Non-coding intergenic DNA sequences have lost the connotation of “junk DNA” as their role in gene regulation has become increasingly apparent. Elegant studies by Dr. Ralph Stadhouders and colleagues of Erasmus Medical Center in Rotterdam, The Netherlands, demonstrate how sequence variations in one such intergenic region (HBS1L-MYB) influences the expression of fetal hemoglobin (HbF) and other erythrocyte characteristics.
Red blood cell (RBC) parameters measured during a routine complete blood count provide important information for diagnosing and monitoring disease. It has long been known that there is significant variation in RBC parameters in individuals and in population groups, and genome-wide association studies (GWAS) have identified inherited DNA sequence variants linked to these clinically important traits. Of particular interest is the association of several erythroid parameters, including erythrocyte count, volume, hemoglobin concentration and HbF levels with single nucleotide polymorphisms (SNPs) in the q23 region of chromosome 6. More precise localization studies revealed that these SNPs cluster in a 126kb area between the HBS1L and MYB genes, establishing this intergenic area as a quantitative trait locus. The HBS1L gene encodes a member of the GTP-binding elongation factor family, but its function in erythroid biology is unknown. In contrast, the MYBgene plays an essential role in erythropoiesis as the encoded c-MYB protein is a transcription factor that influences expression of a number of erythoid specific genes. Regulation of the MYBgene in erythroid cells has not been completely characterised, but appears to involve microRNA-mediated control and a proximal promoter.
The functional roles of intergenic areas are difficult to assess because they are non-coding elements, but recent bioinformatic and technological advances have provided new tools to assess chromatin-protein interactions. In the current study, Dr. Stadhouders and colleagues used some of these techniques to investigate the interplay between sequence variants in the HBS1L-MYB intergenic region and the MYBgene. The authors identified a 24-kb sequence that contained a cluster of core erythroid-specific enhancers located 84 (-84) and 71 (-71) kb upstream of the MYB promoter that are involved in long-range control of the MYB gene. Several key SNPs in close proximity to these enhancers attenuated their function resulting in reduced expression of c-MYB, which correlated with increased expression of HbF. The SNPs changed critical nucleotides or the spacing in transcription factor binding motifs, which decreased the association of transcription factors including GATA-1 and the LDB1 complex. The authors postulate that the cluster of enhancers and bound transcription factors form a MYB active chromatin hub, which promotes transcription. The intergenic SNPs prevent optimal binding of transcription factors and also disrupt chromatin looping, destabilizing the hub and reducing transcription of the MYB gene.
The molecular mechanism whereby c-MYB regulates HbF and other erythrocyte traits is still unknown. It is speculated that the cell cycle may become dysregulated by the binding of c-MYB to cell-cycle components, resulting in premature termination of proliferation. Such a process would be expected to produce younger red cells with a higher mean cell volume and increased HbF levels. Alternatively, c-MYB may activate key γ-globin repressor genes and thereby exert control on HbF production.
In Brief
Inducing HbF in adults has therapeutic implications as doing so would ameliorate the severity of β-thalassemia and sickle cell disease. Targeting transcription factors is challenging, but this study provides evidence that intergenic enhancers play a key regulatory role and thus may offer an alternative target. Ingenious genome editing techniques have recently been developed, including the clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated protein (Cas) (CRISPR-Cas) system, which could be used to modify the -84 and/or -71 MYB enhancer sequences to diminish c-MYB expression and thereby augment HbF production.
This study provides insight into the complexity of long-range molecular mechanisms underlying MYBgene expression and establishes a framework for the development of novel therapeutic strategies.
Competing Interests
Dr. Coetzer reported no relevant conflicts of interest.
