Genome wide association studies have identified two quantitative trait loci outside of the β-globin cluster associated with fetal hemoglobin (HbF) levels, number of F cell and β-thalassemia severity: the HBS1L-MYB intergenic region and the BCL11A gene. In order to understand the functional role of the associated variants at these loci we applied “Genome Wide Chromosome Conformation Capture” (Hi-C), followed by a novel technique for a selective enrichment at these target regions, to characterize whether they are involved in long range physical interactions able to modulate HBS1L-MYB and BCL11A expression. As a first step we optimized a Hi-C protocol in the K562 fetal erythroid cell line and we set up the conditions for the new method based on the selective enrichment of target regions. We were able to validate the new capture system analyzing the β-globin locus, as a control model, detecting all the interactions found by other “3C-like” technologies with a higher resolution. We then analyzed the data at the HBS1L-MYB and BCL11A loci; the most significant detected interactions involved four HBS1L-MYB intergenic regions, three of which contain the GWAS SNPs, the HBS1L and MYB genes. We hypothesized a contact model where the associated variants could exert their regulatory role likely altering transcription factors binding sites and thus DNA long-range interactions resulting in different levels of MYB expression. Indeed, although we can not exclude the implication of HBS1L gene in this mechanism, MYB represents the best candidate in modulating HbF levels given its role in the erythropoiesis kinetics. Our results highlighted the power of the new capture system, able to identify chromatin interactions with very high resolution simultaneously at different loci. Finally, we discovered new chromatin interactions that support the transcription factor MYB as a potential good candidate to develop new targeted therapeutic strategies to treat β-thalassemia patients.
Identificazione e analisi funzionale di fattori regolatori dei geni globinici
DESOGUS, ALESSIA
2014-05-30
Abstract
Genome wide association studies have identified two quantitative trait loci outside of the β-globin cluster associated with fetal hemoglobin (HbF) levels, number of F cell and β-thalassemia severity: the HBS1L-MYB intergenic region and the BCL11A gene. In order to understand the functional role of the associated variants at these loci we applied “Genome Wide Chromosome Conformation Capture” (Hi-C), followed by a novel technique for a selective enrichment at these target regions, to characterize whether they are involved in long range physical interactions able to modulate HBS1L-MYB and BCL11A expression. As a first step we optimized a Hi-C protocol in the K562 fetal erythroid cell line and we set up the conditions for the new method based on the selective enrichment of target regions. We were able to validate the new capture system analyzing the β-globin locus, as a control model, detecting all the interactions found by other “3C-like” technologies with a higher resolution. We then analyzed the data at the HBS1L-MYB and BCL11A loci; the most significant detected interactions involved four HBS1L-MYB intergenic regions, three of which contain the GWAS SNPs, the HBS1L and MYB genes. We hypothesized a contact model where the associated variants could exert their regulatory role likely altering transcription factors binding sites and thus DNA long-range interactions resulting in different levels of MYB expression. Indeed, although we can not exclude the implication of HBS1L gene in this mechanism, MYB represents the best candidate in modulating HbF levels given its role in the erythropoiesis kinetics. Our results highlighted the power of the new capture system, able to identify chromatin interactions with very high resolution simultaneously at different loci. Finally, we discovered new chromatin interactions that support the transcription factor MYB as a potential good candidate to develop new targeted therapeutic strategies to treat β-thalassemia patients.
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