The regulation of gene expression of B lymphocytes is being investigated using two genes as models: the human k light chain immunoglobulin gene and the human J chain gene. The expression of the k gene is regulated by a transcriptional enhancer located in the J-C intron. We were able to detect evidence of in vivo interactions between the 5' region of the enhancer and B cell nuclear proteins. More recently, we have been examining in vitro interactions between the enhancer and nuclear proteins (extracted from B cell nuclei) using an exonuclease protection method. These experiments define two binding domains in the enhancer: a 5' domain that includes an enhancer """"""""core' element and an octamer consensus sequence (kE1); and a 3' domain that includes another occurrence of the octamer sequence (kE2). Using the exonuclease protection protocol as an assay, we have been purifying a protein that binds to the 3' domain. The partially purified protein has been used to characterize the binding site by in vitro DNAase I footprinting. To study features of gene regulation that may be common to genes specifically expressed in B cells, we have been investigating the human J chain gene. A search for an enhancer in a 15 kb segment spanning the genomic J chain gene has not demonstrated a region able to support transcription from either an SV40 or a J chain promoter sequence. Sequence analysis of the J chain promote revealed motifs resembling regions of immunoglobulin promoters and enhancers. To assess the functional significance of these motifs, the J chain promoter - with or without varying 5' deletions - was linked to a marker gene and an immunoglobulin enhancer and tested in transient transfection assays. These experiments suggest that, apart from these motifs, additional elements are important for the function of the J chain promoter in vivo. We are continuing the investigation of this promoter by in vitro mutagenesis and further deletion experiments as well as by analysis of in vitro binding B cell nuclear proteins. We have recently initiated a collaboration with Drs. T Rado and J. Mestecky to study expression of the J chain protein using J chain cDNA constructs derived in our lab.
