Developmental regulation of the immunoglobulin kappa 3' enhancer: Characterization of negative regulatory elements and cloning of a transcriptional activator/repressor, NF -E1
The immunoglobulin kappa 3$\sp\prime$ enhancer ($\kappa$E3$\sp\prime$) activity is developmentally regulated during B-cell development. It is inactive at the pre-B cell stage but becomes active at the B-cell and plasma cell stages. The mechanism underlying the silencing of the $\kappa$E3$\sp\prime$ enhancer at the pre-B cell stage is unclear. This study was initiated to identify cis- and trans-acting factors that are involved in the developmental regulation of $\kappa$E3$\sp\prime$ enhancer activity. Transfection of $\kappa$E3$\sp\prime$ enhancer deletion constructs into pre-B cells showed that the 132 bp core of the enhancer is active at this stage, but it is repressed by multiple negative-acting flanking DNA sequences. Deletion of either of these flanking sequences results in activation of the $\kappa$E3$\sp\prime$ enhancer in pre-B cells. A nuclear factor, NF-E1, that binds to negative-acting DNA sequences on the 3$\sp\prime$ side of the enhancer core was identified and is identical to the protein that binds to the $\mu$E1 site of the immunoglobulin heavy chain enhancer. Human cDNA clones encoding NF-E1 were isolated by oligonucleotide screening of a $\lambda$gt11 expression library prepared from human B-cells. Analysis of the predicted amino acid sequence revealed that NF-E1 is a zinc finger protein which belongs to the Kruppel family and contains amino acid sequences common to both transcriptional activators and transcriptional repressors. Electrophoretic mobility shift assays with NF-E1 protein prepared in E. coli indicate that eukaryotic specific protein modifications are not necessary for sequence specific DNA binding activity, but the intact zinc finger domain of the NF-E1 protein is necessary for its DNA binding. The presence of NF-E1 binding sites in both negative and positive regulatory elements suggests that it may have a dual function. Consistent with this expectation, cotransfection experiments showed that NF-E1 functions as an activator as well as a repressor in a dose-dependent manner. Dissection of the NF-E1 protein showed that the region of the protein responsible for repression lies near the carboxy terminus encompassing the zinc finger domain and is distinct from the region necessary for activation. Identification of multiple negative regulatory elements and isolation of NF-E1 have furthered our understanding of how the $\kappa$E3$\sp\prime$ enhancer may be regulated during B-cell development, and how a single transcription factor can result in either transcriptional activation or repression.
Park, Kyoungsook, "Developmental regulation of the immunoglobulin kappa 3' enhancer: Characterization of negative regulatory elements and cloning of a transcriptional activator/repressor, NF -E1" (1992). Dissertations available from ProQuest. AAI9235186.