Sequential activation of the immunoglobulin heavy (IgH)chain gene and the kappa light chain gene during B cell development is governed, in part, by enhancer sequences located in the JC intron of these genes. The long-term objectives of this application are two-fold: first, to use enhancers as a means to identify the differentiation signals that trigger B cell development and second, to understand how a tissue-specific enhancer works. At the simplest level, enhancers """"""""work"""""""" by binding nuclear proteins that carry out the specific functions characteristic of enhancers. In principle, the two long-term objectives can be addressed by identifying developmentally regulated factors and by studying the function of the DNA bound proteins. However, in practice these goals are hindered by the observation that most enhancers contain multiple protein-binding sites that are functionally redundant, thereby making it difficult to distinguish which proteins are more important than others. The experiments outlined in Specific Aims 1 and 2 are aimed at investigating the biochemical and biological properties of a domain of the Ig mu heavy chain gene enhancer. This domain has no redundant motifs, and all three sites present are simultaneously required for enhancer function. These sites bind ETS domain proteins and bHLH/Zip proteins, whose interactions with each other will be studied by a combination of in vitro and in vivo experiments. In addition, the role of enhancers in affecting chromatin structure will be assessed by binding studies with nucleosome reconstituted DNA and accessibility to V(D)J recombinase in cells. In the Specific Aim 3, NF-kappaB induction will be characterized. The composition of inducible non-DNA binding nuclear NF-kappaB complexes will be evaluated by immunoblotting and immunoprecipitation experiments using extracts from cells stimulated in different ways. An activator of inhibited NF-kappaB and p50 associated p28 protein will be purified from nuclear extracts by a combination of conventional and affinity chromatography, with the ultimate objective of cloning the genes encoding the proteins. These molecules are likely to be considerable of interest as modulators of NF-kappaB functions. The combined biochemical and genetic experiments are expected to provide insights into the processes involved in lymphocyte development and activation, as well as oncogenesis by Rel and ETS family of proteins.
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| Ishii, Haruhiko; Du, Hansen; Zhang, Zhaoqing et al. (2009) Mi2beta shows chromatin enzyme specificity by erasing a DNase I-hypersensitive site established by ACF. J Biol Chem 284:7533-41 |
