The regulation of gene expression plays an essential role in cellular function and behavior. Heart conditions that arise as a result of response to various forms of stress, as well as altered cholesterol levels are both associated with changes in transcriptional programs. Transcriptional regulation is therefore a crucial step before the progression of any cellular response to stress. Recent work has shown that the localization of genes in the nucleus plays an important role in regulating transcription; a gene’s location can impact its transcriptional activity and its chromatin structure. In brewer’s yeast, many genes are targeted from the nucleoplasm to the nuclear periphery, through association with the Nuclear Pore Complex (NPC), upon activation. Similar phenomena have been reported in metazoans. Targeting to the NPC promotes transcription and is controlled by cis-acting “DNA zip codes” in the promoters of these genes. Interestingly, the two zip-code harboring genes we have closely analyzed are both induced as a response to various forms of stress. To determine the molecular basis of zip code-mediated targeting, we have identified the transcription factor Put3 as a DNA zip code binding protein. We seek to determine how the DNA binding properties of Put3 affect the movement of chromosomal loci to the NPC, and how this singular transcription factor can have two distinct roles in the yeast nucleus: the activation of PUT genes, and relocalizing genomic loci by binding to zip codes. By assaying for the binding properties of Put3 to various zip codes in both in vitro and in vivo contexts, we have mapped the important residues within and flanking the DNA zip code core sequence. A number of upstream zip code residues play an important role for Put3-based gene targeting, as demonstrated by live yeast cell microscopy. The two nuclear roles for Put3 suggest that this protein associates with distinct groups of other proteins for each of its functions, and possibly in different conformations. Mutations within Put3 can separate these two functions. On the broader scale, this work aims to provide important fundamental insight into how eukaryotic genomes regulate their expression through alteration of their spatial organization within the nucleus.