An Intergenic Silencer Region Regulates HBG Expression
The most common genetic disorders worldwide are the ß-hemoglobinopathies, including sickle cell disease and ß-thalassemia caused by HBB gene mutations. Five functional ß-like globin genes on chromosome 11 are expressed during development in a tissue and stage-specific manner. The HBG genes encoding g-globin polypeptide chains to form fetal hemoglobin (HbF), undergo gene silencing during the first year of life. Our study aimed to characterize an upstream DNA regulatory region that can be targeted for HBG reactivation during adult development. We investigated the intergenic region between HBE1 and HBG2 which we termed the fetal chromatin domain (FCD) to define its role in globin gene regulation. Using normal human CD34+ stem cells to establish erythroid progenitors, we characterized the binding of different transcription factors to the FCD, which facilitated long-range chromatin looping between the FCD and locus control region and HBG. Using CRISPR/Cas9 genome editing technology, the FCD was knocked out in KU812 and CD34+ stem cells. We observed enhanced HBG transcription and  HbF expression in both lines. For FCD knockout CD34+ stem cells, flow cytometry showed increased HbF positive cells and HbF expression and enhanced long-range interactions between the locus control and HBG region.  Single cell RNA-sequencing revealed normal erythropoiesis in the FCD-KO line and partial reversal of HBG silencing. These results support a putative silencer role of the FCD in regulating HBG transcription and HbF expression. Overall design: The FCD knockout or AAVS1 edited controls were generated in CD34+ stem cells by CRISPR/Cas9 in the Hematopoietic Progenitor/Stem Cell Gene Editing core of the Fred Hutchinson Cancer Center. CD34+ stem cells were then grown in primary culture system.  and harvested on Day 10 for scRNA-seq. Cells were resuspended in ice-cold cryopreservation solution comprised of 50% fetal bovine serum, 40% growth media, and 10% DMSO and store at -80°C until shipping to Active Motif (Carlsbad, CA). Clustering algorithms are applied to identify distinct cell populations and further characterization of gene expression patterns within these clusters was performed. The resulting data were used to compare gene expression patterns across erythroid cell populations and to identify differentially expressed genes.