Transcription is regulated by sequence-specific transcription factors (TFs) that bind to short genomic DNA elements that can be located in promoters, enhancers and other cis-regulatory modules. Determining which TFs bind where requires techniques that enable the ab initio identification of TF-DNA interactions. These techniques can either be "TF-centered" (protein-to-DNA), where regions of DNA bound by a TF of interest are identified, or "gene-centered" (DNA-to-protein), where TFs that bind a DNA sequence of interest are identified. Here, we describe gene-centered yeast one-hybrid (Y1H) assays. Briefly, in Y1H assays, a DNA fragment is cloned upstream of two different reporters, and these reporter constructs are integrated into the genome of a yeast strain. Next, plasmids expressing TFs as hybrid proteins (hence the name of the assay) fused with the strong transcriptional activation domain (AD) of the yeast TF Gal4 are introduced into the yeast strain. When a TF interacts with the DNA fragment of interest, the AD moiety activates reporter expression in yeast regardless of whether the TF is an activator or repressor in vivo. Sequencing the plasmid in the colonies that exhibit reporter activation reveals the identity of the TFs that can bind the DNA fragment. We have shown Y1H to be a robust method for detecting interactions between a variety of DNA elements and multiple families of TFs.