A major aim of this proposal is to gain insight into the role of enhancer sequences in the establishment and maintenance of a tissue-specific pattern of gene expression. Immunoglobulin (Ig) mu and kappa transcripts is temporally regulated during differentiation of lymphocytes. We have previously demonstrated that enhancer sequences are required for the developmentally regulated expression of a mu gene introduced into the mouse germ line. Although transcriptional activation is accompanied by alterations in chromatin structure, it is still unclear whether these represent a cause or effect. To examine whether enhancer sequences can confer chromatin accessibility independent of transcription, we will link the IgH enhancer to a promoter of RNA polymerase T7 and introduce the DNA construct into the mouse germ line. WE will derive lymphoid and non- lymphoid cell lines from the transgenic mice and examine the accessibility of the T7 promoter in isolated cell nuclei by adding exogenous T7 RNA polymerase. We will also investigate the molecular basis for the temporal regulation of kappa gene expression and for the B cell-specific inducibility of kappa enhancer function. To examine factor binding in vivo, we will examine the protection of specific nucleotides in the kappa enhancer from methylation in intact B and non-B cells from transgenic mice. Finally, we will examine the dependence of Ig gene expression on enhancer sequences during terminal cell differentiation. WE will delete the IgH enhancer from a stably transfected mu gene in a late-stage B cell line using gene replacement by homologous recombination. WE will also identify sites in the IgH locus that become hypersensitive to DNaseI digestion in late-stage B cells and examine these DNA sequence elements for their putative function of conferring the active transcriptional state upon Ig genes in the absence of the IgH enhancer. These experiments will shed some light on the molecular basis for some B cell malignancies which appear to be the result of translocation of the c-myc proto-oncogene into the vicinity of an Ig locus.
| 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 |
