Abstract
During human ontogeny, high-level transcription within the beta-globin gene cluster switches sequentially from embryonic-to-fetal-to-adult genes. Beta-thalassemias and sickle-cell disease are manifested by reduced or mutated expression of the adult-stage, beta-globin gene. Research is aimed toward the eventual therapeutic goal of safely preventing or reversing the fetal-to-adult hemoglobin switch among these patient populations.
To identify genes that may be involved in regulation of the fetal-to-adult erythroid switch, purified CD34(+) cells from six umbilical cord (fetal) and six adult peripheral blood samples were cultured in serum-free medium, and gene expression libraries were prepared and sequenced from CD71(+), CD235a(+) erythroblast mRNA. In total, 546 million paired-end reads with a length of 101bp were generated for a comparison of cord and adult erythroblast transcriptomes. Reads were aligned to the human reference genome (hg19), and differential gene expression was identified [false discovery rate ≤ 0.05, fold change ≥ 1.5, and reads per kilobase per million mapped reads (RPKM) ≥ 0.5]. A total of 145 genes were differentially expressed according to these criteria, with four of the top five encoding targets of the let-7 family of microRNAs. The topmost gene was insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which is normally involved in transcriptome regulation and developmental timing. IGF2BP1 expression was 770-fold increased in the fetal erythroblasts (RPKM > 3.0) compared with low background levels in adult erythroblasts (RPKM < 0.01). IGF2BP1 protein is present in fetal tissues including fetal liver; however, it is not detected in adult human bone marrow.
A potential role for adult-stage IGF2BP1 over-expression (IGF2BP1-OE) in the regulation of globin genes and proteins was explored using lentiviral vectors designed for let-7 resistant, erythroid-specific expression of IGF2BP1 protein. IGF2BP1-OE transduced CD34(+) cells expressed the transgenic protein and maintained their ability to differentiate, accumulate hemoglobin, and enucleate ex vivo in the presence of erythropoietin. Globin mRNA and protein levels were investigated. While alpha-globin mRNA remained unchanged, gamma-globin mRNA became predominant [90% of (gamma + beta) mRNA] in IGF2BP1-OE samples [Control (empty vector) = 3.2E+06 ± 8.2E+05 copies/ng; IGF2BP1-OE = 2.0E+07 ± 5.9E+06 copies/ng; p < 0.05], and beta-globin mRNA decreased to minor levels [Control (empty vector) = 2.2E+07 ± 4.0E+06 copies/ng; IGF2BP1-OE = 2.2E+06 ± 6.2E+05 copies/ng; p < 0.05]. IGF2BP1-OE caused a pan-cellular HbF distribution by flow cytometry. Cellular fetal hemoglobin percentages [HbF/(HbF + HbA)] were measured as 5.3 ± 0.4% in donor matched control cells versus 80.3 ± 3.7% in IGF2BP1-OE cells (p < 0.05). HPLC tracings revealed that the minor HbA2 peak, composed of alpha and delta globin chains, was reduced or absent in IGF2BP1-OE. Also, IGF2BP1-OE suppressed the expression of related genes including the transcription factor BCL11A. These data demonstrate that erythroblast IGF2BP1 is silenced in humans during fetal-to-adult ontogeny, and that IGF2BP1 in adult erythroblasts reverses the developmentally related switch in beta-like globin gene and protein expression patterns.
Disclosures
No relevant conflicts of interest to declare.
Analysis of the adult chicken beta-globin gene. Nucleotide sequence of the locus, microheterogeneity at the 5'-end of beta-globin mRNA, and aberrant nuclear RNA species.