MYCN recruits the nuclear exosome complex to RNA polymerase II to prevent transcription-replication conflicts.

The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.

MYC multimers shield stalled replication forks from RNA polymerase.

Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.

Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies.

Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.

Recruitment of BRCA1 limits MYCN-driven accumulation of stalled RNA polymerase.

MYC is an oncogenic transcription factor that binds globally to active promoters and promotes transcriptional elongation by RNA polymerase II (RNAPII)1,2. Deregulated expression of the paralogous protein MYCN drives the development of neuronal and neuroendocrine tumours and is often associated with a particularly poor prognosis3. Here we show that, similar to MYC, activation of MYCN in human neuroblastoma cells induces escape of RNAPII from promoters. If the release of RNAPII from transcriptional pause sites (pause release) fails, MYCN recruits BRCA1 to promoter-proximal regions. Recruitment of BRCA1 prevents MYCN-dependent accumulation of stalled RNAPII and enhances transcriptional activation by MYCN. Mechanistically, BRCA1 stabilizes mRNA decapping complexes and enables MYCN to suppress R-loop formation in promoter-proximal regions. Recruitment of BRCA1 requires the ubiquitin-specific protease USP11, which binds specifically to MYCN when MYCN is dephosphorylated at Thr58. USP11, BRCA1 and MYCN stabilize each other on chromatin, preventing proteasomal turnover of MYCN. Because BRCA1 is highly expressed in neuronal progenitor cells during early development4 and MYC is less efficient than MYCN in recruiting BRCA1, our findings indicate that a cell-lineage-specific stress response enables MYCN-driven tumours to cope with deregulated RNAPII function.

Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA damage repair.

N-terminal sequences are important sites for post-translational modifications that alter protein localization, activity, and stability. Dipeptidyl peptidase 9 (DPP9) is a serine aminopeptidase with the rare ability to cleave off N-terminal dipeptides with imino acid proline in the second position. Here, we identify the tumor-suppressor BRCA2 as a DPP9 substrate and show this interaction to be induced by DNA damage. We present crystallographic structures documenting intracrystalline enzymatic activity of DPP9, with the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in its active site. Intriguingly, DPP9-depleted cells are hypersensitive to genotoxic agents and are impaired in the repair of DNA double-strand breaks by homologous recombination. Mechanistically, DPP9 targets BRCA2 for degradation and promotes the formation of RAD51 foci, the downstream function of BRCA2. N-terminal truncation mutants of BRCA2 that mimic a DPP9 product phenocopy reduced BRCA2 stability and rescue RAD51 foci formation in DPP9-deficient cells. Taken together, we present DPP9 as a regulator of BRCA2 stability and propose that by fine-tuning the cellular concentrations of BRCA2, DPP9 alters the BRCA2 interactome, providing a possible explanation for DPP9's role in cancer.

MDM2 Associates with Polycomb Repressor Complex 2 and Enhances Stemness-Promoting Chromatin Modifications Independent of p53.

The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53.

MicroRNA-101 Suppresses Tumor Cell Proliferation by Acting as an Endogenous Proteasome Inhibitor via Targeting the Proteasome Assembly Factor POMP.

Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tumor cells often emerge. Here we show that the microRNA-101 (miR-101) targets the proteasome maturation protein POMP, leading to impaired proteasome assembly and activity, and resulting in accumulation of p53 and cyclin-dependent kinase inhibitors, cell cycle arrest, and apoptosis. miR-101-resistant POMP restores proper turnover of proteasome substrates and re-enables tumor cell growth. In ERα-positive breast cancers, miR-101 and POMP levels are inversely correlated, and high miR-101 expression or low POMP expression associates with prolonged survival. Mechanistically, miR-101 expression or POMP knockdown attenuated estrogen-driven transcription. Finally, suppressing POMP is sufficient to overcome tumor cell resistance to the proteasome inhibitor bortezomib. Taken together, proteasome activity can not only be manipulated through drugs, but is also subject to endogenous regulation through miR-101, which targets proteasome biogenesis to control overall protein turnover and tumor cell proliferation.

Therapeutische Nanobodies gegen SARS-CoV-2.

Monoclonal immunoglobulins are widely successful as therapeutics and have also been effective in treating COVID-19. However, their production in mammalian cells is expensive and cannot be scaled to meet the demand in a global pandemic. Camelid VHH antibodies (also called nanobodies), however, can be manufactured cost-efficiently in bacteria or yeast. Here we highlight our progress in developing nanobodies that effectively neutralize SARS-CoV-2 and its variants.

Chromatin modifiers Mdm2 and RNF2 prevent RNA:DNA hybrids that impair DNA replication.

The p53-Mdm2 system is key to tumor suppression. We have recently reported that p53 as well as Mdm2 are capable of supporting DNA replication fork progression. On the other hand, we found that Mdm2 is a modifier of chromatin, modulating polycomb repressor complex (PRC)-driven histone modifications. Here we show that, similar to Mdm2 knockdown, the depletion of PRC members impairs DNA synthesis, as determined in fiber assays. In particular, the ubiquitin ligase and PRC1 component RNF2/Ring1B is required to support DNA replication, similar to Mdm2. Moreover, the Ring finger domain of Mdm2 is not only essential for its ubiquitin ligase activity, but also for proper DNA replication. Strikingly, Mdm2 overexpression can rescue RNF2 depletion with regard to DNA replication fork progression, and vice versa, strongly suggesting that the two ubiquitin ligases perform overlapping functions in this context. H2A overexpression also rescues fork progression upon depletion of Mdm2 or RNF2, but only when the ubiquitination sites K118/K119 are present. Depleting the H2A deubiquitinating enzyme BAP1 reduces the fork rate, suggesting that both ubiquitination and deubiquitination of H2A are required to support fork progression. The depletion of Mdm2 elicits the accumulation of RNA/DNA hybrids, suggesting R-loop formation as a mechanism of impaired DNA replication. Accordingly, RNase H overexpression or the inhibition of the transcription elongation kinase CDK9 each rescues DNA replication upon depletion of Mdm2 or RNF2. Taken together, our results suggest that chromatin modification by Mdm2 and PRC1 ensures smooth DNA replication through the avoidance of R-loop formation.

Mdm2: Open questions.

The Mdm2 oncoprotein and its association with p53 were discovered 30 years ago, and a cornucopia of activities and regulatory pathways have been associated with it. In this review, we will raise questions about Mdm2 and its cousin Mdm4 that we consider worth pursuing in future research, reaching from molecular structures and intracellular activities all the way to development, evolution, and cancer therapy. We anticipate that such research will not only close a few gaps in our knowledge but could add new dimensions to our current view. This compilation of questions contributes to the preparation for the 10th Mdm2 Workshop in Tokyo.