Hidesato Ogawa

Mechanism of asymmetric ovarian development in chick embryos

In most animals, the gonads develop symmetrically, but most birds develop only a left ovary. A possible role for estrogen in this asymmetric ovarian development has been proposed in the chick, but the mechanism underlying this process is largely unknown. Here, we identify the molecular mechanism responsible for this ovarian asymmetry. Asymmetric PITX2 expression in the left presumptive gonad leads to the asymmetric expression of the retinoic-acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptors Ad4BP/SF-1 and estrogen receptor α in the right ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These data suggest that early asymmetric expression of PITX2 leads to asymmetric ovarian development through up- or downregulation of RALDH2, Ad4BP/SF-1, estrogen receptor α and cyclin D1.

Glycolytic genes are targets of the nuclear receptor Ad4BP/SF-1

Genetic deficiencies in transcription factors can lead to the loss of certain types of cells and tissue. The steroidogenic tissue-specific nuclear receptor Ad4BP/SF-1 (NR5A1) is one such gene, because mice in which this gene is disrupted fail to develop the adrenal gland and gonads. However, the specific role of Ad4BP/SF-1 in these biological events remains unclear. Here we use chromatin immunoprecipitation sequencing to show that nearly all genes in the glycolytic pathway are regulated by Ad4BP/SF-1. Suppression of Ad4BP/SF-1 by small interfering RNA reduces production of the energy carriers ATP and nicotinamide adenine dinucleotide phosphate, as well as lowers expression of genes involved in glucose metabolism. Together, these observations may explain tissue dysgenesis as a result of Ad4BP/SF-1 gene disruption in vivo. Considering the function of estrogen-related receptor α, the present study raises the possibility that certain types of nuclear receptors regulate sets of genes involved in metabolic pathways to generate energy carriers.

Transcriptional Suppression by Transient Recruitment of ARIP4 to Sumoylated Nuclear Receptor Ad4BP/SF-1

The small ubiquitin-like modifier SUMO conjugates transcription factors and suppresses their respective activation of target genes. Although various SUMO-modified transcription factors have been isolated, mechanisms whereby sumoylated-substrates modulate transcription remain unknown. Here, we purified ARIP4 (AR interacting protein 4, a Rad54 family member and a SNF2 chromatin remodeling factor), which interacts with sumoylated Ad4BP/SF-1 through two SUMO-interacting motifs and one Ad4BP/SF-1-binding region. Remarkably, ARIP4 also interacts selectively with other sumoylated nuclear receptors including LRH-1, AR, and GR. Interestingly, the ATPase activity of ARIP4 was stimulated in the presence of sumoylated Ad4BP/SF-1 and the Ad4BP/SF-1-binding site containing double-stranded DNA. ChIP assays and siRNA studies strongly suggested that ARIP4 temporally suppresses Ad4BP/SF-1-mediated transcription through its transient recruitment to target genes. These findings suggest that ARIP4 may be a cofactor that modulates SUMO-mediated fine-tuning of transcriptional suppression.

Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9

Sox9 is a transcription factor that is required for tissue development in mammals. In general, such transcription factors require co-regulators for precise temporal and spatial control of the activation and inactivation of the numerous genes necessary for precise development during embryogenesis. Here we identify a new Sox9 co-regulator: Using affinity chromatography with immobilized Sox9 protein, we identified exportin 4 (Exp4) as an interacting protein of Sox9 in human cultured cells. Interaction between endogenous Exp4 and Sox9 proteins was confirmed in the human osteosarcoma U2OS cells by immunoprecipitation experiments using anti-Sox9 antibody. siRNA depletion of Exp4 enhanced transcription of Sox9 target genes in U2OS cells, but did not affect nuclear localization of Sox9. These results suggest that Exp4 regulates Sox9 activity in the nucleus. Furthermore we found that the HMG box of Sox9 was responsible for binding to Exp4, and the HMG box was required for suppression of Sox9-mediated transcription. This contrasts with the known Sox9 co-regulators which bind to its transcriptional activation domain. Chromatin immunoprecipitation analyses revealed that Exp4 prevents Sox9 binding to the enhancers of its target genes. These results demonstrate that Exp4 acts as a Sox9 co-regulator that directly regulates binding of Sox9 to its target genes.

Generation of Rat Monoclonal Antibodies Specific for Ad4BPSF-1

Ad4BP/SF-1 (adrenal4 binding protein/steroidogenic factor-1[NR5A1]) is an essential nuclear receptor required for animal reproduction and endocrine regulation. The present study reports on monoclonal antibodies (MAbs) directed against mouse Ad4BP/SF-1, which were produced by the hybridization of mouse myeloma cells with lymph node cells of an immunized rat. The produced MAbs reacted with both recombinant and endogenous Ad4BP/SF-1. These MAbs will be useful in immunolocalization and immunoblotting experiments conducted on different tissue types to determine the levels of expression of Ad4BP/SF-1 throughout development, as well as further analyses of the biological function and cellular dynamics of this protein.

Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells

Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here, we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62‐knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA‐mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery.

Expression analysis of Baf60c during heart regeneration in axolotls and neonatal mice.

Some organisms, such as zebrafish, urodele amphibians, and newborn mice, have a capacity for heart regeneration following injury. However, adult mammals fail to regenerate their hearts. To know why newborn mice can regenerate their hearts, we focused on epigenetic factors, which are involved in cell differentiation in many tissues. Baf60c (BRG1/BRM‐associated factor 60c), a component of ATP‐dependent chromatin‐remodeling complexes, has an essential role for cardiomyocyte differentiation at the early heart development. To address the function of Baf60c in postnatal heart homeostasis and regeneration, we examined the detailed expression/localization patterns of Baf60c in both mice and axolotls. In the mouse heart development, Baf60c was highly expressed in the entire heart at the early stages, but gradually downregulated at the postnatal stages. During heart regeneration in neonatal mice and axolotls, Baf60c expression was strongly upregulated after resection. Interestingly, the timing of Baf60c upregulation after resection was consistent with the temporal dynamics of cardiomyocyte proliferation. Moreover, knockdown of Baf60c downregulated proliferation of neonatal mouse cardiomyocytes. These data suggested that Baf60c plays an important role in cardiomyocyte proliferation in heart development and regeneration. This is the first study indicating that Baf60c contributes to the heart regeneration in vertebrates.

Ess2 bridges transcriptional regulators and spliceosomal complexes via distinct interacting domains

Transcription and pre-mRNA splicing are complex, coupled processes that involve transcriptional co-regulators. Ess2 (also termed Dgcr14) is a nuclear protein that enhances the transcriptional activity of retinoic acid receptor-related orphan receptor gamma/gamma-t (Rorγ/γt). Ess2 is also a component of the spliceosomal C complex (containing U2, U5 and U6 snRNAs). However, the domains in Ess2 that function in splicing and transcription have not been identified. To elucidate the roles of Ess2 in splicing and transcription, we performed RNA immunoprecipitation (RIP) assays to detect Ess2-interacting snRNAs. We found that Ess2 associated with U6 snRNA as well as U1 and U4 snRNAs. Experiments using Ess2 deletion mutants showed that a C-terminus deletion mutant of Ess2 (1-399 a. a.) lost its ability to associate with snRNAs, whereas the N-terminus domain of Ess2 (1-200 a. a.) associated with Rorγ/γt, but not with snRNAs. Interestingly, experiments using anti-ROR common antibody showed that Rors also associated with U4 and U6 snRNAs. Ess2 knockdown in a T cell hybridoma (68-41 cells) abrogated the interaction between spliceosomes and Rors. An Ess2-dependent association was also found between an lncRNA (Rmrp) and Rors. We thus propose that Ess2 associates with both transcriptional factors and spliceosomal complexes and modulates splicing reactions coupled with transcription factors.

Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation

Transcriptional coregulators contribute to several processes involving nuclear receptor transcriptional regulation. The transcriptional coregulator androgen receptor-interacting protein 4 (ARIP4) interacts with nuclear receptors and regulates their transcriptional activity. In this study, we identified p62 as a major interacting protein partner for ARIP4 in the nucleus. Nuclear magnetic resonance analysis demonstrated that ARIP4 interacts directly with the ubiquitin-associated (UBA) domain of p62. ARIP4 and ubiquitin both bind to similar amino acid residues within UBA domains; therefore, these proteins may possess a similar surface structure at their UBA-binding interfaces. We also found that p62 is required for the regulation of ARIP4 protein levels under nutrient starvation conditions. We propose that p62 is a novel binding partner for ARIP4, and that its binding regulates the cellular protein level of ARIP4 under conditions of metabolic stress.

Shelterin promotes tethering of late replication origins to telomeres for replication-timing control

DNA replication initiates at many discrete loci on eukaryotic chromosomes, and individual replication origins are regulated under a spatiotemporal program. However, the underlying mechanisms of this regulation remain largely unknown. In the fission yeast Schizosaccharomyces pombe, the telomere‐binding protein Taz1, ortholog of human TRF1/TRF2, regulates a subset of late replication origins by binding to the telomere‐like sequence near the origins. Here, we showed using a lacO/LacI‐GFP system that Taz1‐dependent late origins were predominantly localized at the nuclear periphery throughout interphase, and were localized adjacent to the telomeres in the G1/S phase. The peripheral localization that depended on the nuclear membrane protein Bqt4 was not necessary for telomeric association and replication‐timing control of the replication origins. Interestingly, the shelterin components Rap1 and Poz1 were required for replication‐timing control and telomeric association of Taz1‐dependent late origins, and this requirement was bypassed by a minishelterin Tpz1‐Taz1 fusion protein. Our results suggest that Taz1 suppresses replication initiation through shelterin‐mediated telomeric association of the origins at the onset of S phase.