A single 3' alpha hs1,2 enhancer in the rabbit IgH locus

Area of Science:

• Immunology
• Genetics
• Molecular Biology

Background:

• Immunoglobulin heavy chain (IgH) gene expression is regulated by cis-acting elements.
• The 3'alpha enhancer (3'alphaE), composed of hypersensitive sites, is crucial for IgH gene regulation in mice and humans.
• Rabbits possess multiple Calpha genes, necessitating an investigation into their associated regulatory elements.

Purpose of the Study:

• To determine if a 3'alphaE is associated with each of the rabbit Calpha genes.
• To identify and characterize the hs1,2 enhancer region in the rabbit genome.
• To assess the enhancer activity of the identified rabbit hs1,2 region.

Main Methods:

• Development of a rabbit hs1,2 probe for identifying enhancer homologues.
• Screening a genomic fosmid library to locate potential enhancer regions.
• Genomic Southern blot analysis to confirm the number of enhancer regions.
• Transient transfection assays using luciferase reporter genes to test enhancer activity with different promoters (SV40, V(H), Ialpha).

Main Results:

• A single hs1,2 enhancer fragment was identified 8-kb downstream of the presumed 3'-most Calpha gene (Calpha13).
• A novel Calpha gene, Calpha14, was discovered upstream of Calpha13.
• Genomic Southern blot confirmed only one hs1,2 enhancer region in the rabbit genome.
• The identified hs1,2 enhancer significantly enhanced SV40 and V(H) promoter activity and slightly enhanced the Ialpha promoter activity in transfection assays.

Conclusions:

• The rabbit genome contains a single hs1,2 enhancer located at the 3' end of the IgH gene cluster.
• This unique hs1,2 enhancer is a potential cis-acting element involved in regulating rabbit IgH gene expression.
• The findings contribute to understanding the regulatory mechanisms of IgH gene expression across different species.