding motif of chromatin-looping transcription factor CTCF under ATAC- seq peaks enriched in stress-BFU-E
To define potential differences in active regulatory DNA
elements across the genome, we performed the assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis18 on the same stress- and steady-state BFU-E pop- ulations as used for transcriptional profiling. ATAC-seq peaks (open chromatin regions) were detected using ENCODE ATAC-seq pipeline (https://github.com/kundaje- lab/atac_dnase_pipelines). K-means clustering demonstrated a relatively large overlap in chromatin availability between the two populations, with some of the peaks being enriched in stress- (orange) or steady-state (blue) BFU-E (Figure 5D and Online Supplementary Figure S9). The absolute majority of peaks with differential accessibility were located at distal elements (>1,000 bp away from the transcription start site) with only 5% situated around the promoter regions in both stress- and steady-state BFU-E, indicating that transcriptional differences between stress- and steady-state BFU-E are likely to be regulated by distal chromatin interactions.
To identify DNA-binding factors with differential chro- matin availability in stress-erythropoiesis we performed motif analysis using peak files extracted from the heatmap clusters. This detected the transcriptional regulator CTCF (CCCTC-binding factor, CTCFL; testis-specific paralog) as the most significantly enriched DNA-binding factor in stress-BFU-E peaks followed by ERG (Figure 5E), whereas steady-state BFU-E peaks most significantly enriched for SFBPI1 and RUNX2 (Figure 5F). Notably, only for CTCF was the motif distributed closely around the peak center (Figure 5G). CTCF is known to regulate the formation of chromatin loops by binding together strands of DNA, and also constitutes a primary part of insulators by blocking the interaction between enhancers and promoters (reviewed by Phillips et al.).29 Interestingly, CTCF has been shown to mark active promoters and boundaries of repressive chro- matin domains in primary human erythroid cells,30 and LDB1-CTCF enhancer looping has recently been shown to underlie activation of a substantial fraction of erythroid genes.31 Furthermore, CTCF has been shown to regulate growth and erythroid differentiation of human myeloid leukemia cells (K562 cell line).32 Although both steady-state and stress-BFU-E enriched for the CTCF motif, the frequen- cy was considerably higher in sBFU-E, where 24,3% of the enriched peaks marked a CTCF motif, compared to 8,5% in steady-state BFU-E (Figure 5H). In conclusion, while the transcriptional and chromatin landscape of stress- and steady-state BFU-E display a high degree of similarity, dis- tinctions in gene expression patterns and in the epigenetic landscape might underlie the unique capacity of sBFU-E to rapidly make large numbers of erythroid cells in response to anemia.
Discussion
The identity of stress-erythroid progenitors has remained largely elusive, and their precise identification is important to understand the mechanisms governing recovery from anemia. Previous studies have identified stress-progenitors to be cKit+CD71low/Ter119ow,14 and after culture the BFU-E potential is found in the CD34–CD133– fraction of the Sca1+cKit+CD71low/Ter119low population. However, only a small fraction of these cells (0.12-0.2%) gave rise to BFU-E colonies. Here we demonstrate that the differential expression of the surface markers CD150, CD9 and Sca1 provide fractionation and definition of a
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