Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an “ancient” RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4⁺ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.

The Wnt, TGF-β, and Notch signaling pathways are essential for the regulation of cellular polarity, differentiation, proliferation, and migration. Differential activation and mutual crosstalk of these pathways during animal development are crucial instructive forces in the initiation of the body axis and the development of organs and tissues. Due to the ability to initiate cell proliferation, these pathways are vulnerable to somatic mutations selectively producing cells, which ultimately slip through cellular and organismal checkpoints and develop into cancer. The architecture of the Wnt, TGF-β, and Notch signaling pathways is simple. The transmembrane receptor, activated by the extracellular stimulus, induces nuclear translocation of the transcription factor, which subsequently changes the expression of target genes. Nevertheless, these pathways are regulated by a myriad of factors involved in various feedback mechanisms or crosstalk. The most prominent group of regulators is the ubiquitin–proteasome system (UPS). To open the door to UPS-based therapeutic manipulations, a thorough understanding of these regulations at a molecular level and rigorous confirmation in vivo are required. In this quest, mouse models are exceptional and, thanks to the progress in genetic engineering, also an accessible tool. Here, we reviewed the current understanding of how the UPS regulates the Wnt, TGF-β, and Notch pathways and we summarized the knowledge gained from related mouse models.

SKP1-CUL1-F-box protein (SCF) ubiquitin ligases are versatile protein complexes that mediate the ubiquitination of protein substrates. The direct substrate recognition relies on a large family of F-box-domain-containing subunits. One of these substrate receptors is FBXO38, which is encoded by a gene found mutated in families with early-onset distal motor neuronopathy. SCF ^FBXO38 ubiquitin ligase controls the stability of ZXDB, a nuclear factor associated with the centromeric chromatin protein CENP-B. Loss of FBXO38 in mice results in growth retardation and defects in spermatogenesis characterized by deregulation of the Sertoli cell transcription program and compromised centromere integrity. Moreover, it was reported that SCF ^FBXO38 mediates the degradation of PD-1, a key immune-checkpoint inhibitor in T cells. Here, we have re-addressed the link between SCF ^FBXO38 and PD-1 proteolysis. Our data do not support the notion that SCF ^FBXO38 directly or indirectly controls the abundance and stability of PD-1 in T cells.

The ubiquitin ligase SCF^FBXO38 controls centromeric chromatin by promoting the degradation of the ZXDB protein. To determine the importance of this pathway during development, Fbxo38-deficient mice were generated. The loss of FBXO38 resulted in growth retardation affecting several organs, including the male reproductive system. A detailed analysis of the mutant testes revealed pathological changes in the seminiferous tubules, accompanied by a significant decrease in sperm production and reduced fertility. In adult testes, FBXO38 was specifically expressed in Sertoli cells, a somatic population essential for spermatogenesis initiation and progression. Sertoli cells lacking FBXO38 exhibited stabilized ZXDB protein and upregulated centromeric chromatin. Furthermore, the gene expression profile revealed that the absence of FBXO38 led to a defect in Sertoli cell maturation, specifically characterized by dysregulation in genes controlling retinoic acid metabolism and intercellular communication. Consequently, we documented significant changes in their ability to initiate spermatogonial differentiation. In conclusion, we show that FBXO38 acts as a Sertoli cell maturation factor, affecting the Sertoli cell transcription program, centromere integrity, and, subsequently, the ability to control spermatogenesis.

Alterations in the gene encoding the E3 ubiquitin ligase substrate receptor FBXO38 have been associated with several diseases, including early-onset motor neuronopathy. However, the cellular processes affected by the enzymatic action of FBXO38 are not yet known. Here, we identify the zinc finger proteins ZXDA/B as its interaction partners. FBXO38 controls the stability of ZXDA/B proteins via ubiquitination and proteasome-dependent degradation. We show that ZXDA/B proteins associate with the centromeric protein CENP-B and that the interaction between ZXDA/B and FBXO38 or CENP-B is mutually exclusive. Functionally, ZXDA/B factors control the protein level of chromatin-associated CENP-B. Furthermore, their inappropriate stabilization leads to upregulation of CENP-A and CENP-B positive centromeric chromatin. Thus we demonstrate a previously unknown role of cullin-dependent protein degradation in the control of centromeric chromatin integrity.

The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5^tg) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5^tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.