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Meiotic recombination enables reciprocal exchange of genetic information between parental chromosomes and is essential for fertility. MEIOB, a meiosis-specific ssDNA-binding protein, regulates early meiotic recombination. Here we report that the human infertility-associated missense mutation (N64I) in MEIOB causes protein degradation and reduced crossover formation in mouse testes. Although the MEIOB N64I substitution is associated with human infertility, the point mutant mice are fertile despite meiotic defects. Meiob mutagenesis identifies serine 67 as a critical residue for MEIOB. Biochemically, these two mutations (N64I and S67 deletion) cause self-aggregation of MEIOB and sharply reduced protein half-life. Molecular genetic analyses of both point mutants reveal an important role for MEIOB in crossover formation in late meiotic recombination. Furthermore, we find that the MEIOB protein levels directly correlate with the severity of meiotic defects. Our results demonstrate that MEIOB regulates meiotic recombination in a dosage-dependent manner., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Triple-negative breast cancer (TNBC) is characterized by a high degree of immune infiltrate in the tumour microenvironment, which may influence the fate of TNBC cells. We reveal that loss of the tumour suppressive transcription factor Elf5 in TNBC cells activates intrinsic interferon-γ (IFN-γ) signalling, promoting tumour progression and metastasis. Mechanistically, we find that loss of the Elf5-regulated ubiquitin ligase FBXW7 ensures stabilization of its putative protein substrate IFN-γ receptor 1 (IFNGR1) at the protein level in TNBC. Elf5 low tumours show enhanced IFN-γ signalling accompanied by an increase of immunosuppressive neutrophils within the tumour microenvironment and increased programmed death ligand 1 expression. Inactivation of either programmed death ligand 1 or IFNGR1 elicited a robust anti-tumour and/or anti-metastatic effect. A positive correlation between ELF5 and FBXW7 expression and a negative correlation between ELF5, FBXW7 and IFNGR1 expression in the tumours of patients with TNBC strongly suggest that this signalling axis could be exploited for patient stratification and immunotherapeutic treatment strategies for Elf5 low patients with TNBC.

Prolactin controls the development and function of milk-producing breast epithelia but also supports growth and differentiation of breast cancer, especially luminal subtypes. A principal signaling mediator of prolactin, Stat5, promotes cellular differentiation of breast cancer cells in vitro, and loss of active Stat5 in tumors is associated with antiestrogen therapy failure in patients. In luminal breast cancer, progesterone induces a cytokeratin-5 (CK5)-positive basal cell-like population. This population possesses characteristics of tumor stem cells including quiescence, therapy resistance and tumor-initiating capacity. Here we report that prolactin counteracts induction of the CK5-positive population by the synthetic progestin (Pg) R5020 in luminal breast cancer cells both in vitro and in vivo. CK5-positive cells were chemoresistant as determined by fourfold reduced rate of apoptosis following docetaxel exposure. Pg-induction of CK5 was preceded by marked upregulation of BCL6, an oncogene and transcriptional repressor critical for the maintenance of leukemia-initiating cells. Knockdown of BCL6 prevented induction of CK5-positive cell population by Pg. Prolactin suppressed Pg-induced BCL6 through Jak2-Stat5 but not Erk- or Akt-dependent pathways. In premenopausal but not postmenopausal patients with hormone receptor-positive breast cancer, tumor protein levels of CK5 correlated positively with BCL6, and high BCL6 or CK5 protein levels were associated with unfavorable clinical outcome. Suppression of Pg-induction of CK5-positive cells represents a novel prodifferentiation effect of prolactin in breast cancer. The present progress may have direct implications for breast cancer progression and therapy as loss of prolactin receptor-Stat5 signaling occurs frequently and BCL6 inhibitors currently being evaluated for lymphomas may have value for breast cancer.

The p53 homologue p73 efficiently activates p53-responsive genes. The well documented over-expression of p73 spliced forms in a wide variety of tumor types promoted us to elucidate the mechanisms underlying p73-mediated transcription. Using the luciferase reporter gene driven by Mdm2-minimal promoter in p53 null cells, we demonstrate that the weak transcriptional activity mediated by p73alpha was increased by the mutant form p73beta292, which by itself is transcriptionally inactive. Similarly, cooperation between p73beta and an inactive form of p73alpha increased p73beta-mediated transcriptional activities. Conversely, p73beta elicited a silencing effect on a gain of function mutant, p53(281), which by itself mediated efficient transactivation of the MDR promoter. Neither anisomycin nor actinomycin D altered p73-mediated transcriptional activities, whereas sorbitol profoundly inhibited them through a rapid proteasome-dependent degradation of p73. Our observations point to plausible scenarios in which p73, through cooperation between p73 spliced forms and suppression of gain of function mutant p53 may elicit changes in the transcription of p53 target genes that play key roles in cell growth and death.

Activation of the transcription factor NF-kappa B in response to proinflammatory stimuli requires the phosphorylation-triggered and ubiquitin-dependent degradation of the NF-kappa B inhibitor, I kappa B alpha. Here, we show the in vitro reconstitution of the phosphorylation-dependent ubiquitination of I kappa B alpha with purified components. ROC1, a novel SCF-associated protein, is recruited by cullin 1 to form a quatemary SCFHOS-ROC1 holenzyme (with Skp1 and the beta-TRCP homolog HOS). SCFHOS-ROC1 binds IKK beta-phosphorylated I kappa B alpha and catalyzes its ubiquitination in the presence of ubiquitin, E1, and Cdc34. ROC1 plays a unique role in the ubiquitination reaction by heterodimerizing with cullin 1 to catalyze ubiquitin polymerization.

