Chromosomal Loop Editing Reveals New Epigenomic Landscape In The Beta-Globin Locus With Implications For Hemoglobinopathy Therapies

Human β-globin locus consists of at least six genes encoding components of the oxygen transport protein hemoglobin and an upstream locus control region (LCR) containing five DNase I hypersensitive sites. In addition, there are at least four conserved CTCF insulator elements surrounding the locus, which form dynamic chromatin interaction patterns across different cell types in the course of hematopoietic stem and progenitor cells (HSPCs) development. Chromatin conformation of the β-globin locus in normal adult CD34+ HSPCs reveals the formation of three topological associated domains (TADs), in which individual TAD boundaries are demarcated by CTCF sites (Figure 1). By comparing chromatin loop interactions between CD34+ HSPCs and its differentiated erythroid progenitors, we identify a chromatin loop that forms in erythroid progenitors and is not evident in the CD34+ HSPCs. Detailed examination of this loop shows that a DNase I hypersensitivity site, also called 3'HS1, overlaps with a CTCF site that forms a loop with another CTCF site adjacent to OR52A5 gene (Figure 2). To investigate the role of this specific chromatin loop in the regulation of hemoglobin gene expression, we knock out the 3'HS1 CTCF motif in adult CD34+ HSPCs under erythroid differentiation medium by CRISPR/Cas9-mediated gene editing. We find that deletion of 3'HS1 CTCF results in a 2-fold decrease of β-globin (HBB) and a 4-fold increase of the fetal hemoglobin gene encoded by γ-globin (HBG1/2) in erythroid colonies of edited CD34+ cells (Figure 3). Elevation of fetal hemoglobin upon 3'HS1 CTCF deletion was also confirmed in human umbilical cord blood-derived erythroid progenitor-2 cells (HUDEP-2), which results in a 12-fold increase of γ-globin expression (Figure 4). These results suggest that the 3'HS1 CTCF plays a crucial role in regulating the expression of fetal hemoglobin gene, however it remains unclear whether changes in chromatin structure is responsible for these changes. CTCF looping interactions have been described to form under convergent directionality. To validate the role of 3'HS1 CTCF in establishing chromatin interactions at the β-globin locus, we aim to invert this binding motif and evaluate how it disrupts chromatin organization and gene expression at this locus. Deciphering the underlying mechanisms will shed light on how three-dimensional chromatin structure is reorganized in differentiating erythroid cells as it undergoes nuclear condensation. Furthermore, elevation of fetal hemoglobin expression can potentially be a new therapeutic gene editing strategy to treat sickle cell disease and some cases of β-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.