Masako ASAHINA et al. (2002) European Worm Meeting "Expression and function of ftz-f1/nhr-25 in Caenorhabditis elegans"

Susumu Hirose, Marie SILHANKOVA, Marek JINDRA, Masako ASAHINA

[European Worm Meeting, 2002]

FTZ-F1 is one of the ancient nuclear receptors and its ortholog is termed NHR-25 in C. elegans. The important roles of this molecule were identified genetically in mice (differentiation of primary steroidogenic glands and synthesis of steroid hormones) and in Drosophila melanogaster (embryonic segmentation, larval molting, metamorphosis). nhr-25/ftz-f1 mRNA expression was detected by in situ hybridization throughout embryonic development. Zygotic expression was seen particularly in the gut and epidermal precursor cells. Epidermal expression persisted until L2 stage, when the developing gonadal cells took over the expression. Adult gonads were loaded with the nhr-25 transcript (Asahina et al. 2000). Activities of transgenic nhr-25::gfp constructs mirrored the embryonic and the larval epidermal mRNA pattern. Two nhr-25::gfp constructs showed different expression profiles, indicating elements for stage specific regulation of nhr-25. We have previously isolated an nhr-25 deletion mutant (jm2389) which is recessive embryonic lethal at the 1.5-fold stage (the time of cuticle synthesis and elongation of the embryo). To address a possible NHR-25/FTZ-F1 role in postembryonic stages, we performed RNA interference by injection. RNAi confirmed the embryonic lethality and in addition showed molting defects at L1 and L2 (Asahina et al. 2000; Gissendanner and Sluder, 2000), and gonadal defects from L3 to adult stage. The gonad was typically tumorous, misshapen and undifferentiated. These worms were vulvaless and sterile as adults, and showed abnormal cuticle. To resolve temporal requirements for nhr-25, we applied RNAi also via feeding and soaking of larval stages. The prolonged exposure to dsRNA resulted in pronounced late phenotypes (Vul) and newly observed L4 to adult molting defects. These results suggest that NHR-25/FTZ-F1 acts in a stage specific manner in different tissues. Finally, we wanted to exclude the possibility of non-specific RNAi effects due to the conservation of the zinc finger DNA binding domain (DBD). We found no difference whether RNAi was performed with dsRNA including or lacking the DBD region. A polyclonal antibody was raised against recombinant bacterial NHR-25. It recognized a single band on immunoblots of worm extracts. Immunocytochemistry performed on gonads showed striking NHR-25 expression in the nuclei of the adult somatic gonadal cells, namely the distal tip cells (DTC) and the sheath cells. These cells are known to be important for the gonadal guidance and the differentiation of the germline, both processes dependent on NHR-25. NHR-25 expression and loss-of-function phenotypes are in a good correlation and suggest various important roles of NHR-25 throughout the worm development.

Masako ASAHINA et al. (2003) International Worm Meeting "A highly conserved yet non-essential coactivator MBF1 may play a role in oxidative stress in C. elegans"

Masako ASAHINA, Marek JINDRA, Susumu Hirose

[International Worm Meeting, 2003]

A small helix-turn-helix protein MBF1 has been maintained throughout the evolution of eukaryotes. Our previous genetic evidence from yeast and Drosophila implicates MBF1 in transcriptional activation mediated by bZIP transcription factors [1,2]. To understand the role of a C. elegans homolog of mbf1, we began to analyze its expression and function. The expression was visualized using an C. elegans mbf1(Cembf1)::GFP reporter and a polyclonal antibody against the Drosophila protein, revealing constitutive and ubiquitous presence of the gene product. Deletions of the transgenic GFP fusion construct showed that a relatively short (223 bp) upstream region was sufficient for Cembf1 expression. Functional conservation of mbf1 was tested in yeast (S. cerevisiae) mbf1 disruptant that is viable but sensitive to histidine starvation [1]. The growth of the mbf1 mutant yeast was partially restored by transformation with the C. elegans mbf1 homolog, indicating that the mechanism of MBF1 action could be conserved from unicellular to multicellular organisms. RNA interference silencing of mbf1 in C. elegans showed fairly wild type phenotype although the Cembf1 dsRNA effectively suppressed GFP in the Cembf1::GFP transgenic worms. This suggests that, as in yeast and flies, mbf1 is not an essential gene in C. elegans. The question then arises of what makes mbf1 important enough to be conserved during evolution. Our preliminary data show that the mbf1 RNAi treated worms are more sensitive to hydrogen peroxide, implying that MBF1 is required to withstand oxidative stress. A microarray analysis is underway to find what genes might be affected in mbf1 RNAi background during the hydrogen peroxide challenge. [1] Takemaru K, Harashima S, Ueda H and Hirose S. (1998) Mol. Cell Biol. 18, 4971-76 [2] Liu QX, Jindra M, Ueda H, Hiromi Y, Hirose S. (2003) Development 130, 719-28

Masako Asahina et al. (2006) Development & Evolution Meeting "Crosstalk between Nuclear Receptor NHR-25 and β-Catenin Signaling Decides Cell Fates in the C. elegans Somatic Gonad"

Tomas Valenta, Masako ASAHINA, Vladimir Korinek, Marek JINDRA, Marie SILHANKOVA

[Development & Evolution Meeting, 2006]

