RNF4 and VHL regulate the proteasomal degradation of SUMO-conjugated Hypoxia-Inducible Factor-2α

Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1α and HIF-2α are continuously synthesized in cells and degraded via the ubiquitin–proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels. HIF proteins are tightly regulated via post-translational modifications including phosphorylation, acetylation, prolyl-hydroxylation and ubiquitination. Here we show for the first time that exogenous and endogenous HIF-2α are also regulated via the ubiquitin-like modifier small ubiquitin-like modifiers (SUMO). Using mutational analysis, we found that K394, which is situated in the sumoylation consensus site LKEE, is the major SUMO acceptor site in HIF-2α. Functionally, sumoylation reduced the transcriptional activity of HIF-2α. Similar to HIF-1α, HIF-2α is regulated by the SUMO protease SENP1. The proteasome inhibitor MG132 strongly stabilized SUMO-2-conjugated HIF-2α during hypoxia but did not affect the total level of HIF-2α. The ubiquitin E3 ligases von Hippel–Lindau and RNF4 control the levels of sumoylated HIF-2α, indicating that sumoylated HIF-2α is degraded via SUMO-targeted ubiquitin ligases

WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis

Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial. Here, we observe that the BRCA1/BARD1 ubiquitin E3 activity is not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identify substrates for BRCA1/BARD1. We find that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results provide additional insight about the importance of BRCA1/BARD1 E3 activity in Homologous Recombination.Research and publication of this work was funded by the EMERGIA 2020 program (EMERGIA20_00276) from the Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía, Spain to R.G.-P. Research was additionally supported by a Young Investigator Grant from the Dutch Cancer Society (KWF-KIG 11367/2017-2) and Plan Propio de Investigación VI-PP-A.Talento-IV.2 from the University of Sevilla, grants CNS2022-135216 funded by MICIU/AEI/10.13039/501100011033 and by European Union NextGenerationEU/PRTR and PID2021-122361NA-I00 by MICIU/AEI/10.13039/501100011033 and by European Union to R.G.-P. Work in the laboratory of A.C.O.V. has been supported by the European Research Council (ERC; grant 310913) and the Dutch Research Council (NWO; grant 724.016.003). N.G.-R. was supported by the EMERGIA 2021 program (EMERGIA21_00057) from the Consejería de Universidad, Investigación e Innovación, Andalusian Regional Government- Junta de Andalucía to NG-R.Peer reviewe

Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome

Supporting Information for A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair

Supporting Text: Extended Methods Figures S1 to S6 Tables S1 to S4 SI References (16) Other supporting materials for this manuscript include the following: Datasets / spreadsheet S1Peer reviewe

Uncovering Ubiquitin and Ubiquitin-like Signaling Networks

Microscopic imaging and technolog

Ubiquitin-dependent and independent roles of SUMO in proteostasis

Cellular proteomes are continuously undergoing alterations as a result of new production of proteins, protein folding, and degradation of proteins. The proper equilibrium of these processes is known as proteostasis, implying that proteomes are in homeostasis. Stress conditions can affect proteostasis due to the accumulation of misfolded proteins as a result of overloading the degradation machinery. Proteostasis is affected in neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, and multiple polyglutamine disorders including Huntington's disease. Owing to a lack of proteostasis, neuronal cells build up toxic protein aggregates in these diseases. Here, we review the role of the ubiquitin-like posttranslational modification SUMO in proteostasis. SUMO alone contributes to protein homeostasis by influencing protein signaling or solubility. However, the main contribution of SUMO to proteostasis is the ability to cooperate with, complement, and balance the ubiquitin-proteasome system at multiple levels. We discuss the identification of enzymes involved in the interplay between SUMO and ubiquitin, exploring the complexity of this crosstalk which regulates proteostasis. These enzymes include SUMO-targeted ubiquitin ligases and ubiquitin proteases counteracting these ligases. Additionally, we review the role of SUMO in brain-related diseases, where SUMO is primarily investigated because of its role during formation of aggregates, either independently or in cooperation with ubiquitin. Detailed understanding of the role of SUMO in these diseases could lead to novel treatment options

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)

Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR

RNF4 and VHL regulate the proteasomal degradation of SUMO-conjugated Hypoxia-Inducible Factor-2a

Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1a and HIF-2a are continuously synthesized in cells and degraded via the ubiquitin–proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels. HIF proteins are tightly regulated via post-translational modifications including phosphorylation, acetylation, prolyl-hydroxylation and ubiquitination. Here we show for the first time that exogenous and endogenous HIF-2a are also regulated via the ubiquitin-like modifier small ubiquitin-like modifiers (SUMO). Using mutational analysis, we found that K394, which is situated in the sumoylation consensus site LKEE, is the major SUMO acceptor site in HIF-2a. Functionally, sumoylation reduced the transcriptional activity of HIF-2a. Similar to HIF-1a, HIF-2a is regulated by the SUMO protease SENP1. The proteasome inhibitor MG132 strongly stabilized SUMO-2-conjugated HIF-2a during hypoxia but did not affect the total level of HIF-2a. The ubiquitin E3 ligases von Hippel–Lindau and RNF4 control the levels of sumoylated HIF-2a, indicating that sumoylated HIF-2a is degraded via SUMO-targeted ubiquitin ligases