Kubota Y et al. (2022) Genes Cells "The PAF1 complex cell-autonomously promotes oogenesis in Caenorhabditis elegans."

Ota N, Takatsuka H, Unno T, Ito M, Kubota Y, Onami S, Sugimoto A

[Genes Cells, 2022]

The RNA polymerase II-associated factor 1 complex (PAF1C) is a protein complex that consists of LEO1, RTF1, PAF1, CDC73, and CTR9, and has been shown to be involved in RNA polymerase II-mediated transcriptional and chromatin regulation. Although it has been shown to regulate a variety of biological processes, the precise role of the PAF1C during germ line development has not been clarified. In this study, we found that reduction in the function of the PAF1C components, LEO-1, RTFO-1, PAFO-1, CDC-73, and CTR-9, in Caenorhabditis elegans affects oogenesis. Defects in oogenesis were also confirmed using an oocyte maturation marker, OMA-1::GFP. While four to five OMA-1::GFP-positive oocytes were observed in wild-type animals, their numbers were significantly decreased in pafo-1 mutant and leo-1(RNAi), pafo-1(RNAi), and cdc-73(RNAi) animals. Expression of a functional PAFO-1::mCherry transgene in the germline significantly rescued the oogenesis-defective phenotype of the pafo-1 mutants, suggesting that expression of the PAF1C in germ cells is required for oogenesis. Notably, overexpression of OMA-1::GFP partially rescued the oogenesis defect in the pafo-1 mutants. Based on our findings, we propose that the PAF1C promotes oogenesis in a cell-autonomous manner by positively regulating the expression of genes involved in oocyte maturation.

Kubota Y et al. (2006) Biol Chem "C. elegans as a model system to study the function of the COG complex in animal development."

Nishiwaki K, Kubota Y

[Biol Chem, 2006]

The conserved oligomeric Golgi (COG) complex is an octameric protein complex associated with the Golgi apparatus and is required for proper sorting and glycosylation of Golgi resident enzymes and secreted proteins. Although COG complex function has been extensively studied at the cellular and subcellular levels, its role in animal development mostly remains unknown. Recently, mutations in the components of the COG complex were found to cause abnormal gonad morphogenesis in Caenorhabditis elegans. In C. elegans, the COG complex acts in the glycosylation of an ADAM (a disintegrin and metalloprotease) family protein, MIG-17, which directs migration of gonadal distal tip cells to lead gonad morphogenesis. This is the first link between the COG complex and the function of an ADAM protease that is directly involved in organ morphogenesis, demonstrating the potential of C. elegans as a model system to study COG function in animal development.

Kubota Y et al. (2014) Dev Biol "The PAF1 complex is involved in embryonic epidermal morphogenesis in Caenorhabditis elegans."

Maruyama R, Sugimoto A, Tsuyama K, Iida N, Haruta N, Takabayashi Y, Kubota Y

[Dev Biol, 2014]

The PAF1 complex (PAF1C) is an evolutionarily conserved protein complex involved in transcriptional regulation and chromatin remodeling. How the PAF1C is involved in animal development is still not well understood. Here, we report that, in the nematode Caenorhabditis elegans, the PAF1C is involved in epidermal morphogenesis in late embryogenesis. From an RNAi screen we identified the C. elegans ortholog of a component of the PAF1C, CTR-9, as a gene whose depletion caused various defects during embryonic epidermal morphogenesis, including epidermal cell positioning, ventral enclosure and epidermal elongation. RNAi of orthologs of other four components of the PAF1C (PAFO-1, LEO-1, CDC-73 and RTFO-1) caused similar epidermal defects. In these embryos, whereas the number and cell fate determination of epidermal cells were apparently unaffected, their position and shape were severely disorganized. PAFO-1::mCherry, mCherry::LEO-1 and GFP::RTFO-1 driven by the authentic promoters were detected in the nuclei of a wide range of cells. Nuclear localization of GFP::RTFO-1 was independent of other PAF1C components, while PAFO-1::mCherry and mCherry::LEO-1 dependent on other components except RTFO-1. Epidermis-specific expression of mCherry::LEO-1 rescued embryonic lethality of the leo-1 deletion mutant. Thus, although the PAF1C is universally expressed in C. elegans embryos, its epidermal function is crucial for the viability of this animal.

