A major goal of this proposal is to gain insight into two aspects of enhancer function: assembly of proteins into a higher-order nucleoprotein complex and regulation of accessibility of DNA in nuclear chromatin. Enhancers associated with the T cell receptor (TCR) alpha and immunoglobulin (Ig) mu genes confer transcriptional activation specifically in lymphocytes. The minimal TCRalpha enhancer is a paradigm for a regulatory region that is dependent on a particular arrangement of binding sites for nuclear factors that synergize functionally. The Ig mu enhancer region represents a locus control region that mediates the correct developmental pattern of gene expression in germ line transformations. In a first set of experiments, we will study by quantitative DNase l footprint experiments the assembly of a higher-order nucleoprotein complex at the TGRalpha enhancer using purified proteins. In particular, we will address the question of how specific members of large families of proteins that share DNA sequence recognition are selected for recruitment into an enhancer complex. We will compare different protein family members for their potential to interact with other enhancer-bound proteins and to form a stable nucleoprotein structure in vitro. Another aim will be the characterization of an activation domain of the transcriptional regulator, LEF-l, that functions only in a specific context of other factor binding sites. We will study the role of proteins chat interact with this context- dependent activation domain. The second set of experiments will address the mechanisms by which the Ig mu enhancer mediates short-range and long-range accessibility in nuclear chromatin. In particular, we will attempt to uncouple changes in chromatin structure from transcription. We plan to link the mu enhancer core alone or in combination with flanking nuclear matrix attachment regions (MARs) to a proximal or distal promoter for prokaryotic T7 RNA polymerase and introduce the gene constructs into the mouse germ line. We will examine accessibility of the T7 promoter by isolating nuclei from transformed transgenic B cells, adding 17 RNA polymerase and measuring 17 specific transcripts. Based on our previous finding that both MARs and the enhancer core are essential for mu gene expression in transgenic mice, we will also study the molecular basis of the synergy of the MARs and enhancer by genomic footprint assays and by alterations in the arrangement or identity of these regulatory elements. Finally we will address the question of why MAR function can be detected only in germ line transformations but not in stable transfection assays. We will examine whether this can be accounted for by differences in the methylation status of the integrated DNA. Together, these experiments will shed light on the molecular basis for some lymphoid malignancies that involve alterations in the structure and function of transcript ion factors or are based on chromosomal translocations and rearrangement of gene loci.
| Tsukamoto, A S; Grosschedl, R; Guzman, R C et al. (1988) Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice. Cell 55:619-25 |
