| This thesis is an investigation of the novel protein, Luman Repression and Recruitment Factor (LRF), identified by a yeast two-hybrid screen through its interaction with Luman/LZIP/CREB3, an endoplasmic reticulum (ER)-bound cellular transcription factor. Luman has been implicated in the mammalian unfolded protein response, as well as herpes simplex virus reactivation from latency in sensory neurons. Similar to Luman, LRF is a basic-region leucine zipper protein that is tightly regulated by the protcasome, with an estimated half-life of less than 20 minutes. I confirmed that LRF interacts with Luman both in vitro and in the cell, and mapped the Luman-binding domain to a C-terminal region that contains two leucine zipper-like motifs. I found that LRF recruited the nuclear form of Luman to discrete nuclear foci that overlap with nuclear receptor coactivator GRIP1 bodies. Mapping of the subnuclear targeting sequences responsible for transport to these specific nuclear punetates identified the central region containing the first leucine zipper (a.a.415-519) as essential, however, finer mapping suggested that multiple independent sequences within this region may he responsible for protein trafficking. Characterization of the tissue distribution of LRF mRNA revealed high levels of LRF expression in brain, thymus, lung, spleen and testicular tissue, facilitating the discovery of LRF protein in spermatozoa. Chemical treatment of rat glial cells with the ER stress inducers tunicamycin and thapsigargin or the proteasomal inhibitor MC132 induced LRF and Luman mRNA expression, while co-expression of these proteins triggered a repression in Luman-mediated transcriptional activation from the unfolded protein response element. I propose that LRF is a co-factor that represents another level of regulation for Luman following its proteolytic cleavage on the ER and nuclear translocation, and that LRF represses the transactivation potential of Luman by recruiting it to special subnucicar domains, sequestering Luman from key co-factors (such as HCF-1) that are required for transcriptional activation. |