Kubota Y et al. (2004) Curr Biol "A fibulin-1 homolog interacts with an ADAM protease that controls cell migration in C. elegans."

Kuroki R, Nishiwaki K, Kubota Y

[Curr Biol, 2004]

ADAM (a disintegrin and metalloprotease) family proteins play important roles in animal development and pathogenesis. In C. elegans, a secreted ADAM protein, MIG-17, acts from outside the gonad to control the migration of gonadal distal tip cells (DTCs) that promote gonad morphogenesis. Here, we report that dominant mutations in the fbl-1 gene encoding fibulin-1 spliced isoforms, which are calcium binding extracellular matrix proteins, bypass the requirement for MIG-17 activity in directing DTC migration. Specific amino acid substitutions in the third EGF-like motif of one of the two isoforms, FBL-1C, which corresponds to mammalian fibulin-1C, suppress mig-17 mutations. FBL-1C is synthesized in the gut cells and localizes strongly to the gonadal basement membrane in a MIG-17-dependent manner. Localization of mutant FBL-1C is weaker than that of the wild-type protein and is insensitive to MIG-17 activity, suggesting that it gains a novel function that compensates for its reduced molecular density. We propose that proteolysis by MIG-17 recruits FBL-1C to the gonadal basement membrane, where it is required for the guidance of DTCs, and that mutant FBL-1C acts in a manner that mimics the downstream events of MIG-17-mediated proteolysis.

Kubota Y et al. (2006) Development "The conserved oligomeric Golgi complex acts in organ morphogenesis via glycosylation of an ADAM protease in C. elegans."

Goda S, Suzuki N, Sano M, Kubota Y, Nishiwaki K

[Development, 2006]

In C. elegans, the gonad acquires two U-shaped arms through directed migration of gonadal distal tip cells (DTCs). A member of the ADAM (a disintegrin and metalloprotease) family, MIG-17, is secreted from muscle cells and localizes to the gonadal basement membrane where it functions in DTC migration. Mutations in cogc-3 and cogc-1 cause misdirected DTC migration similar to that seen in mig-17 mutants. Here, we report that COGC-3 and COGC-1 proteins are homologous to mammalian COG-3/Sec34 and COG-1/ldlBp, respectively, two of the eight components of the conserved oligomeric Golgi (COG) complex required for Golgi function. Knockdown of any of the other six components by RNA interference also produces DTC migration defects, suggesting that the eight components function in a common pathway. COGC-3 and COGC-1 are required for the glycosylation and gonadal localization of MIG-17, but not for secretion of MIG-17 from muscle cells. Furthermore, COGC-3 requires MIG-17 activity for its action in DTC migration. Our findings demonstrate that COG complex-dependent glycosylation of an ADAM protease plays a crucial role in determining organ shape.

Kubota Y et al. (2012) Genetics "Tissue architecture in the Caenorhabditis elegans gonad depends on interactions among fibulin-1, type IV collagen and the ADAMTS extracellular protease."

Kubota Y, Nagata K, Sugimoto A, Nishiwaki K

[Genetics, 2012]

Molecules in the extracellular matrix (ECM) regulate cellular behavior in both development and pathology. Fibulin-1 is a conserved ECM protein. The Caenorhabditis elegans ortholog, FBL-1, regulates gonad-arm elongation and expansion by acting antagonistically to GON-1, an ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family protease. The elongation of gonad arms is directed by gonadal distal tip cells (DTCs). Here we report that a dominant mutation in the EMB-9/type IV collagen 1 subunit can compensate for loss of FBL-1 activity in gonadogenesis. A specific amino acid substitution in the noncollagenous 1 (NC1) domain of EMB-9 suppressed the fbl-1 null mutant. FBL-1 was required to maintain wild-type EMB-9 in the basement membrane (BM), whereas mutant EMB-9 was retained in the absence of FBL-1. EMB-9 (either wild type or mutant) localization in the BM enhanced PAT-3/-integrin expression in DTCs. In addition, overexpression of PAT-3 partially rescued the DTC migration defects in fbl-1 mutants, suggesting that EMB-9 acts in part through PAT-3 to control DTC migration. In contrast to the suppression of fbl-1(tk45), mutant EMB-9 enhanced the gonadal defects of gon-1(e1254), suggesting that it gained a function similar to that of wild-type FBL-1, which promotes DTC migration by inhibiting GON-1. We propose that FBL-1 and GON-1 control EMB-9 accumulation in the BM and promote PAT-3 expression to control DTC migration.

