Title

Transcriptional regulation of the human lambda light chain immunoglobulin locus: Characterization of the 3' enhancer

Date of Award

1994

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Bonnie B. Blomberg, Committee Chair

Abstract

The first transcriptional enhancer for the human $\lambda$ light chain immunoglobulin locus was recently identified 11.7 kb downstream of C$\lambda$7, the most 3$\sp\prime$ constant region gene. In this study, the cis-acting elements which comprise this enhancer as well as the trans-acting factors which bind to it were examined. By deletion mapping and CAT assays, the complete human $\lambda$ enhancer was determined to reside on a 311 bp PstI-SstI fragment with partial activity retained on a 124 bp PstI-SmaI core element. The complete human $\lambda$ enhancer shares 65% identity with the murine $\lambda$ enhancers, while the core fragment is 70-73% identical. By methylation interference analysis, protein interaction at the well conserved $\lambda{\rm A}$ and $\lambda{\rm B}$ motifs was identified. In addition, protein interaction was observed at a site which is unique to the human $\lambda$ enhancer. This site has been named the Human Enhancer Lambda Protein (HELP) site. From electrophoretic mobility shift assays, a protein factor(s), HELP, has been shown to specifically bind to this site. This motif is located immediately 3$\sp\prime$ of the core enhancer and augments the activity of the core enhancer element. By EMSA analysis, this protein(s) is present primarily in hematopoietic cells but not restricted to B cells. Thus it most likely interacts with another, B cell-restricted protein(s) to play a role in the tissue specific activation of the human $\lambda$ enhancer.The 311 bp human $\lambda$ enhancer is active at the pre-B cell stage, but a larger fragment containing the flanking regions is inactive in pre-B cells whereas it is active in B cells. This suggests the presence of negative elements both upstream and downstream of the complete human $\lambda$ enhancer which are developmentally controlled. By methylation interference analysis, protein interaction at sites in regions 5$\sp\prime$ and 3$\sp\prime$ of the complete human $\lambda$ enhancer were identified which may be responsible for the developmental stage specific activity of this enhancer. The binding site sequence is conserved in both the 5$\sp\prime$ and 3$\sp\prime$ elements, and by cross-competition EMSA the same protein(s) binds to both elements.

Keywords

Biology, Molecular; Health Sciences, Immunology

Link to Full Text

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