An erythroid-specific ATP2B4 enhancer mediates red blood cell hydration and malaria susceptibility
Samuel Lessard, … , Daniel E. Bauer, Guillaume Lettre
Samuel Lessard, … , Daniel E. Bauer, Guillaume Lettre
Published July 17, 2017
Citation Information: J Clin Invest. 2017;127(8):3065-3074. https://doi.org/10.1172/JCI94378.
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Research Article Genetics Hematology

An erythroid-specific ATP2B4 enhancer mediates red blood cell hydration and malaria susceptibility

Abstract

The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4–/– mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.

Authors

Samuel Lessard, Emily Stern Gatof, Mélissa Beaudoin, Patrick G. Schupp, Falak Sher, Adnan Ali, Sukhpal Prehar, Ryo Kurita, Yukio Nakamura, Esther Baena, Jonathan Ledoux, Delvac Oceandy, Daniel E. Bauer, Guillaume Lettre

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Figure 3

Genome editing at the ATP2B4 erythroid enhancer.

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Genome editing at the ATP2B4 erythroid enhancer. (A) Upper panel shows d...
(A) Upper panel shows deletions engineered around the ATP2B4 regulatory region. Lower panel shows sgRNA cleavage sites inside the 98-bp core enhancer. The alternative allele of rs10751451 creates a GATA1 motif (purple box). Boxes separated by dashed lines correspond to GATA1-TAL1 motifs. (B) ATP2B4 expression measured in HUDEP-2 cells. Combined analysis of 2 sgRNA pairs that result in overlapping 927-bp and 889-bp deletions. Nondeletion (n = 13), monoallelic deletion (n = 13), and biallelic deletion (n = 14) are clones with, respectively, 0, 1, or 2 ATP2B4 enhancer alleles deleted. None of these clones has an inversion allele. One clone was identified with 1 deletion allele and 1 inversion allele. Four clones exposed to nontargeting sgRNAs are shown for comparison. Gene expression is normalized to mean of nondeletion clones for the same sgRNA pair. Bars and whiskers show mean and SD. The difference in ATP2B4 expression levels between nondeletion and nontargeting clones is not significant. (C) ATP2B4 expression in 293T cell clones exposed to enhancer targeting sgRNA pairs, but without deletion (n = 4) or with biallelic deletion (n = 3). (D) ATP2B4 expression in HUDEP-2 cells with 98-bp core enhancer deletion. Control (n = 2) refers to 1 nontargeting control clone and unedited parental cells as compared with biallelic deletion clones (n = 11). (E) ATP2B4 expression in HUDEP-2 cells with individual sgRNAs specifying cleavages within the 98-bp core enhancer. Each dot indicates an edited bulk population that is an independent transduction of cells. Mean and SD are plotted for each of 4 biological replicates. Gene expression is normalized to unedited cells. In B and E, we used 1-way ANOVA with Bonferroni’s correction for the number of comparisons tested. In C and D, we used Welch’s t test. All P values are 2 tailed. **P < 0.01; ****P < 0.0001.