Kubota Y et al. (2019) Genetics "The Role of Tissue Inhibitors of Metalloproteinases in Organ Development and Regulation of ADAMTS Family Metalloproteinases in Caenorhabditis elegans."

Ito M, Nishiwaki K, Sugimoto A, Kubota Y

[Genetics, 2019]

Remodeling of the extracellular matrix supports tissue and organ development by regulating cellular morphology and tissue integrity. However, proper extracellular matrix remodeling requires spatiotemporal regulation of extracellular metalloproteinase activity. Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family, including MIG-17 and GON-1, are evolutionarily conserved, secreted zinc-requiring metalloproteinases. Although these proteases are required for extracellular matrix remodeling during gonadogenesis in <i>Caenorhabditis</i><i>elegans</i>, their <i>in vivo</i> regulatory mechanisms remain to be delineated. Therefore, we focused on the <i>C. elegans</i> tissue inhibitors of metalloproteinases, TIMP-1 and CRI-2. Analysis of the transcription and translation products for GFP/Venus fusions with TIMP-1 or CRI-2 indicated that these inhibitors were secreted and localized to the basement membrane of gonads and the plasma membrane of germ cells. A <i>timp-1</i> deletion mutant exhibited gonadal growth defects and sterility, and the phenotypes of this mutant were fully rescued by a TIMP-1::Venus construct but not by a TIMP-1(C21S)::Venus mutant construct, in which the inhibitor coding sequence had been mutated. Moreover, genetic data suggested that TIMP-1 negatively regulates proteolysis of the 1-chain of type-IV collagen. We also found that the loss of function observed for the mutants <i>timp-1</i> and <i>cri-2</i> involves a partial suppression of gonadal defects found for the mutants <i>mig-17/ADAMTS</i> and <i>gon-1/ADAMTS</i>, and this suppression was canceled upon overexpression of <i>gon-1</i> or <i>mig-17</i>, respectively. Based on these results, we propose that both TIMP-1 and CRI-2 act as inhibitors of MIG-17 and GON-1 ADAMTSs to regulate gonad development in a non-cell-autonomous manner.

Kubota, Y. et al. (2017) International Worm Meeting "Tissue inhibitors of metalloproteinases (TIMPs) genetically interact with ADAMTS proteases and regulate gonadal development in C. elegans."

Nishiwaki, K., Sugimoto, A., Kubota, Y.

[International Worm Meeting, 2017]

During development, extracellular matrix (ECM) supports proper development of tissues and organs by regulating cellular behaviors and tissue integrity. For proper ECM remodeling, spatiotemporal regulation of matrix metalloproteinase (matrix protease) activity is required. The ADAMTS family proteases are evolutionally conserved secreted zinc matrix proteases. In C. elegans, ADAMTS proteases, GON-1 and MIG-17 are required for gonadal morphogenesis (Blelloch R and Kimble J, 1999; Nishiwaki K et al, 2000). However, regulatory mechanisms of ADAMTS proteases in vivo remain to be explored. Here, we focus on tissue inhibitors of metalloproteinases (TIMPs) that are small secretory proteins with a netrin domain, which have been shown to negatively regulate ADAMTS proteases in mammalian in vitro studies. The C. elegans genome encodes two TIMPs, K07C11.3/TIMP-1 and K07C11.5/CRI-2. Transcriptional and translational GFP/Venus fusion analyses indicated that TIMP-1 and CRI-2 were secreted and localized to the gonadal basement membrane and plasma membrane of the germ cells. The timp-1(tk71) deletion mutant, but not the cri-2(gk314) deletion mutant exhibited gonadal growth defects and sterility. These phenotypes of the timp-1(tk71) mutant was almost completely rescued by TIMP-1::Venus but not by TIMP-1(C21S)::Venus in which an essential Cys residue for inhibitor activity was substituted by Ser, suggesting that the protease inhibitor function of TIMP-1 is required for gonadogenesis and germ cell proliferation. RNAi knockdown and mutations of the timp-1 and cri-2 partially suppressed gonadal morphogenesis defects of the gon-1/ADAMTS and mig-17/ADAMTS mutants. In timp-1(tk71) mutant, localization of mCherry-tagged EMB-9, a1 chain type IV collagen, in the gonadal basement membrane was significantly decreased, and EMB-9::mCherry overexpression partially rescued the gonadal defects of the timp-1(tk71) mutant. Based on these observations, we propose that C. elegans TIMPs, TIMP-1 and CRI-2 act as negative regulators of the ADAMTS proteases to regulate basement membrane remodeling and gonadal development.

