No abstract

Cyclin G-associated kinase (GAK) is an association partner of clathrin heavy chain (CHC) and is essential for clathrinmediated membrane trafficking. Here, we report two novel functions of GAK: maintenance of proper centrosome maturation and of mitotic chromosome congression. Indeed, GAK knockdown by siRNA caused cell-cycle arrest at metaphase, which indicates that GAK is required for proper mitotic progression. We found that this impaired mitotic progression was due to activation of the spindle-assembly checkpoint, which senses protruded, misaligned or abnormally condensed chromosomes in GAK-siRNA-treated cells. GAK knockdown also caused multi-aster formation, which was due to abnormal fragmentation of pericentriolar material, but not of the centrioles. Moreover, GAK and CHC cooperated in the same pathway and interacted in mitosis to regulate the formation of a functional spindle. Taken together, we conclude that GAK and clathrin function cooperatively not only in endocytosis, but also in mitotic progression. Supplementary material available online at

Sirtuins are NAD + -dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis.

PIWI‐interacting RNAs (piRNAs) are germ cell‐specific small RNAs essential for retrotransposon gene silencing and male germ cell development. In piRNA biogenesis, the endonuclease MitoPLD/Zucchini cleaves long, single‐stranded RNAs to generate 5′ termini of precursor piRNAs (pre‐piRNAs) that are consecutively loaded into PIWI‐family proteins. Subsequently, these pre‐piRNAs are trimmed at their 3′‐end by an exonuclease called Trimmer. Recently, poly(A)‐specific ribonuclease‐like domain‐containing 1 (PNLDC1) was identified as the pre‐piRNA Trimmer in silkworms. However, the function of PNLDC1 in other species remains unknown. Here, we generate Pnldc1 mutant mice and analyze small RNAs in their testes. Our results demonstrate that mouse PNLDC1 functions in the trimming of both embryonic and post‐natal pre‐piRNAs. In addition, piRNA trimming defects in embryonic and post‐natal testes cause impaired DNA methylation and reduced MIWI expression, respectively. Phenotypically, both meiotic and post‐meiotic arrests are evident in the same individual Pnldc1 mutant mouse. The former and latter phenotypes are similar to those of MILI and MIWI mutant mice, respectively. Thus, PNLDC1‐mediated piRNA trimming is indispensable for the function of piRNAs throughout mouse spermatogenesis.

We have comprehensively identified the genes whose expressions are augmented in bone marrow-derived mononuclear cells (BMMC) from patients with Rheumatoid Arthritis (RA) as compared with BMMCs from Osteoarthritis (OA) patients, and named them AURA after augmented in RA. Both stepwise subtractive hybridization and microarray analyses were used to identify AURA genes, which were confirmed by northern blot analysis and/or reverse transcription polymerase chain reaction (RT-PCR). We also assessed their expression levels in individual patients by quantitative real-time RT-PCR. Of 103 AURA genes we have identified, the mRNA levels of the following 10 genes, which are somehow related to immune responses, were increased in many of the RA patients: AREG (=AURA9), FK506-binding protein 5 (FKBP5 = AURA45), C-type lectin superfamily member 9 (CLECSF9 = AURA24), tyrosylprotein sulfotransferase 1 (TPST1 = AURA52), lymphocyte G0/G1 switch gene (G0S2 = AURA8), chemokine receptor 4 (CXCR4 = AURA86), nuclear factor-kappa B (NF-kappaB = AURA25) and two genes of unknown function (FLJ11106 = AURA1, BC022398 = AURA2 and XM_058513 = AURA17). Since AREG was most significantly increased in many of the RA patients, we subjected it to further analysis and found that AREG-epidermal growth factor receptor signaling is highly activated in synovial cells isolated from RA patients, but not in OA synoviocytes. We propose that the expression profiling of these AURA genes may improve our understanding of the pathogenesis of RA.

TISP40, a mouse spermatid‐specific gene, encodes a CREB/CREM family transcription factor that is predominantly expressed during spermiogenesis. We report here that TISP40 generates two types of proteins, Tisp40α and Tisp40β, both of which contain a transmembrane domain and localize to the endoplasmic reticulum (ER). In contrast, mutant proteins lacking the transmembrane domain (Tisp40α/βΔTM) primarily localize to the nucleus. Endoglycosidase H treatment shows that the C‐terminus of Tisp40α/β is glycosylated. Protease experiments demonstrate that Tisp40α/β are Type II transmembrane proteins that are released into the nucleus by a two‐step cleavage mechanism called ‘regulated intramembrane proteolysis’ (Rip). Unlike previously published observations, Tisp40α does not bind to the NF‐κB site; instead, it specifically binds to the unfolded protein response element (UPRE). Luciferase assays reveal that Tisp40βΔTM activates transcription through UPRE. Northern blot analysis shows that Tisp40α/βΔTM proteins up‐regulate EDEM (ER degradation of enhancing α‐manosidase‐like protein) mRNA. These observations unveil a novel event in mouse spermiogenesis and show that the final stage of trans‐criptional regulation is controlled by the Rip pathway.

Mammalian spermiogenesis, a process where haploid male germ cells differentiate to become mature spermatozoa, entails dramatic morphological and biochemical changes including remodeling of the germ cell chromatin. Proteins that contain one or more plant homeodomain (PHD) fingers have been implicated in the regulation of chromatin structure and function. Pygopus 2 (Pygo2) belongs to a family of evolutionarily conserved PHD finger proteins thought to act as co-activators of Wnt signaling effector complexes composed of beta-catenin and LEF/TCF transcription factor. Here we analyze mice containing hypomorphic alleles of pygopus 2 (Pygo2 or mpygo2) and uncover a beta-catenin-independent involvement of the Pygo2 protein in spermiogenesis. Pygo2 is expressed in elongating spermatids at stages when chromatin remodeling occurs, and block of Pygo2 function leads to spermiogenesis arrest and consequent infertility. Analysis of spermiogenesis in Pygo2 mutants reveals reduced expression of select post-meiotic genes including protamines, transition protein 2, and H1fnt, all of which are required for germ cell chromatin condensation, and drastically altered pattern of histone H3 hyperacetylation. These findings suggest that Pygo2 is involved in the chromatin remodeling events that lead to nuclear compaction of male germ cells.

During the development of mammalian embryonic germ cells, global demethylation and de novo DNA methylation take place. In mouse embryonic germ cells, two PIWI family proteins, MILI and MIWI2, are essential for the de novo DNA methylation of retrotransposons, presumably through PIWI-interacting RNAs (piRNAs). Although piRNA-associated MIWI2 has been reported to play critical roles in the process, its molecular mechanisms have remained unclear. To identify the mechanism, transgenic mice were produced; they contained a fusion protein of MIWI2 and a zinc finger (ZF) that recognized the promoter region of a type A LINE-1 gene. The ZF-MIWI2 fusion protein brought about DNA methylation, suppression of the type A LINE-1 gene, and a partial rescue of the impaired spermatogenesis of MILI-null mice. In addition, ZF-MIWI2 was associated with the proteins involved in DNA methylation. These data indicate that MIWI2 functions as an effector of de novo DNA methylation of the retrotransposon.