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Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor responsible for mounting an anti-oxidation gene expression program to counter oxidative stress. Under unstressed conditions, Kelch-like ECH-associated protein 1 (KEAP1), an adaptor protein for CUL3 E3 ubiquitin ligase, mediates NRF2 ubiquitination and degradation. We show here that the deubiquitinase USP25 directly binds to KEAP1 and prevents KEAP1’s own ubiquitination and degradation. In the absence of Usp25 or if the DUB is inhibited, KEAP1 is downregulated and NRF2 is stabilized, allowing the cells to respond to oxidative stress more readily. In acetaminophen (APAP) overdose-induced oxidative liver damage in male mice, the inactivation of Usp25 , either genetically or pharmacologically, greatly attenuates liver injury and reduces the mortality rates resulted from lethal doses of APAP. The redox status of a cell is regulated through a number of mechanisms, chief among these is the KEAP1-mediated ubiquitination and degradation of NRF2. Here the authors show that KEAP1 itself is ubiquitinated and degraded in a process that is opposed by the ubiquitin-specific protease USP25.

Postnatal growth of mammalian oocytes is accompanied by a progressive gain of DNA methylation, which is predominantly mediated by DNMT3A, a de novo DNA methyltransferase 1 , 2 . Unlike the genome of sperm and most somatic cells, the oocyte genome is hypomethylated in transcriptionally inert regions 2 – 4 . However, how such a unique feature of the oocyte methylome is determined and its contribution to the developmental competence of the early embryo remains largely unknown. Here we demonstrate the importance of Stella, a factor essential for female fertility 5 – 7 , in shaping the oocyte methylome in mice. Oocytes that lack Stella acquire excessive DNA methylation at the genome-wide level, including in the promoters of inactive genes. Such aberrant hypermethylation is partially inherited by two-cell-stage embryos and impairs zygotic genome activation. Mechanistically, the loss of Stella leads to ectopic nuclear accumulation of the DNA methylation regulator UHRF1 8 , 9 , which results in the mislocalization of maintenance DNA methyltransferase DNMT1 in the nucleus. Genetic analysis confirmed the primary role of UHRF1 and DNMT1 in generating the aberrant DNA methylome in Stella-deficient oocytes. Stella therefore safeguards the unique oocyte epigenome by preventing aberrant de novo DNA methylation mediated by DNMT1 and UHRF1. Stella, a factor essential for female fertility, protects the oocyte methylome in mice by suppressing de novo DNA methylation mediated by the DNA methyltransferase DNMT1.

Background Triple-negative breast cancer (TNBC) has pronounced stemness that is associated with relapse. N 6 -methyladenosine (m 6 A) plays a crucial role in shaping cellular behavior by modulating transcript expression. However, the role of m 6 A in TNBC stemness, as well as the mechanisms governing its abundance, has yet to be elucidated. Methods We analyzed proteomic and transcriptomic data derived from breast cancer cohorts, with an emphasis on m 6 A regulators. To unravel the role of m 6 A in TNBC, we employed RNA sequencing, methylated RNA immunoprecipitation sequencing, RNA immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assays with mesenchymal stem-like (MSL) TNBC models. The clinical relevance was validated using human tissue microarrays and publicly accessible databases. Results Our findings indicate that the global level of m 6 A modification in MSL TNBC is downregulated primarily due to the loss of methyltransferase-like 14 ( METTL14 ) . The diminished m 6 A modification is crucial for the maintenance of TNBC stemness, as it increases the expression of yes-associated protein 1 (YAP1) by blocking YTH domain-containing family protein 2 (YTHDF2)-mediated transcript decay, thereby promoting the activation of Hippo-independent YAP1 signaling. YAP1 is essential for sustaining the stemness regulated by METTL14. Furthermore, we demonstrated that the loss of METTL14 expression results from lysine-specific demethylase 1 (LSD1)-mediated removal of histone H3 lysine 4 methylation at the promoter region, which is critical for LSD1-driven stemness in TNBC. Conclusion These findings present an epi-transcriptional mechanism that maintains Hippo-independent YAP1 signaling and plays a role in preserving the undifferentiated state of TNBC, which indicates the potential for targeting the LSD1-METTL14 axis to address TNBC stemness.

Overexpression of ubiquitin‐specific protease 28 (USP28) is found in hepatic carcinoma. It is unclear whether the deubiquitinase plays a role in hepatocarcinogenesis. Deregulation of the Wnt signaling pathway is frequently associated with liver cancer. Transcription factor 7‐like 2 (TCF7L2) is an important downstream transcription factor of the Wnt/β‐catenin signaling pathway, but the mechanisms by which TCF7L2 itself is regulated have not yet been revealed. Here, we report that USP28 promotes the activity of the Wnt signaling pathway through maintaining the stability of TCF7L2. We further show that FBXW7 is the E3 ubiquitin ligase for TCF7L2. By regulating the levels of TCF7L2, USP28 modulates the Wnt/β‐catenin signaling in liver cancer and USP28 depletion or inhibition by a small molecule inhibitor leads to a halt of growth in liver cancer cells. These results suggest that USP28 could be a potential therapeutic target for liver cancer. we reported that USP28 promotes the activity of the Wnt signaling pathway through maintaining the stability of TCF7L2. We further showed that FBXW7 is the E3 ubiquitinatin ligase for TCF7L2. By regulating the levels of TCF7L2, USP28 modulates the Wnt/β‐catenin signaling pathway in liver cancer. As a result, liver cancer cells are sensitive to USP28 depletion or inhibition by a small molecule inhibitor. Our results suggest that USP28 could be a potential therapeutic target for liver cancer.

