Biology subjects

Boontanrart, M. Y.

Publications and source records attributed to Boontanrart, M. Y..

2 recordsLinked to original sources

bioRxiv2022

Engineering of the Endogenous HBD promoter increases HbA2

The {beta}-hemoglobinopathies, such as sickle cell disease and {beta}-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of {beta}-globin subunits in adult hemoglobin. Many gene editing efforts to treat the {beta}-hemoglobinopathies attempt to correct {beta}-globin mutations or increase {gamma}-globin for fetal hemoglobin production. {delta}-globin, the subunit of adult hemoglobin A2, has high homology to {beta}-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of {delta}-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of {delta}-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate {delta}-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of {delta}-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total {beta}-like globins. Edited CD34+ hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 35% HBD. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat {beta}-hemoglobinopathies.

genetics↗

bioRxiv2020

ATF4 mediates fetal globin upregulation in response to reduced β-globin

Fetal development and anemias such as {beta}-hemoglobinopathies trigger rapid production of red blood cells in a process known as stress erythropoiesis. Cellular stress prompts differentiating erythroid precursors to express high levels of fetal {gamma}-globin, which has suggested strategies to treat hemoglobinopathies such as thalassemia and sickle cell disease. However, the mechanisms underlying {gamma}-globin production during cellular stress are still poorly defined. Here we use CRISPR-Cas genome editing and CRISPRi transcriptional repression to model the stress caused by reduced levels of adult {beta}-globin. We find that loss of {beta}-globin is sufficient to induce widespread globin compensation, including robust re-expression of {gamma}-globin. Time-course RNA-seq of differentiating isogenic erythroid precursors identified the ATF4 transcription factor as a causal regulator of this response. ChIP-seq of multiple erythroid precursor genotypes and differentiation states revealed that {beta}-globin knockout leads to reduced engagement of ATF4 targets involved in the unfolded protein response. This ATF4 program indirectly regulates the levels of BCL11A, a key repressor of {gamma}-globin. Identification of ATF4 as a key regulator of globin compensation adds mechanistic insight to the poorly understood phenomenon of stress-induced globin compensation and could be relevant for proposed gene editing strategies to treat hemoglobinopathies.

genetics↗