NF-kappa B and Oct-2 synergize to activate the human 3' Igh hs4 enhancer in B cells

In B cells, the Igh gene locus contains several DNase I-hypersensitive (hs) sites with enhancer activity. These include the 3' Igh enhancers, which are located downstream of the Calpha gene(s) in both mouse and human. In vivo experiments have implicated murine 3' enhancers, hs3B and/or hs4...

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Published in:The Journal of immunology (1950) Vol. 172; no. 2; p. 1054
Main Authors: Sepulveda, Manuel A, Emelyanov, Alexander V, Birshtein, Barbara K
Format: Journal Article
Language:English
Published: United States 15.01.2004
Subjects:
3' Untranslated Regions - immunology
Animals
B-Lymphocytes - chemistry
B-Lymphocytes - immunology
B-Lymphocytes - metabolism
Chromatin - metabolism
DNA-Binding Proteins - metabolism
DNA-Binding Proteins - physiology
Electrophoretic Mobility Shift Assay
Enhancer Elements, Genetic - immunology
Host Cell Factor C1
Humans
Immunoglobulin Heavy Chains - genetics
Immunoglobulin Heavy Chains - metabolism
Mice
NF-kappa B - metabolism
NF-kappa B - physiology
Nuclear Proteins
Octamer Transcription Factor-1
Octamer Transcription Factor-2
PAX5 Transcription Factor
Precipitin Tests
Protein Binding - genetics
Protein Binding - immunology
Proteins - isolation & purification
Proteins - metabolism
Proto-Oncogene Proteins - isolation & purification
Trans-Activators - metabolism
Trans-Activators - physiology
Transcription Factor RelB
Transcription Factors - isolation & purification
Transcription Factors - metabolism
Transcription Factors - physiology
Transfection
ISSN:0022-1767
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Summary:In B cells, the Igh gene locus contains several DNase I-hypersensitive (hs) sites with enhancer activity. These include the 3' Igh enhancers, which are located downstream of the Calpha gene(s) in both mouse and human. In vivo experiments have implicated murine 3' enhancers, hs3B and/or hs4, in class switching and somatic hypermutation. We previously reported that murine hs4 was regulated by NF-kappaB, octamer binding proteins, and Pax5 (B cell-specific activator protein). In this study we report that human hs4 is regulated differently. EMSAs and Western analysis of normal B cells before and after stimulation with anti-IgM plus anti-CD40 showed the same complex binding pattern formed by NF-kappaB, Oct-1, and Oct-2 (but not by Pax5). A similar EMSA pattern was detected in mature human B cell lines (BL-2, Ramos, and HS-Sultan) and in diffuse large B cell lymphoma cell lines, although yin yang 1 protein (YY1) binding was also observed. We have confirmed the in vivo association of these transcription factors with hs4 in B cells by chromatin immunoprecipitation assays. The diffuse large B cell lymphoma cell lines had a distinctive slow-migrating complex containing YY1 associated with Rel-B. We have confirmed by endogenous coimmunoprecipitation an association of YY1 with Rel-B, but not with other NF-kappaB family members. Transient transfection assays showed robust hs4 enhancer activity in the mature B cell lines, which was dependent on synergistic interactions between NF-kappaB and octamer binding proteins. In addition, human hs4 enhancer activity required Oct-2 and correlated with expression of Oct coactivator from B cells (OCA-B).
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ISSN:0022-1767
DOI:10.4049/jimmunol.172.2.1054