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

Yong Shen,Yoon Jung Kim,Anindita Basak,Satish K Nandakumar,Jeffrey M Verboon,Richard A Voit,Yuannyu Zhang,Samantha Marglous,Nan Liu,Stuart H Orkin,Ayesha Ejaz,Vijay G Sankaran,Claudia Fiorini,Jian Xu

doi:10.1038/s41467-021-25298-9

Abstract

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.

Highlights

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
Such deletions can be broadly classified into two categories: those that have higher HBG1/2 mRNA and HbF production, termed hereditary persistence of fetal hemoglobin (HPFH) deletions, and those that are characterized by lower HbF production with resultant globin chain imbalance, termed δβ-thalassemia (Fig. 1a and Supplementary Fig. 1a)
We expected that existing genomeediting tools would enable us to recreate each category of the well-characterized rare perturbations that significantly alter HbF, including variants in the proximal promoters of the HBG1/2 genes, deletions involving HBD and HBB, and perturbation of the key trans-acting regulatory factors, BCL11A and ZBTB7A (Fig. 1a and Supplementary Fig. 1b)

Summary

Introduction

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 geneticallynominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A number of studies have provided insights into HbF regulation through the identification and analysis of naturally occurring mutations impacting this process Such variants have been extensively characterized at two distinct loci: (1) in the gene encoding the BCL11A transcription factor and (2) within the β-globin gene locus that harbors the HbF genes, HBG1 and HBG2. We illuminate the value of performing single-cell individual and combinatorial functional perturbations that are inspired by human genetic variation

Methods

Results

Conclusion