A Long Noncoding RNA from the HBS1L-MYB Intergenic Region on Chr6q23 Regulates Human Fetal Hemoglobin Expression

Fetal hemoglobin (HbF) modifies the severity of sickle cell disease (SCD) and β-thalassemia. One of the major quantitative trait loci (QTL) associated with HbF level is the HBS1L-MYB intergenic polymorphisms (HMIP) on chr6q23. MYB regulates proliferation and maturation of erythroid cells, and modulates gene expression within the HBB gene cluster. A distal enhancer located at ~84 kb upstream of MYB has been shown by GWAS (Farrell et al , Blood 2011), insertional mutation (Suzuki et al, Mol Cell Biol 2013), long-range interaction by 3C analysis (Stadhouders et al, J Clin Invest 2014), and gene editing with Cas9 nucleases (Canver et al, Nat Genet 2017). This enhancer encompasses a 3-bp deletion polymorphism (rs66650371), which is surrounded by binding sites for erythroid-specific transcription factors, and is likely the functional motif to account for most of the effect upon HbF level by this HbF QTL. Furthermore, ENCODE datasets annotated RNA polymerase 2 occupancy and a 50-bp RNA transcript adjacent to rs66650371, and led us to hypothesize that this transcript is part of a long noncoding RNA (lncRNA). LncRNAs are greater than 200 nucleotides long, are transcribed throughout the genome, and have broad functionality.

We characterized a novel 1283 bp lncRNA (HMI-LNCRNA) which was transcribed from this MYB enhancer and contained binding sites for TAL1/E47, GATA, and RUNX1. Its expression in 25 human tissues and cells was examined by qPCR. The thymus and testis had by far the highest expression. Increased expression was seen in erythroblasts from cultured cord blood CD34⁺ cells, followed by fetal liver, bone marrow, spleen and K562 cells. Other non-hematopoietic tissues had mostly minimal expression. The expression pattern of HMI-LNCRNA displayed similar features of most lncRNAs, including being located almost entirely within nuclei in erythroid cells, suggesting that it might regulate gene expression; and being less abundant than the protein-coding gene, MYB . HMI-LNCRNA expression was significantly higher in erythroblasts from cultured adult peripheral blood CD34⁺ cells, which expressed more HBB, compared to erythroblasts from cultured cord blood CD34⁺ cells, which expressed much more HBG . Similarly, HMI-LNCRNA expression was higher in HUDEP-2 cells, an immortalized human adult-like erythroid cells that expressed only HBB, compared to HUDEP-1 cells that expressed predominantly HBG .

Transduction of HUDEP-2 cells with HMI-lncRNA shRNA led to downregulation of HMI-LNCRNA expression to 50% of normal level, and resulted in 200-fold increase in HBG mRNA but no change in HBB or HBS1L mRNA. HBG mRNA expression in these cells amounted to 20% of the total HBB and HBG mRNA levels. The changes in HBG mRNA levels were associated with corresponding changes in HBG protein levels documented by Western blot analysis and immunofluorescence staining using anti-HBG antibody. Similar changes in mRNA and protein levels of HBG were seen when HUDEP-2 cells transduced with HMI-lncRNA shRNA were allowed to mature in culture for up to 7 days. There was no change in the expression of BCL11A and other transcription factors known to modulate HBG expression in HUDEP-2 cells transduced with HMI-lncRNA shRNA. FACS analysis of CD71 and CD235 expression showed that 55-65% of HUDEP-2 "naïve" cells or cells transduced with scrambled shRNA were double positive for both markers, while 85% of HUDEP-2 cells transduced with HMI-lncRNA shRNA were double positive, suggesting that downregulation of HMI-LNCRNA promoted maturation of HUDEP-2 cells.

HMI-LNCRNA is likely an enhancer RNA (eRNA), and facilitates the interaction between the distal enhancer with the MYB promoter. Our experimental results indicate that it has an important role in regulating HBG expression. Its downregulation can result in markedly increased HBG expression. HMI-LNCRNA might be a potential therapeutic target for HbF induction treatment in SCD and β-thalassemia.