A unified model of human hemoglobin switching through single-cell genome editing

• Published in: Nature communications Vol. 12; no. 1; p. 4991
• Main Authors: Shen, Yong, Verboon, Jeffrey M., Zhang, Yuannyu, Liu, Nan, Kim, Yoon Jung, Marglous, Samantha, Nandakumar, Satish K., Voit, Richard A., Fiorini, Claudia, Ejaz, Ayesha, Basak, Anindita, Orkin, Stuart H., Xu, Jian, Sankaran, Vijay G.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 17.08.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 38/39; 45; 45/23; 631/136/232/1473; 631/208/199; 631/208/200; 692/699/1541/4036; beta-Globins - genetics; Chromatin; Chromosomes; CRISPR-Cas Systems; DNA-Binding Proteins - metabolism; Editing; Fetal Hemoglobin - genetics; Fetal Hemoglobin - metabolism; Fetuses; Gene Editing; Gene Expression; Genetic analysis; Genetic diversity; Genome editing; Genomes; Globins; Hemoglobin; Hemoglobins - genetics; Hemoglobins - metabolism; Humanities and Social Sciences; Humans; multidisciplinary; Mutation; Regulatory mechanisms (biology); Repressor Proteins; Science; Science (multidisciplinary); Switching; Transcription factors; Transcription Factors - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-25298-9

Key mechanisms of fetal hemoglobin (HbF) regulation and switching have been elucidated through studies of human genetic variation, including mutations in the HBG1/2 promoters, deletions in the β-globin locus, and variation impacting BCL11A. While this has led to substantial insights, there has not been a unified understanding of how these distinct genetically-nominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A key limitation has been the inability to model specific genetic changes in primary isogenic human hematopoietic cells to uncover how each of these act individually and in aggregate. Here, we describe a single-cell genome editing functional assay that enables specific mutations to be recapitulated individually and in combination, providing insights into how multiple mutation-harboring functional elements collectively contribute to HbF expression. In conjunction with quantitative modeling and chromatin capture analyses, we illustrate how these genetic findings enable a comprehensive understanding of how distinct regulatory mechanisms can synergistically modulate HbF expression. Genetic mechanisms underlying fetal hemoglobin (HbF) regulation and switching are not fully understood. Here, the authors develop a single-cell genome editing functional assay to model how effects of mutation-harbouring functional elements contribute to HbF expression.