Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2832898 | Molecular Immunology | 2006 | 8 Pages |
Abstract
Transcription factor-mediated immunoglobulin (Ig) enhancer activation has been analyzed extensively outside the physiological constraints of chromatin. Towards understanding the role sequence-specific DNA binding proteins identified by these methods play in activating Ig genes during B cell development, we have investigated in vivo interaction between the Ig enhancer activator PU.1 and two target elements, the Igμ and κ3â² enhancers, by chromatin immunoprecipitation (ChIP). By using two antibodies recognizing different PU.1 epitopes in murine B cells, these analyses demonstrate that ChIP results may depend on the availability of the epitope(s) targeted by the immunoprecipitating antibody. Specifically, PU.1 epitope availability at the μ and κ3â² enhancers does not accurately quantitate total PU.1 association. This result suggests the nucleoprotein complexes formed at these various active enhancers is cell type-specific. Interestingly, RAG1â/â but not RAG2â/â pro-B cells lack PU.1/κ3â² association, probably due to limited accessibility of the κ locus in the former. The more robust association of PU.1 with the κ3â² versus μ enhancer in all but RAG1â/â B lineage cells is not explained by differences in PCR primer efficiency, but likely reflects the different structures formed by the complexes at μ versus κ3â² enhancers. Finally, PU.1 is not associated with an inaccessible μ or κ3â² enhancer chromatin structure in macrophages, again emphasizing the importance cellular protein context plays in PU.1/Ig enhancer association. The demonstration that changes in epitope availability, hence nucleoprotein structure, can be monitored by ChIP suggests using this technique to monitor biologically important changes in nucleoprotein complex structure/composition in situ.
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Authors
Daniel C. McDevit, Barbara S. Nikolajczyk,