Abstract: Transcription is a highly orchestrated process involving gene-specific transcription factors, general transcriptional machinery and a complex chromatin substrate. Here I describe two chemical prosthetic approaches to study transcriptional regulation. First, I describe a strategy to develop small-molecule control over transcription factor-DNA interactions using synthetic nucleotides and phage display technology. In this approach, a small molecule is synthetically appended to DNA blocking the wild-type transcription factor binding site. I refer to this appendage as a chemical prosthetic since it adds new functionality to the DNA. These modified DNA strands can be used to isolate mutants of the transcription factor with space at the protein-DNA interface to accommodate the prosthetic. Such mutants may serve as a predisposed set to then engineer transcription factors that bind to DNA only in the presence of a small molecule. I report progress using this approach in the context of homeodomain-DNA interactions including the engineering of engrailed homeodomain to recognize an unnatural nucleotide. This project includes the synthesis of novel nucleosides and their incorporation into DNA using solid phase DNA synthesis. Furthermore, a new phage display selection was developed to enrich for mutant homeodomains with desired DNA-binding activities. Characterization of phage-derived homeodomain mutants, including x-ray crystallography, affords fundamental insights into both engineered and wild-type homeodomains. Second, I describe a new approach to study chromatin using methyl lysine analogues installed into recombinant histories. This approach allows for the clean installation of monomethyl, dimethyl and trimethyl lysine analogues into any position throughout the entire histone sequence. This chemistry is based on the well established aminoethylation reaction, in which cysteine is alkylated with an electrophilic ethylamine to generate a lysine analogue, aminoethyl-cysteine. Here I report the extension of the aminoethylation reaction to install analogues of methylated lysine into recombinant histones, and demonstrate that these analogues are functionally similar to their natural counterparts. Given the importance of lysine methylation in epigenetic regulation, the methyl lysine analogue approach is a simple and powerful means to study the impact of specific methyl lysine residues on the regulation of chromatin structure and function.