Chromatin modifications play a major role in regulating genome function. Perturbations in such modifications can contribute to neoplastic processes. We will focus on a specific chromatin modification: histone H2B monoubiquitylation. The significance of monoubiquitylated H2B (H2Bub) will be studied by manipulating RNF20, the major E3 ubiquitin ligase responsible for H2B ubiquitylation as part of a heteromeric complex with RNF40.In one major line of research, we will assess the biochemistry of RNF20/H2Bub. The effects of RNF20/H2B on gene expression will be explored through identification of proteins that interact with H2Bub and through in vitro incorporation of H2Bub into nucleosomes. Effects of H2Bub on transcription elongation will be studied by a new high resolution ChIP-seq method (NET-seq). Based on recent ChIP-seq data, we will also explore links between H2B and regulation of splicing. Furthermore, we will investigate the regulatory crosstalk between H2Bub and microRNAs.The other major line of research will explore the biology of RNF20/H2Bub, with particular emphasis on cancer-related processes. This will be addressed through a combination of cell culture models and mouse models, including constitutive and conditional RNF20 knockout mice. The contribution of RNF20/H2Bub to various differentiation programs, with particular emphasis on embryonic stem cell differentiation, will also be investigated. In addition, we will study the impact of RNF20/H2Bub on NF-kB activity and on inflammatory responses; this will combine in vitro and in vivo systems, with emphasis on inflammation-related cancer. Finally, we will monitor changes in RNF20, RNF40 and H2Bub status in human tumors and investigate underlying mechanisms.