The immunoglobulin Kappa locus is transcriptionally controlled by two developmental stage-specific enhancers. In addition to transcription, these enhancers have been implicated in the processes of somatic rearrangement and somatic mutation, necessary for proper B cell development. Therefore, understanding the mechanisms of kappa locus enhancer activity is important. The intron enhancer lies within the intron separating the joining from constant region exons and is largely controlled by the availability of the active form of NF-kappa B. The 3 prime enhancer lies 8.5 kilobases downstream of the constant region and is controlled by the interplay between positive-and negative-acting sequences. We have identified many of the proteins that bind to the 3 prime enhancer to control its activity. The transcription factors PU.1, PIP, E2A, c-fos, c-jun, ATF1, and CREM bind to the central 132 basepair core of the enhancer. These proteins can assemble on enhancer DNA sequences as a higher order complex (the enhanceosome). We have identified some of the protein contacts between the various enhancer binding proteins. Some of these contacts are necessary for enhanceosome formation and for transcriptional activity. In experiments proposed here, we will determine the mechanism of specific protein interactions between various enhancer binding proteins. Specifically, interactions between PU.1, PIP, c-fos, c-jun, ATF1, CREM, and E2A will be characterized. Mutants of the PU.1 protein will be assayed for their ability to physically interact with other enhancer binding proteins. The consequences of mutations that disrupt protein interactions for enhanceosome formation and for enhancer activity will be determined. We will also study various physical properties of the enhanceosome complex. We will also study the role of protein interactions by PU.1 on hematopoietic development. Mice deficient in PU.1 lack B cells, T cells, and myeloid cells. We will insert various Pu.1 mutants with altered abilities to make various protein contacts into embryonic stem (ES) cells deficient in PU.1. The 'corrected' ES cells will be used to prepare chimeric mice and their contribution to various hematopoietic lineages will be determined. Finally, we will study the role of various positive-acting, inducible, and negative-acting enhancer DNA sequences on the developmental control of enhancer activity in transgenic mice. Many of the proteins that bind to the 3 prime enhancer are encoded by proto-oncogenes. Therefore, these studies will also be useful for understanding the roles of these proteins in oncogenesis.
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