BRCA1 is an important mediator of the DNA damage response, which promotes homologous recombination (HR) and antagonizes 53BP1-dependent non-homologous end joining in S/G2 phase. But how this is achieved remains unclear. Here, we report that the E3 ubiquitin ligase UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) directly participates in the interplay between BRCA1 and 53BP1. Mechanistically, UHRF1 is recruited to DNA double-strand breaks (DSBs) by BRCA1 in S phase, which requires the BRCT domain of BRCA1 and phosphorylated Ser674 of UHRF1. Subsequently, UHRF1 mediates K63-linked polyubiquitination of RIF1, and results in its dissociation from 53BP1 and DSBs thereby facilitating HR initiation. Thus, UHRF1 is a key regulator of DSB repair choice, which is separate from its role in heterochromatin formation and epigenetic regulator. BRCA1 is a key regulator of DNA double-strand break repair, functioning to promote homologous recombination and repress non-homologous end-joining. Here the authors show that the ubiquitin ligase UHRF1 is recruited to breaks by BRCA1, where it targets RIF1 and thereby facilitates recombination.

FTO (fat mass and obesity associated) was identified as an obesity-susceptibility gene by several independent large-scale genome association studies. A cluster of SNPs (single nucleotide polymorphism) located in the first intron of FTO was found to be significantly associated with obesity-related traits, such as body mass index, hip circumference, and body weight. FTO encodes a protein with a novel C-terminal α-helical domain and an N-terminal double-strand β-helix domain which is conserved in Fe(II) and 2-oxoglutarate-dependent oxygenase family. In vitro, FTO protein can demethylate single-stranded DNA or RNA with a preference for 3-methylthymine or 3-methyluracil. Its physiological substrates and function, however, remain to be defined. Here we report the generation and analysis of mice carrying a conditional deletion allele of Fto. Our results demonstrate that Fto plays an essential role in postnatal growth. The mice lacking Fto completely display immediate postnatal growth retardation with shorter body length, lower body weight, and lower bone mineral density than control mice, but their body compositions are relatively normal. Consistent with the growth retardation, the Fto mutant mice have reduced serum levels of IGF-1. Moreover, despite the ubiquitous expression of Fto, its specific deletion in the nervous system results in similar phenotypes as the whole body deletion, indicating that Fto functions in the central nerve system to regulate postnatal growth.

The RAD51 recombinase is evolutionarily conserved critical for homologous recombination (HR)‐mediated repair of DNA double‐strand breaks. It binds to single strand DNA to form protein‐DNA filaments for homology searching and pairing during HR repair. RFWD3 is an E3 ubiquitin ligase shown to remove RAD51 at the completion of HR repair through ubiquitination and degradation of RAD51. However, it remains elusive what prevents RFWD3 from attacking RAD51 in the absence of DNA damage and early on during the repair process. Here, we show that it is UHRF1 that protects RAD51, and it does so by acting as an E3 ubiquitin ligase of RFWD3 is demonstrated. Interestingly, RAD51 also protects RFWD3 from UHRF1, thereby establishing a negative feedback circuit that regulates the protein levels of RFWD3 and RAD51. Furthermore, it is shown that the ubiquitination of RFWD3 is regulated by phosphorylation status of UHRF1, and that phosphatase PP4 is important for modulating UHRF1 activity. Altogether, these regulatory mechanisms ensure that the recombinase RAD51 is maintained at appropriate levels for HR repair. We demonstrate here that the recombinase RAD51 is protected by UHRF1 through ubiquitinating RFWD3, an E3 for RAD51, and this very process is inhibited by RAD51, leading to a triple negative feedback circuit that ensures appropriate levels of RFWD3 and RAD51 for DNA damage response.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.

Noncoding polymorphisms in the fat mass and obesity-associated (FTO) gene represent common alleles that are strongly associated with effects on food intake and adiposity in humans. Previous studies have suggested that the obesity-risk allele rs8050136 in the first intron of FTO alters a regulatory element recognized by the transcription factor CUX1, thereby leading to decreased expression of FTO and retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L). Here, we evaluated the effects of rs8050136 and another potential CUX1 element in rs1421085 on expression of nearby genes in human induced pluripotent stem cell-derived (iPSC-derived) neurons. There were allele-dosage effects on FTO, RPGRIP1L, and AKT-interacting protein (AKTIP) expression, but expression of other vicinal genes, including IRX3, IRX5, and RBL2, which have been implicated in mediating functional effects, was not altered. In vivo manipulation of CUX1, Fto, and/or Rpgrip1l expression in mice affected adiposity in a manner that was consistent with CUX1 influence on adiposity via remote effects on Fto and Rpgrip1l expression. In support of a mechanism, mice hypomorphic for Rpgrip1l exhibited hyperphagic obesity, as the result of diminished leptin sensitivity in Leprb-expressing neurons. Together, the results of this study indicate that the effects of FTO-associated SNPs on energy homeostasis are due in part to the effects of these genetic variations on hypothalamic FTO, RPGRIP1L, and possibly other genes.

The spindle assembly checkpoint (SAC) is essential for proper sister chromatid segregation. Defects in this checkpoint can lead to chromosome missegregation and aneuploidy. An increasing body of evidence suggests that aneuploidy can play a causal role in tumorigenesis. However, mutant mice that are prone to aneuploidy have only mild tumor phenotypes, suggesting that there are limiting factors in the aneuploidy-induced tumorigenesis. Here we provide evidence that p53 is such a limiting factor. We show that aneuploidy activates p53 and that loss of p53 drastically accelerates tumor development in two independent aneuploidy models. The p53 activation depends on the ataxia-telangiectasia mutated (ATM) gene product and increased levels of reactive oxygen species. Thus, the ATM-p53 pathway safeguards not only DNA damage but also aneuploidy.