Session: 112. Thalassemia and Globin Gene Regulation I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Sickle Cell Disease, Translational Research, Thalassemia, Hemoglobinopathies, Diseases
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.
Disclosures: Blobel: Fulcrum Therapeutics: Research Funding; Pfizer: Consultancy.
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