14q32 and let-7 microRNAs regulate transcriptional networks in fetal and adult human erythroblasts

Abstract

In humans, fetal erythropoiesis takes place in the liver whereas adult erythropoiesis occurs in the bone marrow. Fetal and adult erythroid cells are not only produced at different sites, but are also distinguished by their respective transcriptional program. In particular, whereas fetal erythroid cells express γ-globin chains to produce fetal hemoglobin (HbF), adult cells express β-globin chains to generate adult hemoglobin. Understanding the transcriptional regulation of the fetal-to-adult hemoglobin switch is clinically important as re-activation of HbF production in adult erythroid cells would represent a promising therapy for the hemoglobin disorders sickle cell disease and β-thalassemia. We used RNA-sequencing to measure global gene and microRNA (miRNA) expression in human erythroblasts derived ex vivo from fetal liver (n = 12 donors) and bone marrow (n = 12 donors) hematopoietic stem/progenitor cells. We identified 7829 transcripts and 402 miRNA that were differentially expressed (false discovery rate <5%). The miRNA expression patterns were replicated in an independent collection of human erythroblasts using a different technology. By combining gene and miRNA expression data, we developed transcriptional networks which show substantial differences between fetal and adult human erythroblasts. Our analyses highlighted the miRNAs at the imprinted 14q32 locus in fetal erythroblasts and the let-7 miRNA family in adult erythroblasts as key regulators of stage-specific erythroid transcriptional programs. Altogether, our results provide a comprehensive resource to prioritize genes that may modify clinical severity in red blood cell (RBC) disorders, or genes that might be implicated in erythropoiesis by genome-wide association studies of RBC traits.

Figure 1.

 Differential gene expression between fetal and adult human erythroblast. Fetal and adult erythroblasts were differentiated ex vivo from CD34+ HSPCs collected from the fetal liver (FL) and adult bone marrow (BM). (A) PC analysis of the 500 most variable genes. The first PC captures the developmental stage, whereas the second PC is explained by the sex of the donors (genes expressed on the X-chromosome). (B) DE genes that are up-regulated in fetal (red) and adult (blue) erythroblasts (FDR <5%). The y-axis represents the log₂ of fetal-to-adult expression FC. The x-axis represents the mean normalized gene counts calculated by DESeq2. N.S., not significant. (C) Expression of the top 20 most significantly DE genes in order of FC. (D) Mean FPKM of genes at the α- and β-globin loci. Error bars represent the S.E.M.

Figure 2.

 Many miRNAs are DE between fetal and adult human erythroblast. Fetal and adult erythroblasts were differentiated ex vivo from CD34+ HSPCs collected from the fetal liver (FL) and adult bone marrow (BM). (A) PC analysis of the 500 most variable miRNAs. The first PC captures the developmental stage of the erythroblasts. (B) DE miRNAs that are up-regulated in fetal (red) and adult (blue) erythroblasts (FDR < 5%). The y-axis represents the log₂ of fetal-to-adult expression FC. The x-axis represents the mean normalized gene counts calculated by DESeq2. Most of the outstanding miRNA up-regulated in fetal erythroblasts are from the 14q32 locus. N.S., not significant. (C) Expression of the top 10 most up-regulated miRNAs in fetal erythroblasts, and of the 10 most up-regulated miRNAs in adult erythroblasts. Expression is reported in counts normalized using DESeq2’s regularized log₂ transformation. (D) Manhattan plot of DE miRNAs P-values (y-axis) based on the genomic location of their precursor sequences (x-axis).

Figure 3.

 Validation of DE miRNAs by qPCR. (A) Correlation between miRNA expression FCs obtained by RNA-seq (N_adult = 12, N_fetal = 12) and qPCR (N_adult=3, N_fetal = 3). Only DE miRNA in the RNA-seq experiment are included. Red and blue dots represent DE miRNAs with one-tailed P < 0.05 in the qPCR experiment that are up-regulated in fetal and adult erythroblasts, respectively. (B) qPCR log₂ FC for the 20 most DE miRNAs in the qPCR experiment (all one-tailed P < 5 × 10⁻³). Each bar represents the mean log₂ FC for adult (blue) and fetal (red) erythroblasts. Error bars represent the S.E.M. Bars marked with # correspond to miRNAs from the let-7 family (blue bars) or the 14q32 locus (red bars).

Figure 4.

 miRNA target networks. Using validated and predicted connections between miRNAs and their target genes, we identified genes that are down-regulated when their targeting miRNA is up-regulated. Gray and purple circles correspond to DE target genes and miRNAs, respectively. (A) The network in adult erythroblasts includes 18 DE miRNAs. Most adult-stage miRNAs target the same genes, consistent with an over-representation of members of the let-7 family. (B) Network of 34 up-regulated fetal miRNAs enriched for down-regulated targets.