Influence of the polymorphisms of the α-major regulatory element HS-40 on in vitro gene expression

Author(s)

D.M. Ribeiro ^[1] ; T.R. Zaccariotto ^[2] ; M.N.N. Santos ^[3] ; F.F. Costa ^[4] ; M.F. Sonati ^[5]

Affiliation

^[1] Universidade Estadual de Campinas. Departamento de Patologia Clínica

^[2] Universidade Estadual de Campinas. Departamento de Patologia Clínica

^[3] Universidade Estadual de Campinas. Departamento de Patologia Clínica

^[4] Universidade Estadual de Campinas. Faculdade de Ciências Médicas. Hemocentro e Departamento de Clínica Médica - Brasil

^[5] Universidade Estadual de Campinas. Departamento de Patologia Clínica

• Document type: Journal article
• Source: Brazilian Journal of Medical and Biological Research; v. 42, n. 9, p. 783-786, 2009-07-31.

Abstract

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression. (AU)

• FAPESP's process: 02/13801-7 - Hereditary hemoglobin disorders: molecular genetics, clinical features and animal models with the production of transgenic animals
• Grantee: Fernando Ferreira Costa
• Support Opportunities: Research Projects - Thematic Grants