Chromatin conformation underlies accessibility of enzymes that participate in transcription, replication, recombination, and repair. Many chromatin-regulatory mechanisms ensure that enzymes such as RNA polymerase II gain access to and efficiently transcribe chromatin. One of the mechanisms of chromatin regulation is based on the posttranslational modification of histones. Chemical groups are deposited, read and removed by specific families of enzymes to regulate chromatin structure. These modifications rarely function in isolation, and there is often crosstalk between them that results in a coordinated transcriptional output. One of the prime examples of such crosstalk is seen in transcription elongation, in which histones H3 and H4 are acetylated, H2B is ubiquitinated, and histone H3 is methylated at lysine 4 and 79 (H3K4 and H3K79). COMPASS/MLL and Dot1 are the enzymes that catalyze methylation of H3K4 and H3K79 respectively and are evolutionarily conserved. They play essential roles in processes such as transcription elongation, cell cycle control, and DNA repair. Homologs are found, among others, in yeast, and mammals. Deregulation of Dot1L and MLL1, has been found in several cancers, especially in leukemias, and Dot1L and MLL1 inhibition has emerged as a promising therapeutic strategy. Trypanosome Dot1 is involved in evasion of the human immune system contributing to devastating diseases such as African sleeping sickness. Understanding the mechanisms underlying Dot1 and COMPASS/MLL1 functions is critical to discovery of novel strategies to fight diseases associated with their deregulation. This proposal centers on finding and characterizing novel mechanisms of Dot1 and COMPASS/MLL1 regulation. We will use biochemical, biophysical and structural methods to study these histone methyltransferases. We will validate our mechanistic hypotheses in cells and in vivo. Using this integrative structural and functional approach and different model systems will allow us to determine evolutionarily conserved and organism-specific biological functions and modes of regulation of Dot1 and COMPASS/MLL1 which can be applied to therapeutic strategies for a variety of diseases.