Examining the Role of Individual Baf Chromatin Remodeling Complex Components in Globin Gene Regulation

Regulation of the switch from fetal (HbF, α₂γ₂) to adult (HbA,α₂β₂) hemoglobin production is an important area of study as reversal of the switch is a key therapeutic approach for treatment of sickle cell disease and β-thalassemia. While multiple specific repressors of γ-globin gene transcription have been identified, there has not been a parallel finding of a γ-globin specific transcriptional activator. The mammalian BAF (BRG1/BRM-associated factor) complex is a multi-subunit, ATP-dependent, chromatin remodeling complex of the SWI/SNF family, with diverse composition and roles in regulation of gene expression. BRG1/SMARCA4 is a core component of the complex with ATPase catalytic activity. Prior studies showed that BRG1 is essential for proper β-globin expression and regulates long-range chromatin interactions between the locus control region (LCR) and the β-globin gene promoter. Studies in K562 cells, where γ-globin expression is predominant, have similarly shown the requirement for BRG1 for transcriptional activation. To date, the role of the BAF complex in regulation of γ-globin versus β-globin gene expression has not been well characterized. Moreover, the considerable diversity in the subtypes of this complex (such as polybromo-associated BAF or PBAF and non-canonical BAF), with subunit composition and functions that differ across cell types and developmental stages, suggests the hypothesis that specific subtypes or subunits of the BAF complex have specific roles in γ-globin gene regulation.

We recently identified BRG1 as a potential activator of γ-globin production in a HUDEP2 CRISPR screen targeting ATPase and helicase domains. In validation experiments in HUDEP2 and primary human erythroid cultures, loss of BRG1 led to a marked decrease in γ-globin containing F-cells by flow cytometry and γ-globin protein by western blot, along with a smaller decrease in β-globin production. Because the BAF subunits have significant heterogeneity in expression and function, we asked whether any components may have specificity for γ-globin versus β-globin gene transcription in adult erythroid cells. Using CRISPR-Cas9 targeting in a HUDEP2 sub-clone with elevated fetal hemoglobin levels, we systematically ablated 12 individual BAF/PBAF complex components and assayed fetal hemoglobin by flow cytometry, HPLC, and quantitative western blots. We identified 5 components whose loss of function led to a reduction in γ-globin relative to β-globin: BAF53A, BAF155, BAF60A, BAF60B, and BAF57.

We subsequently depleted these five components in primary human erythroid cultures from three independent donors using CRISPR-Cas9 targeting. While loss of BAF53A impaired cell proliferation, loss of remaining components was well tolerated. Loss of BAF155 and BAF60B had minimal effect on globin chain production. Loss of BAF60A had the most potent effect on γ-globin, with up to 80% reduction in γ-globin with no decrease in β-globin production. BAF57 knockdown led to a more modest decrease in γ-globin, but with some decrease in total globin production.

Together these data suggest that the requirements for some specific components of the BAF complex differ for activation of fetal or adult globin gene transcription. Additional studies are under way to characterize chromatin accessibility changes at the β-globin locus in response to alterations in the BAF complex, as well as the response to loss of additional BAF complex components.

Blobel:Fulcrum Therapeutics: Research Funding; Pfizer: Consultancy.