Role of chromosomal 6q23 intergenic region in the modulation of haemoglobin F production

Abstract

The HBS1L-MYBintergenic region on chromosome 6q23 is a quantitative trait locus (QTL) of haemoglobin F (Hb F) expression. It shows characteristics of a distal regulatory regionwhich may affect expression of neighbouring genes including MYB. However, the exact functional role of this region on Hb F level is still unclear. Recently, a 3-bp deletion site in this intergenic region was found to be linked with high Hb F level and possess motifs for gene regulation, making it a candidate functional site of this QTL. Here we further investigate this region for its functional role in Hb F modulation.

Erythroid cultures were used to profile MYBand GYPA expression from human cord blood of different 6q23 haplotypes. Regulatory functions of the 3-bp deletion region were assessed by luciferase assays. Identification of proteins binding to this region was done through electrophoretic mobility shift assay (EMSA) with subsequent mass spectrometry. Results were confirmed by chromatin immunoprecipitation (ChIP).

High Hb F haplotype at 6q23 was associated with lower MYB expression and accelerated erythroid maturation when compared to low Hb F haplotype. The 3-bp deletion linked to a high HbF haplotype showed a site-specific suppressive effect on gene expression in luciferase assays. An architectural DNA-bending/looping chromosomal protein, high mobility group protein B1 (HMGB1), was found to bind differentially to this site depending on the presence or absence of the 3-bp deletion.

These functional studies confirm a regulatory role of the Hb F QTL at HBS1L-MYB intergenic region on chromosome 6q23. The suppressive effect of high Hb F haplotypes can lead to reduced MYB expression and accelerated erythroid maturation that is known to be associated with a high Hb F level. Identification of HMGB1 binding to this 3-bp deletion site provides a potential mechanism for the action of this QTL on expression of MYB and other important genes in this chromosomal region.