Inducible DNA-protein interactions of the murine kappa immunoglobulin enhancer in intact cells: comparison with in vitro interactions.
The large intron of the kappa immunoglobulin gene contains a cis-acting enhancer element, which is important in the tissue-specific expression of the gene. We have confirmed the binding activity of a sequence-specific factor present in lymphoid extracts derived from cell lines expressing, or induced to express, the kappa gene. We have extended these studies to show the binding activity is present in normal activated splenic B cells as well as lambda producing cells, and have demonstrated by DNAse footprint analysis full protection of a sequence containing the 11 bp homology to the SV-40 core enhancer. We have compared these in vitro binding studies with an analysis of protein-DNA interactions in intact murine cell lines using genomic sequencing techniques. We demonstrate significant alterations in DMS reactivity of DNA in the murine 70Z/3 cell line after it is induced to kappa expression. These alterations occur at guanine residues which are part of the the 11 bp core sequence, and are identical to those observed in cells constitutively expressing kappa. This provides direct evidence for the induced binding of the tissue specific factor to intact chromatin. In intact chromatin we also observed significant alteration in the reactivity of a guanine, 3' of the core sequence, which is part of a potential secondary DNA structure, and protection of four residues that are part of a region homologous to the heavy chain enhancer.
Hromas
R
R
Department of Biochemistry, University of Iowa, Iowa City 52242.
Pauli
U
U
Marcuzzi
A
A
Lafrenz
D
D
Nick
H
H
Stein
J
J
Stein
G
G
Van Ness
B
B
D000818:Animals; D001402:B-Lymphocytes / Q000032:analysis; D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005803:Genes, Immunoglobulin; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics
D002843:Chromatin; D004268:DNA-Binding Proteins; D007145:Immunoglobulin kappa-Chains