Ring finger 20/ring finger 40/WW domain-containing adaptor with coiled-coil complex interacts with p53 to regulate gene transcription in DNA damage response.

p53 is one of the most important tumor suppressor genes, and its primary function is to act as a transcriptional activator to control cell cycle arrest, DNA repair and cellular metabolism by recognizing and binding to specific DNA sequences. Defects in the ring finger (RNF)20/RNF40/WW domain-containing adaptor with coiled-coil (WAC) complex, one of the histone H2B ubiquitination E3 ligases, have been reported to be a key factor in oncogenesis, cancer cell migration and invasion. Histone H2B mono-ubiquitination has been demonstrated to be essential for maintaining the functionality of the p53 tumor suppressor protein. The aim of the present study was to identify any sites in the p53 DNA-binding domain (DBD) specific to the RNF20/RNF40/WAC complex that may be involved in the gene regulation in DNA damage response. The results demonstrated that p53 and the RNF20/RNF40/WAC complex interacted with each other, and the coiled-coil regions in RNF20, RNF40 and WAC were identified to directly interact with p53. The R282 site in the p53 DBD, one of the frequent missense mutations associated with p53 mutation-dependent cancer, was demonstrated to be the key binding site for the RNF20/RNF40/WAC complex. Furthermore, knockout of RNF20/RNF40 suppressed the expression levels of p53 and its target genes in HCT116 cells compared with those in wild-type HCT116 cells. Consistent with these results, the R282W mutation in p53 inhibited the expression levels of p53 and its downstream genes by inactivating the interaction between p53 and RNF20/RNF40 compared with those in wild-type HCT116 cells. In conclusion, the results of the present study revealed the molecular mechanism of the interaction between the RNF20/RNF40/WAC complex and p53, and demonstrated that these proteins regulated gene transcription in the DNA damage response.

Meng D ，Guo K ，Zhang D ，Zhao C ，Sun C ，Zhang F ... -  《-》

WAC, a functional partner of RNF20/40, regulates histone H2B ubiquitination and gene transcription.

Histone H2B ubiquitination plays an important role in regulating chromatin organization during gene transcription. It has been shown that RNF20/40 regulates H2B ubiquitination. Here, using protein affinity purification, we have identified WAC as a functional partner of RNF20/40. Depletion of WAC abolishes H2B ubiquitination. WAC interacts with RNF20/40 through its C-terminal coiled-coil region and promotes RNF20/40 s E3 ligase activity for H2B ubiquitination. The N-terminal WW domain of WAC recognizes RNA polymerase II. During gene transcription, WAC targets RNF20/40 to associate with RNA polymerase II complex for H2B ubiquitination at active transcription sites, which regulates transcription. Moreover, WAC-dependent transcription is important for cell-cycle checkpoint activation in response to genotoxic stress. Taken together, our results demonstrate an important regulator for transcription-coupled histone H2B ubiquitination.

Zhang F ，Yu X 《-》

Structure and Function of the RING Domains of RNF20 and RNF40, Dimeric E3 Ligases that Monoubiquitylate Histone H2B.

Monoubiquitylation of histone H2B is a post-translational mark that plays key roles in regulation of transcription and genome stability. In humans, attachment of ubiquitin to lysine 120 of histone H2B depends on the activity of the E2 ubiquitin-conjugating enzyme, Ube2B, and the really interesting new gene (RING) E3 ligases, RING finger protein (RNF) 20 and RNF40. To better understand the molecular basis of this modification, we have solved the crystal structure of the RNF20 RING domain and show that it is a homodimer that specifically interacts with the Ube2B~Ub conjugate. By mutating residues at the E3-E2 and E3-ubiquitin interfaces, we identify key contacts required for interaction of the RNF20 RING domain with the Ube2B~Ub conjugate. These mutants were used to generate a structure-based model of the RNF20-Ube2B~Ub complex that reveals differences from other RING-E2~Ub complexes, and suggests how the RNF20-Ube2B~Ub complex might interact with its nucleosomal substrate. Additionally, we show that the RING domains of RNF20 and RNF40 can form a stable heterodimer that is active. Together, our studies provide new insights into the mechanisms that regulate RNF20-mediated ubiquitin transfer from Ube2B.

Foglizzo M ，Middleton AJ ，Day CL 《-》

LIM-domain transcription complexes interact with ring-finger ubiquitin ligases and thereby impact islet β-cell function.

Diabetes is characterized by a loss of β-cell mass, and a greater understanding of the transcriptional mechanisms governing β-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 β-cell function, but few proteins are known to interact with this complex in β-cells. To identify additional components, here we performed SSBP3 reverse-cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse β-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 β-cell function. We observed that Rnf20 and Rnf40 depletion reduces β-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 β-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.

Wade AK ，Liu Y ，Bethea MM ，Toren E ，Tse HM ，Hunter CS ... -  《-》

The tumor suppressor CDC73 interacts with the ring finger proteins RNF20 and RNF40 and is required for the maintenance of histone 2B monoubiquitination.

Hahn MA ，Dickson KA ，Jackson S ，Clarkson A ，Gill AJ ，Marsh DJ ... -  《-》