ABSTRACT The PHD finger (Plant Homeodomain) module is a signature chromatin-associated protein motif. This module is present throughout eukaryotic proteomes, and mutations in the PHD fingers of many proteins are associated with cancers, immunodeficiency and mental retardation syndromes, and other genetic disorders. We previously demonstrated that a subset of PHD fingers act as high affinity binding modules for histone H3 trimethylated at lysine 4 (H3K4me3). We linked H3K4me3 to multiple different functions via its recognition by discrete PHD finger nuclear proteins, including providing the first evidence that disrupting the read-out of a histone modification can cause an inherited human disease. Our long-term goal is to develop a comprehensive understanding of how PHD domain-containing proteins impact on chromatin dynamics and the relationship of such activities to fundamental nuclear functions and human disease processes. Here we focus on the biology and function of the multiple PHD domain-containing proteins NSD2 (also named MMSET and WHSC1) and NSD3 (also named WHSC1L), two histone lysine methyltransferases implicated in cancer pathogenesis. However, the molecular mechanisms by which these enzymes regulate chromatin and the relationship of their enzymatic activities to disease pathogenesis in vitro and in vivo is not well understood. We hypothesize that NSD2 and NSD3, via regulation of H3K36 methylation dynamics, govern nuclear and epigenetic programs important for normal and oncogenic cellular behaviors. In Aim 1, we characterize the molecular mode of action of H3K36. We continue our studies of H3K36me- binding proteins and through further development refine a proteomic platform for discovery of new proteins that preferentially recognize H3K36me2. We test the hypothesis that these proteins transduce NSD2 activity at chromatin to downstream biological outcomes. The goal of Aim 2 is to explore the mode of action of NSD2 in cancer in vivo, which has not previously been done. We will use a novel conditional NSD2 overexpression allele to test the hypothesis that in a wild-type microenvironment increased NSD2 expression and enzymatic activity reprograms the epigenome to stimulate cancer development. In Aim 3, we focus on elucidating the physiologic catalytic activity of NSD3 and its relationship to chromatin and disease regulation. We will use a combination of strategies and approaches to help characterize the mechanisms underlying NSD3 activity in normal and cancer cells. Together these studies will provide important new insights into how PHD finger proteins and histone methylation dynamics regulates fundamental nuclear processes and the relationship of these activities to the pathogenesis of human diseases.