SPT16 ubiquitylation by DCAF14-CRL4 regulates FACT binding to histones.

The histone chaperone complex FACT comprises SPT16 and SSRP1 and contributes to DNA replication, transcription, and repair, but how it plays such various roles is unclear. Here, we show that human SPT16 is ubiquitylated at lysine-674 (K674) by the DCAF14-CRL4 ubiquitin ligase. K674 is located in the middle domain of SPT16, and the corresponding residue of the yeast ortholog is critical for binding to histone H3.

SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility in mice.

Calcineurin is a calcium-dependent phosphatase that plays roles in a variety of biological processes including immune responses. In spermatozoa, there is a testis-enriched calcineurin composed of PPP3CC and PPP3R2 (sperm calcineurin) that is essential for sperm motility and male fertility. Because sperm calcineurin has been proposed as a target for reversible male contraceptives, identifying proteins that interact with sperm calcineurin widens the choice for developing specific inhibitors.

Genetic loss of importin α4 causes abnormal sperm morphology and impacts on male fertility in mouse.

Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4 ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3 ) were fertile.

Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility.

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF.

CRISPR/Cas9-based genome editing in mice uncovers 13 testis- or epididymis-enriched genes individually dispensable for male reproduction†.

Developing a safe and effective male contraceptive remains a challenge in the field of medical science. Molecules that selectively target the male reproductive tract and whose targets are indispensable for male reproductive function serve among the best candidates for a novel non-hormonal male contraceptive method. To determine the function of these genes in vivo, mutant mice carrying disrupted testis- or epididymis-enriched genes were generated by zygote microinjection or electroporation of the CRISPR/Cas9 components.

Analysis of the sperm flagellar axoneme using gene-modified mice.

Infertility is a global health issue that affects 1 in 6 couples, with male factors contributing to 50% of cases. The flagellar axoneme is a motility apparatus of spermatozoa, and disruption of its structure or function could lead to male infertility. The axoneme consists of a "9+2" structure that contains a central pair of two singlet microtubules surrounded by nine doublet microtubules, in addition to several macromolecular complexes such as dynein arms, radial spokes, and nexin-dynein regulatory complexes.

CIB4 is essential for the haploid phase of spermatogenesis in mice†.

Spermatogenesis is a complex developmental process that involves the proliferation of diploid cells, meiotic division, and haploid differentiation. Many genes are shown to be essential for male fertility using knockout (KO) mice; however, there still remain genes to be analyzed to elucidate their molecular mechanism and their roles in spermatogenesis. Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitously expressed protein that possesses three paralogs: CIB2, CIB3, and CIB4.

Pathogenic mutations in the ALS gene CCNF cause cytoplasmic mislocalization of Cyclin F and elevated VCP ATPase activity.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease characterized by a progressive decline in motor function. Genetic analyses have identified several genes mutated in ALS patients, and one of them is Cyclin F gene (CCNF), the product of which (Cyclin F) serves as the substrate-binding module of a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex. However, the role of Cyclin F in ALS pathogenesis has remained unclear.

The ubiquitin ligase subunit β-TrCP in Sertoli cells is essential for spermatogenesis in mice.

β-TrCP is the substrate recognition subunit of an SCF-type ubiquitin ligase. We recently showed that deletion of the genes for both β-TrCP1 and β-TrCP2 paralogs in germ cells of male mice resulted in accumulation of the transcription factor DMRT1 and spermatogenic failure, whereas systemic β-TrCP1 knockout combined with β-TrCP2 knockdown had previously been shown to lead to disruption of testicular organization and accumulation of the transcription factor SNAIL. Here we investigated β-TrCP function in Sertoli cells by generating mice with targeted deletion of the β-TrCP2 gene in Sertoli cells on a background of whole-body β-TrCP1 knockout.

Chimeric analysis with newly established EGFP/DsRed2-tagged ES cells identify HYDIN as essential for spermiogenesis in mice.

The CRISPR/Cas9 system can efficiently introduce biallelic mutations in ES cells (ESCs), and its application with fluorescently-tagged ESCs enables phenotype analysis in chimeric mice. We have utilized ESCs that express EGFP in the cytosol and acrosome [EGR-G101 129S2 × (CAG/Acr-EGFP) B6] in previous studies; however, the EGFP signal in the sperm cytosol is weak and the signal in the acrosome is lost after the acrosome reaction, precluding analysis between wild type and ESC derived spermatozoa. In this study, we established an ESC line from RBGS (Red Body Green Sperm) transgenic mice [B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb] whose spermatozoa exhibit green fluorescence in the acrosome and red fluorescence in the mitochondria within the flagellar midpiece that is retained after the acrosome reaction.

Transforming Growth Factor β-Induced Proliferative Arrest Mediated by TRIM26-Dependent TAF7 Degradation and Its Antagonism by MYC.

Recognition of gene promoters by RNA polymerase II is mediated by general transcription factor IID (TFIID), which has been thought to be a static complex and to play a passive role in the regulation of gene expression under the instruction of gene-specific transcription factors. Here we show that transforming growth factor β (TGF-β) induced degradation of the TFIID subunit TAF7 in cultured mouse mammary epithelial cells and that this effect was required for proliferative arrest in response to TGF-β stimulation. TGF-β stimulated transcription of the gene for the ubiquitin ligase TRIM26, which was shown to ubiquitylate TAF7 and thereby to target it for proteasomal degradation.

Generation of tetracycline-inducible conditional gene knockout cells in a human Nalm-6 cell line.

Conditional gene knockout by homologous recombination combined with an inducible gene expression system is a powerful approach for studying gene function, although homologous recombination in human cells occurs infrequently. The tetracycline-regulated gene expression (Tet-Off) system is a convenient method for achieving conditional gene knockout, but it is not always promising in Nalm-6, a rare human cell line highly effective for gene targeting. Here we modified the Tet-Off system and applied it to the Nalm-6 cell line successfully by using an internal ribosome entry site to drive a selectable marker from the same tetracycline-responsive promoter for the transgene.

A germ cell specific gene of the ARGONAUTE family is essential for the progression of premeiotic mitosis and meiosis during sporogenesis in rice.

The rice (Oryza sativa) genome contains 18 copies of genes of the ARGONAUTE (AGO) family. Although AGO members play important roles in RNA-mediated silencing during plant development, a family member that is specifically involved in sexual reproduction has not been identified in plants. We identified the rice AGO gene MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1) from the analysis of seed-sterile mutants.