Kubota Y et al. (2008) Proc Natl Acad Sci U S A "MIG-17/ADAMTS controls cell migration by recruiting nidogen to the basement membrane in C. elegans."

Kubota Y, Nishiwaki K, Nagata K, Tamai KK, Ohkura K

[Proc Natl Acad Sci U S A, 2008]

Mutations in the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) family of secreted proteases cause diseases linked to ECM abnormalities. However, the mechanisms by which these enzymes modulate the ECM during development are mostly unexplored. The Caenorhabditis elegans MIG-17/ADAMTS protein is secreted from body wall muscle cells and localizes to the basement membrane (BM) of the developing gonad where it controls directional migration of gonadal leader cells. Here we show that specific amino acid changes in the ECM proteins fibulin-1C (FBL-1C) and type IV collagen (LET-2) result in bypass of the requirement for MIG-17 activity in gonadal leader cell migration in a nidogen (NID-1)-dependent and -independent manner, respectively. The MIG-17, FBL-1C and LET-2 activities are required for proper accumulation of NID-1 at the gonadal BM. However, mutant FBL-1C or LET-2 in the absence of MIG-17 promotes NID-1 localization. Furthermore, overexpression of NID-1 in mig-17 mutants substantially rescues leader cell migration defects. These results suggest that functional interactions among BM molecules are important for MIG-17 control of gonadal leader cell migration. We propose that FBL-1C and LET-2 act downstream of MIG-17-dependent proteolysis to recruit NID-1 and that LET-2 also activates a NID-1-independent pathway, thereby inducing the remodeling of the BM required for directional control of leader cell migration.

Kubota Y et al. (2021) Genes Genomics "Overlapping and non-overlapping roles of the class-I histone deacetylase-1 corepressors LET-418, SIN-3, and SPR-1 in Caenorhabditis elegans embryonic development."

Kubota Y, Hamasaki T, Fahmi M, Kitagawa H, Ohnishi Y, Ito M, Ota N, Esaki A, Yasui G

[Genes Genomics, 2021]

BACKGROUND: Histone deacetylase (HDAC)-1, a Class-I HDAC family member, forms three types of complexes, the nucleosome remodeling deacetylase, Sin3, and CoREST complexes with the specific corepressor components chromodomain-helicase-DNA-binding protein 3 (Mi2/CHD-3), Sin3, and REST corepressor 1 (RCOR1), respectively, in humans. OBJECTIVE: To elucidate the functional relationships among the three transcriptional corepressors during embryogenesis. METHODS: The activities of HDA-1, LET-418, SIN-3, and SPR-1, the homologs of HDAC-1, Mi2, Sin3, and RCOR1 in Caenorhabditis elegans during embryogenesis were investigated through measurement of relative mRNA expression levels and embryonic lethality given either gene knockdown or deletion. Additionally, the terminal phenotypes of each knockdown and mutant embryo were observed using a differential-interference contrast microscope. Finally, the functional relationships among the three corepressors were examined through genetic interactions and transcriptome analyses. RESULTS: Here, we report that each of the corepressors LET-418, SIN-3, and SPR-1 are expressed and have essential roles in C. elegans embryonic development. Our terminal phenotype observations of single mutants further implied that LET-418, SIN-3, and SPR-1 play similar roles in promoting advancement to the middle and late embryonic stages. Combined analysis of genetic interactions and gene ontology of these corepressors indicate a prominent overlapping role among SIN-3, SPR-1, and LET-418 and between SIN-3 and SPR-1. CONCLUSION: Our findings suggest that the class-I HDAC-1 corepressors LET-418, SIN-3, and SPR-1 may cooperatively regulate the expression levels of some genes during C. elegans embryogenesis or may have some similar roles but functioning independently within a specific cell.