Mechanism of e47-Pip interaction on DNA resulting in transcriptional synergy and activation of immunoglobulin germ line sterile transcripts.
E47 and Pip are proteins crucial for proper B-cell development. E47 and Pip cooperatively bind to adjacent sites in the immunoglobulin kappa chain 3' enhancer and generate a potent transcriptional synergy. We generated protein-DNA computer models to visualize E47 and Pip bound to DNA. These models predict precise interactions between the two proteins. We tested predictions deduced from these models by mutagenesis studies and found evidence for novel direct interactions between the E47 helix-loop-helix domain (Arg 357 or Asp 358) and the Pip N terminus (Leu 24). We also found that precise spatial alignment of the binding sites was necessary for transcriptional synergy and cooperative DNA binding. A Pip dominant negative mutant that cannot synergize with E47 inhibited enhancer activity in plasmacytoma cells and could not activate transcription in pre-B cells. Using electrophoretic mobility shift assays, we found that Pip can bind to the heavy-chain intron enhancer region. In addition, we found that in fibroblasts Pip greatly increased E47 induction of germ line I micro transcripts associated with somatic rearrangement and isotype class switching. However, a Pip dominant negative mutant inhibited germ line I micro transcripts. The importance of these results for late B-cell functions is discussed.
Nagulapalli
Sujatha
S
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Goheer
Aisha
A
Pitt
Leslie
L
McIntosh
Lawrence P
LP
Atchison
Michael L
ML
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000502:physiology; D019070:Cell Lineage; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D018257:Helix-Loop-Helix Motifs; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D007148:Immunoglobulin mu-Chains / Q000235:genetics*; D050835:Interferon Regulatory Factors; D007438:Introns; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D000099218:Interferon Regulatory Factor-4
D004247:DNA; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D007145:Immunoglobulin kappa-Chains; D007148:Immunoglobulin mu-Chains; D050835:Interferon Regulatory Factors; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D000099218:Interferon Regulatory Factor-4; C544912:TCF7L1 protein, human; C544913:Tcf7l1 protein, mouse