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

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.

Pathogenic BCL11A variants provide insights into the mechanisms of human fetal hemoglobin silencing

“…We extend recent findings that suggest an important role for ZnF5 in enabling DNA binding at the HBG1/2 proximal promoters for effective HbF silencing [ 8 , 9 , 12 ]. We demonstrate how impairment of this DNA binding event is sufficient to derepress HbF in vivo by using a faithful primary cell model.…”

“…We performed chromatin immunoprecipitation in cord blood-derived erythroid cells expressing the wild type or Mut 4 HA-tagged proteins at day 11 of erythroid differentiation. With this approach, we noted robust binding at well-characterized sites known to be bound by BCL11A from prior studies by the wild type, but significantly diminished binding by the Mut 4 ( Fig 4F ) [ 8 , 9 , 12 ]. This was particularly notable at the human β-globin locus on chromosome 11, where we observed reduced binding at the HBG1/2 promoters ( Fig 4F ).…”

“…We have shown that physiologic expression of the XL isoform of BCL11A can effectively silence HBG1/2 expression in erythroid cells derived from primary cord blood CD34 + hematopoietic stem and progenitor cells (HSPCs) [ 12 , 15 ]. We therefore utilized lentiviral transduction of cord blood HSPCs with either the wild type or mutant forms of the BCL11A XL cDNA and promoted semi-synchronous erythroid differentiation using a well-established in vitro culture approach [ 23 , 32 , 33 ] ( Fig 2A ).…”

“…Therefore, Mut 4 is expected to have a reduced binding affinity for the BCL11A motif, such that key DNA binding activities would be altered. This variant would be predicted to thereby alter DNA binding by the XL isoform of BCL11A, including the critical interactions at the HBG1/2 promoters that have been shown to have a role in HbF silencing [ 8 , 9 , 12 ].…”

“…These studies have identified co-factors that cooperate with BCL11A [ 4 , 7 ], DNA binding sites for BCL11A within the HbF-encoding HBG1/2 (γ-globin) promoters [ 8 , 9 ], and potential long-range interactions mediated by BCL11A [ 10 , 11 ]. Recent functional analyses through combinatorial genome editing in single erythroid progenitor cells have revealed how BCL11A acts at both the HBG1/2 promoter elements and through distal interactions to effectively silence HbF [ 12 ]. In addition, transcriptional regulatory elements that are important for erythroid-specific expression of BCL11A [ 13 , 14 ] and upstream developmental regulators of BCL11A mRNA translation have been identified [ 15 ].…”