Ippei Nagamori

Tisp40, a spermatid specific bZip transcription factor, functions by binding to the unfolded protein response element via the Rip pathway

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.

GAK, a regulator of clathrin-mediated membrane traffic, also controls centrosome integrity and chromosome congression.

Cyclin G-associated kinase (GAK) is an association partner of clathrin heavy chain (CHC) and is essential for clathrin-mediated 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.

The testes-specific bZip type transcription factor Tisp40 plays a role in ER stress responses and chromatin packaging during spermiogenesis

We previously reported that the spermatid‐specific transcription factor Tisp40 functions through UPRE and CRE. To investigate Tisp40 function in vivo, we generated TISP40(–/–) mice. TISP40(–/–) mice were born at expected ratios, were healthy, and mutant males bred normally. However, the ER stress‐response protein Grp78/BiP accumulated in the TISP40(–/–) testis and RAMP4 (Ribosome‐associated membrane protein 4) mRNA level was up‐regulated. Disruption of TISP40 caused ER stress and activation of caspase 12 but not caspase 9, leading to apoptosis of meiotic/postmeiotic germ cells. On the other hand, DAPI staining and electron microscopy revealed that epididymal sperm nuclei were abnormally relaxed in the TISP40(–/–) testis, a phenotype that was independent of the expression and maturation of transition proteins and protamines but due to abnormally retained histones. Histones localized to the cytoplasm as well as to the nucleus and were also retained in epididymal sperm. Histones H2A and H4 were dramatically up‐regulated and the acetylation of H2A, H2B and H4 was also enhanced in the TISP40(–/–) testis. Taken together, we conclude that Tisp40 plays an important role in the unfolded protein response of the testis and in regulating the maturation of sperm head nuclei.

The chromatoid body of male germ cells: Epigenetic control and miRNA pathway

Differentiation of germ cells is characterized by a remarkable degree of cellular restructuring and gene regulation that involves complex events of genomic and epigenetic reorganization. The pathways that govern miRNAs have been shown to play an important role in the male germ cell lineage. The chromatoid body is a finely filamentous, lobulated perinuclear granule located in the cytoplasm of male germ cells. The role of the chromatoid body in the mouse has remained elusive for longtime, although it was proposed to be involved in RNA storing and metabolism. Recent findings show that the chromatoid body is related to the RNA processing body (P-body) of somatic cells and that it seems to operate as an intracellular nerve-center of the microRNA pathway. The role of the chromatoid body underscores the importance of post-transcriptional gene regulation and of the microRNA pathway in the control of postmeiotic male germ cell differentiation.

Expression Profiling of PBMC-based Diagnostic Gene Markers Isolated from Vasculitis Patients

Vasculitis (angiitis) is a systemic autoimmune disease that often causes fatal symptoms. We aimed to isolate cDNA markers that would be useful for diagnosing not only vasculitis but also other autoimmune diseases. For this purpose, we used stepwise subtractive hybridization and cDNA microarray analyses to comprehensively isolate the genes whose expressions are augmented in peripheral blood mononuclear cells (PBMCs) pooled from vasculitis patients. Subsequently, we used quantitative real-time polymerase chain reaction (qRT–PCR) to examine the mRNA levels of each candidate gene in individual patients. These analyses indicated that seven genes exhibit remarkably augmented expression in many vasculitis patients. Of these genes, we analyzed G0/G1 switch gene 2 (G0S2) further because G0S2 expression is also enhanced in the PBMCs of patients with systemic lupus erythematodes (SLE). We generated G0S2 transgenic mice that ubiquitously overexpress human G0S2. Although we did not observe any obvious vasculitis-related histopathologic findings in these mice, these mice are unhealthy as they produce only few offspring and showed elevated serum levels of two autoimmunity-related antibodies, anti-nuclear antibody, and anti-double strand DNA antibody. Thus, our large-scale gene profiling study may help finding sensitive and specific DNA markers for diagnosing autoimmune diseases including vasculitis and SLE.

Transcription Factors, cAMP-responsive Element Modulator (CREM) and Tisp40, Act in Concert in Postmeiotic Transcriptional Regulation

We previously isolated 80 TISP (transcript induced in spermiogenesis) genes whose transcription is dramatically induced during spermiogenesis. Our analysis here of the expression of these genes in the testis of the cAMP-responsive element modulator (CREM)-null mouse revealed that 54 TISP genes are under the transcriptional regulation of CREM. One CREM-regulated gene is TISP40, which encodes a basic leucine zipper (bZip)-type transcription factor bearing a transmembrane domain that generates the two proteins Tisp40α and Tisp40β. Both of these proteins function by binding to UPRE (unfolded protein-response element) but do not recognize CRE motifs. We show here that Tisp40α mRNA is generated under the direct transcriptional regulation of CREM. CREMτ and Tisp40 form a heterodimer, which functions through CRE but not through UPRE. Furthermore, binding ability of CREM to CRE is dramatically up-regulated by forming a heterodimer with Tisp40αΔTM, a truncated form of Tisp40α that lacks the transmembrane domain. We confirmed that Tisp40 and CREM actually bind to the Tisp40 promoter in vivo by chromatin immunoprecipitation assay. Finally, we demonstrate that the Tisp40ΔTM-CREMτ heterodimer acts as a recruiter of HIRA, a histone chaperone, to CRE. Taken together, we propose that Tisp40 is an important transcriptional regulator during spermiogenesis.

Isolation and characterization of the TIGA genes, whose transcripts are induced by growth arrest

We report here the isolation of 44 genes that are upregulated after serum starvation and/or contact inhibition. These genes have been termed TIGA, after Transcript Induced by Growth Arrest. We found that there are two kinds of G0 phases caused by serum starvation, namely, the shallow G0 (or G0/G1) and the deep G0 phases. The shallow G0 is induced by only a few hours of serum starvation, while deep G0 is generated after 3 days of serum starvation. We propose that mammalian cells enter deep G0 through a G0 gate, which is only opened on the third day of serum starvation. TIGA1, one of the uncharacterized TIGA genes, encodes a homolog of cyanate permease of bacteria and localizes in mitochondria. This suggests that Tiga1 is involved in the inorganic ion transport and metabolism needed to maintain the deep G0 phase. Ectopic expression of TIGA1 inhibited not only tumor cell proliferation but also anchorage-independent growth of cancer cell lines. A microsatellite marker, ENDL-1, allowed us to detect loss of heterozygosity around the TIGA1 gene region (5q21–22). Further analysis of the TIGA genes we have identified here may help us to better understand the mechanisms that regulate the G0 phase.

Comprehensive DNA Methylation Analysis of Retrotransposons in Male Germ Cells

De novo DNA methylation of retrotransposons is critical for silencing. Here, we use DNA methylation analysis to examine retrotransposons in mouse male germ cells. DNA methylation of long interspersed nuclear elements (LINEs) is dependent on piRNA, and younger LINEs exhibit greater piRNA dependence. In contrast, most long terminal repeat (LTR) retrotransposons produce lower levels of piRNAs and do not show significant piRNA dependence. The relationship between DNA methylation and corresponding piRNA expression of several LTR retrotransposons was reduced in Mili-null cells, but not Miwi2-null cells. These observations raise the possibility of piRNA-dependent DNA methylation without Miwi2. Therefore, it appears that the molecular mechanisms of the gene silencing of retrotransposons are more complicated than previously thought.

MIWI2 as an Effector of DNA Methylation and Gene Silencing in Embryonic 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.

PNLDC1, mouse pre‐piRNA Trimmer, is required for meiotic and post‐meiotic male germ cell development

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.