Thesis

Promoter Characterization of the Mantle Cell Lymphoma Associated Gene, NAP1L1

Promoter Characterization of the Mantle Cell Lymphoma Associated Gene, NAP1L1 By Loni Hands Master of Science in Biology The precise regulation of gene expression within a cell determines the structure and function of a given cell type. Dysregulation of normal gene expression may lead to diseases such as cancer. Differences in gene expression among cancers of similar origin may explain the differing phenotypes between more aggressive cancers and less aggressive cancers. Mantle cell lymphoma (MCL), an aggressive cancer, expresses a higher level of the nucleosome assembly protein 1-like 1 (NAP1L1) than small lymphocytic lymphoma (SLL), a less aggressive cancer of similar origin. NAP1L1 is involved in cell cycle and gene regulation, making it a candidate for involvement in cancer progression. In order to understand the mechanisms behind NAP1L1 expression, the putative regulatory region of NAP1L1 from -1273 bp to +387 bp relative to the transcription start site (TSS, +1) was cloned into the luciferase reporter vector pGL3 Basic. After transient transfections of this construct into human embryonic kidney (HEK293T) cells confirmed the ability of this region to regulate gene expression, deletions of the promoter region were made and revealed the location of a positive regulatory element between -442 bp and -297 bp and a negative regulatory element between -297 bp and -183 bp. Preliminary data from transfections of smaller deletion constructs within each of these regions narrowed down the location of the positive regulatory element to between -367 bp and -335 bp and potentially two negative regulatory elements between -297 bp and -254 bp and between -254 bp and -215 bp. Further research will be necessary to determine the exact identities of these elements. In order to determine whether there is an enhancer element within the first intron of NAP1L1, as there appears to be in mice (Wu et al., 2008), a region from +382 bp to +1915 bp containing the beginning of the first intron was cloned into the pGL3 construct containing the full isolated promoter region of NAP1L1 (-1273 bp to +387 bp). Transient transfections of this construct with the intron in the "forward" direction resulted in increased luciferase activity over 2-fold compared to the construct containing only the full NAP1L1 promoter region. Interestingly, and unexpectedly, the construct containing the full promoter region of NAP1L1 and the intron region in the reverse direction resulted in luciferase activity that was comparable to the construct with the full promoter and no intron. When the transcription factor binding sequence for c-Myc within the intron region was mutated in the construct containing the full promoter region and intron in the forward direction, the results were also similar to those of the construct containing only the full promoter, suggesting a potential role for c-Myc in regulating NAP1L1 in a location-independent, orientation-dependent manner.

Relationships

Items