Subunit structure of cell-specific E box-binding proteins analyzed by quantitation of electrophoretic mobility shift

Area of Science:

• Molecular Biology
• Gene Regulation
• Protein Biochemistry

Background:

• Insulin and immunoglobulin gene expression relies on E boxes in enhancer regions.
• These E boxes bind cell-specific nuclear factors: insulin enhancer factor 1 (IEF1) and lymphoid enhancer factor 1 (LEF1).
• IEF1 and LEF1 are recognized by antibodies to the helix-loop-helix (HLH) protein A1, suggesting a shared component.

Purpose of the Study:

• To characterize the molecular composition of IEF1 and LEF1 complexes.
• To determine the role of E2A gene products in regulating insulin and immunoglobulin genes.
• To identify novel protein factors involved in insulin gene regulation.

Main Methods:

• Electrophoretic mobility shift analysis (EMSA) using in vitro translated DNA-binding proteins.
• Molecular weight estimation of DNA-binding proteins.
• Antibody recognition assays using nuclear extracts and in vitro translated proteins.
• Mixing experiments to analyze protein complex formation.

Main Results:

• IEF1 and LEF1 function as dimeric complexes.
• IEF1 is a heterodimer of a 67-kDa protein (recognized by A1 antibodies) and a 25-kDa protein.
• LEF1 is a homodimer of 67-kDa proteins, consistent with E2A gene products.
• The 25-kDa subunit of IEF1 (IESF1) forms heterodimers with A1 and is specific to certain cell types.

Conclusions:

• E2A gene products are directly involved in regulating insulin and immunoglobulin gene expression.
• Regulation of insulin gene expression requires E2A products and the novel 25-kDa HLH protein, IESF1.
• The distinct subunit composition of IEF1 and LEF1 suggests differential roles in gene regulation.