© 2019 Wade et al. Diabetes is characterized by a loss of-cell mass, and a greater understanding of the transcriptional mechanisms governing -cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA–binding protein 3 (SSBP3) regulates the genes necessary for -cell function, but few proteins are known to interact with this complex in -cells. To identify additional components, here we performed SSBP3 reverse–cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for -cell function. We observed that Rnf20 and Rnf40 depletion reduces -cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate -cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.