title: NF-kappa B and Oct-2 synergize to activate the human 3' Igh hs4 enhancer in B cells.
abstract: 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).
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: Manuel A
authors.initials: MA
authors.lastname: Sepulveda
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Emelyanov
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Birshtein
mesh_terms: D020413:3' Untranslated Regions / Q000276:immunology*; D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D018797:Cell Cycle Proteins; D002843:Chromatin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000276:immunology*; D051863:Host Cell Factor C1; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology*; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; D051757:PAX5 Transcription Factor; D011233:Precipitin Tests; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology*; D051841:Transcription Factor RelB; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; C497693:Hcfc1 protein, mouse; D051863:Host Cell Factor C1; D007143:Immunoglobulin Heavy Chains; D016328:NF-kappa B; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; D050813:Octamer Transcription Factor-2; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human; C494165:POU2F1 protein, human; C058435:POU2F2 protein, human; C497282:Pax5 protein, mouse; C494166:Pou2f1 protein, mouse; C489206:Pou2f2 protein, mouse; D011506:Proteins; D011518:Proto-Oncogene Proteins; C497593:RELB protein, human; C497594:Relb protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; C456659:YY1AP1 protein, human; D051841:Transcription Factor RelB