(A) HEPs from individuals homozygous for the minor allele of the phenotype-associated variants (HMIP-2 LD block variants; SNP/SNP) and WT control individuals (WT/WT) were cultured and assayed for MYB expression at indicated days (left: representative experiment, right: n = 4). (B) Correlation between intergenic genotype and MYB expression was determined using HEPs from 21 individuals (WT/WT, WT/SNP, and SNP/SNP intergenic genotypes; see Methods). Circle represents single data point considered to be an outlier. (C) ChIP-qPCR (n = 3) for GATA1/KLF1 using SNP/SNP and WT/WT HEPs. Enrichments were normalized to IgG and α-globin HS40 values (WT/WT set to 1). (D) Allele-specific measurement of GATA1 binding to rs9494142 (T/C) alleles using SNaPshot on heterozygous individuals (n = 4). rs9494142 C is the phenotype-associated minor allele. (C-allele set to 1). (E) Interaction frequencies between the –84 element and MYB promoter were measured (n = 5) using 3C-qPCR in SNP/SNP and WT/WT HEPs. (F) Allele-specific expression measured by SNaPshot in HEPs from individuals heterozygous (n = 5) or homozygous (n = 5) for the intergenic SNPs; rs210796 SNP (T/A) was used for quantification. (G) Proposed model explaining the effect of trait-associated intergenic SNPs on MYB regulation. Transcription factor-bound regulatory elements cluster around MYB to form an ACH, stimulating transcription (left). Intergenic SNPs reduce TF binding and chromatin looping, partially destabilizing the ACH and reducing MYB transcription (right). Lower MYB levels subsequently affect red cell traits. Error bars display SEM. Statistical significance was determined using linear regression analysis or Student’s t test. *P < 0.05; **P < 0.01.