SCF E3 ubiquitin ligases mediate ubiquitination and proteasome-dependent degradation of phosphorylated substrates. We identified a human F-box/WD40 repeats protein (HOS), which is homologous to Slimb/h betaTrCP. Being a part of SCF complex with Skp1 and Cullin1, HOS specifically interacted with the phosphorylated IkappaB and beta-catenin, targeting these proteins for proteasome-dependent degradation in vivo. This targeting required Cullin1 as expression of a mutant Cullin1 abrogated the degradation of IkappaB and of beta-catenin. Mutant HOS which lacks the F-box blocked TNF alpha-induced degradation of IkappaB as well as GSK3beta-mediated degradation of beta-catenin. This mutant also inhibited NF-kappaB transactivation and increased the beta-catenin-dependent transcription activity of Tcf. These results demonstrate that SCF(HOS) E3 ubiquitin ligase regulate both NF-kappaB and beta-catenin signaling pathways.

Key to p53 ability to mediate its multiple cellular functions lies in its stability. In the present study we have elucidated the mechanism by which Mdm2 regulates p53 degradation. Using in vitro and in vivo ubiquitination assays we demonstrate that Mdm2 association with p53 targets p53 ubiquitination. Exposure of cells to UV-irradiation inhibits this targeting. Mdm2 which is deficient in p53 binding failed to target p53 ubiquitination, suggesting that the association is essential for Mdm2 targeting ability. While mdm2-p53 complex is found in non-stressed cells, the amount of p53-bound mdm2 is decreased after UV-irradiation, further pointing to the relationship between mdm2 binding and p53 level. Similar to Swiss 3T3 cells, the dissociation of mdm2-p53 complex was also found in UV-treated Scid cells, lacking functional DNA-PK, suggesting that DNA-PK is not sufficient for dissociating mdm2 from p53. Together our studies point to the role of Mdm2, as one of p53-associated proteins, in targeting p53 ubiquitination.

Regulatory proteins are often ubiquitinated, depending on their phosphorylation status as well as on their association with ancillary proteins that serve as adapters of the ubiquitination machinery. We previously demonstrated that c-Jun is targeted for ubiquitination by its association with inactive c-Jun NH2-terminal kinase (JNK). Phosphorylation by activated JNK protects c-Jun from ubiquitination, thus by prolonging its half-life. In the study reported here, we determined the ability of JNK to target ubiquitination of its other substrates (Elk1 and activating transcription factor 2 (ATF2)) and associated proteins (ATF2 and JunB). We demonstrate that phosphorylation by JNK protects ATF2, but not Elk1, from JNK-targeted ubiquitination. We also show that association of inactive JNK with JunB or ATF2 is necessary to target them for ubiquitination. Unlike its targeting of c-Jun, JNK requires additional cellular components, yet to be identified, to target the ubiquitination of ATF2. Elk1 is phosphorylated by JNK, but JNK neither associates with nor targets Elk1 for ubiquitination. The implications for the dual role of JNK in the regulation of ubiquitination and stability of c-Jun, ATF2, and JunB in normally growing versus stressed cells are discussed.

To provide better understanding about alterations in transcription factor activities during the promotion and progression stages of lung carcinogenesis we have utilized the A/J mice lung tumor model in which two potent lung carcinogens, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP), were co-administered. Nuclear proteins prepared from lung, brain, kidney, liver and colon of the A/J mice, 19 weeks after the last carcinogen administration, as well as from their respective non-treated controls, were tested for their binding to polyoma enhancer binding protein 2 (PEBP2) target sequence and UV-responsive element (URE). PEBP2 represents a newly identified family of transcription factors that was shown to play a role in cellular differentiation and transformation. URE is similar to CRE and AP1 target sequences, to which the members of ATF/AP1 transcriptional factors family bind. We demonstrate here that there is a marked increase in binding to both PEBP2 and URE sequences in lung, liver, kidney and brain of the treated mice. Such binding appears to be dependent on the mode of carcinogen administration as it was better noticed in the intragastric injected group than in the intraperitoneal group. Taken together, our findings suggest that increased binding to the URE and PEBP2 target sequence reflects changes in transcriptional activities which occur at late stages of lung carcinogenesis in a fashion which appear to depend on mode of carcinogen administration.

Mouse fibroblast cells (WOP) express permissive factors which support polyoma DNA replication. However, electroporation into WOP cells of a mammalian expression vector that encodes the c-jun cDNA results in repression of polyoma DNA replication in a dose-dependent manner. In previous studies we have shown that UV-irradiation is capable of mediating a similar effect on polyoma DNA replication. When c-jun over-expression was combined with ultraviolet (UV)-irradiation, polyoma DNA replication decreased further. The repression of replication mediated by c-jun appears to be mediated by factor(s) that bind to PEBP4/2 target sequences as oligomers bearing the PEBP2/4 target site were capable of restoring polyoma DNA replication when added to UV-treated or c-jun over-expressing cells. The binding to the PEBP2/4 is partially dependent on the availability of AP-1 proteins, since an AP-1 target sequence can efficiently compete one of the three complexes formed with the PEBP2 target site. PEPB2 sequences do not, however, affect binding to the AP1 site. The effect of PEBP2 on polyoma replication is not dependent on the adjacent AP-1 site since PEBP2 could restore replication of polyomavirus which is mutated at the AP-1 sequence. A similar replication pattern was noted in a deletion mutant of polyoma which lacks PEBP4, yet, contains an intact PEBP2 binding sequence, suggesting that PEBP2 is the principle target for mediating repression of polyoma DNA replication.