Bidirectional promoters are abundant in the genome. They are defined as a shared regulatory region that falls in the intergenic space between two oppositely oriented genes that are separated by no more than 1,000 bp. These genes are organized in a head-to-head arrangement and transcribed away from one another. The closely spaced arrangement of the transcription start sites (TSSs) each pair of genes is recognized as a nonrandom event in the genome, proven by the fact that a greater than expected number of promoters have this architecture. One plausible scenario for the creation of bidirectional gene pairs is chromosomal recombination bringing the ends of two TSSs in close proximity. This union is likely to be irreversible because breakage of the intergenic promoter region would interrupt the normal regulation of two genes, profoundly affecting normal genomic function, such as DNA repair. We have mapped bidirectional promoters in numerous vertebrate genomes. In doing so, we are creating the first high-confidence regulatory map of bidirectional promoter sequences shared in vertebrate lineages as well as identifying transcripts that are unique to single vertebrate lineages, including primates. In addition to characterizing promoter regions, we are interested in identifying novel types of elements such as negative regulators of gene expression (NREs;Petrykowska et al, Genome Research 2008). In contrast to the large body of literature on positively acting elements such as enhancers and promoters, cis-acting NREs have not been extensively studied. Despite their scarceness in the literature, these elements are likely to be abundant in the genome. Examples of NREs include silencers, which decrease expression of a gene under their regulation and enhancer-blocking (EB) elements, which prevent the action of an enhancer on a promoter when placed between the two, but not otherwise. By developing a strategy to experimentally test NREs, we have identified novel examples of these functions in the human genome. Exonic splicing enhancers (ESEs) are a third category of regulatory elements affecting gene expression. We have compared all former predictive methods for ESEs to show that some are more precise than others. We have produced an online toolkit to test polymorphisms that occur in coding sequences to assess whether they may affect mRNA splicing (http://research.nhgri.nih.gov/skippy). All of these research projects examine cis-acting elements and converge on the identification of transcription factor binding sites or splicing regulators, which are bound by trans-acting proteins and represent the basic components of gene regulation. Regulatory motifs have been published for each of the projects above. During the course of our work, novel motifs were implicated as silencers and new biological insights were revealed for the regulation of alternative promoters. A collaborative effort has been established to define the regulatory networks involving these motifs, especially in biological pathways implicated in cancer. Moreover, we are building tools to assess epigenetic events that aberrantly affect gene expression in tumors versus normal cells.