Recent findings indicate that nuclear receptor and Wnt/β-catenin signaling pathways intersect during important processes such as cell differentiation, proliferation or malignant growth. In the somatic gonad precursors (SGPs) of Caenorhabditis elegans, a β-catenin pathway plays a crucial role in the proximal-distal cell fate decision. The Wnt nuclear effector TCF/POP-1 and β-catenins WRM-1 and SYS-1 are the key molecules for this decision. Impaired function of all of the respective genes leads to a Sys (Symmetrical sisters) phenotype, when all SGPs adopt the same, proximal fate. Thus, no distal tip cells (DTCs) that would lead germline differentiation and elongation of the gonadal arms are formed in hermaphrodites. Here, we show that loss of the nuclear receptor NHR-25 (a homolog of SF-1, LRH-1 and Ftz-F1) causes an extra DTC phenotype, where up to four DTCs form at the expense of the proximally fated anchor cell. This “all-distal” Sys phenotype is thus opposite to known loss-of-function phenotypes of genes in the β-catenin pathway. Our data show that a balance between the β-catenin/POP-1 activity and the action of NHR-25 is required for the proper establishment of both the distal and proximal cell fates. pop-1(q645) mutants that never form DTCs and consequently lack gonadal arms develop DTCs, extend the gonadal arms, and become fertile when nhr-25 is silenced in them. By using cell transfection techniques, co-immunoprecipitation, yeast two-hybrid and GST pulldown assays, we show that this interaction is coordinated by mutual modulation of NHR-25 and POP-1 activities through direct contacts of NHR-25 with the distinct β-catenins WRM-1 and SYS-1. The crosstalk between nuclear receptor and β-catenin signaling thus establishes the proper axis of the entire organ. The C. elegans gonad can serve as a unique example of how nuclear receptor and β-catenin signaling pathways intersect to govern fate decision of specific cells. Supported by projects Z60220518, Czech Acad. Sci., and 6007665801 from the Czech Ministry of Education.

Masako Asahina et al. (2006) European Worm Meeting "Interplay Between the Nuclear Receptor NHR-25 and ?-Catenin Signaling during Cell Fate Decision in the Gonad of C. elegans"

Marek JINDRA, Masako ASAHINA, Marie SILHANKOVA, Vladimir Korinek, Tomas Valenta

[European Worm Meeting, 2006]

Interplay Between the Nuclear Receptor NHR-25 and ?-Catenin Signaling during Cell Fate Decision in the Gonad of C. elegans. Masako Asahina, Tomas Valenta3, Marie Silhankova2, Vladimir Korinek3 and Marek Jindra. Molecular mechanisms that specify cell fates during asymmetric cell divisions are critical for differentiation. In the somatic gonad precursors (SGPs) of Caenorhabditis elegans, a ?-catenin pathway plays a crucial role in the proximal-distal fate decision. The Wnt nuclear effector TCF/POP-1 and ?-catenins WRM-1 and SYS-1 are the key molecules for this decision. Impaired function of all of these genes leads to a Sys (Symmetrical sisters) phenotype, when all SGPs adopt the same, proximal fate. Thus, no distal tip cells (DTCs) that would lead germline differentiation and elongation of the gonadal arms are formed in hermaphrodites. Here, we show that a loss of the nuclear receptor NHR-25 (a homolog of SF-1 and Ftz-F1) causes an extra DTC phenotype, where up to four DTCs form at the expense of proximally fated the anchor cell. This all-distal Sys phenotype is thus opposite to known loss-of-function phenotypes of genes in the ?-catenin pathway. Our data show that a balance between the ?-catenin/POP-1 activity and the action of NHR-25 is required for the proper establishment of both the distal and proximal cell fates. pop-1(q645) mutants that never form DTCs and consequently lack gonadal arms develop DTCs, extend the gonadal arms, and become fertile when nhr-25 is silenced in them. By using cell transfection techniques, co-immunoprecipitation and yeast two-hybrid assays, we show that this interaction is coordinated by mutual modulation of NHR-25 and POP-1 activities through direct contacts of NHR-25 with the distinct ?-catenins WRM-1 and SYS-1. The crosstalk between nuclear receptor and ?-catenin signaling thus establishes the proper axis of the entire organ.. Supported by projects Z60220518, Czech Acad. Sci., and 6007665801 from the Czech Ministry of Education.

Bojanala, Nagagireesh et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Regulation of the nuclear receptor NHR-25 by sumoylation during C. elegans vulva formation"

Ward, Jordan, Yamamoto, Keith, Bojanala, Nagagireesh, Jindra, Marek, Asahina, Masako

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

During C. elegans development a member of the NR5A family of nuclear receptors, NHR-25, coordinates diverse developmental events such as molting, epidermal cell differentiation, dauer formation, cell fate decisions in the somatic gonad and seam cells, and the formation of vulva. Therefore, the activity of NHR-25 must be tightly regulated both temporally and spatially. Posttranslational modifications of transcription factors play significant biological roles in various organisms, yet to date little is known abou t the posttranslational regulation of the vast number (284) of C. elegans nuclear receptor genes. Studies of the mammalian homolog of NHR-25 (SF-1), revealed sumoylation as an important mechanism regulating its transcriptional activity and we find that NHR-25 contains well-conserved SUMO binding consensus. Deletion of smo-1, the single SUMO gene in C. elegans, causes severe protruding vulva (Pvl) and weak multivulva (Muv) phenotypes. Here we show that the depletion of NHR-25 by RNAi in smo-1 (ok359) worms enhances Muv induction of P3.p, P4.p and P8.p vulval precursor cells (VPCs) similar to synthetic multivulva (SynMuv) genes. Interestingly, daughters of P(5-7).p VPCs are also affected and cannot complete the full program of vulval cell divisions, thereby suppressing the smo-1 Pvl phenotype. Indeed, yeast-two-hybrid assay revealed NHR-25 binds to SMO-1. Taken together, our observations strongly suggest that SMO-1 and NHR-25 function together both physically and genetically to promote proper vulval induction and cell division. These data provide an intriguing mechanism how the sumoylation of nuclear receptors could fine-tune target gene regulation and thus ensure proper development of the organism. Supported by GACR 204/09/H058, 204/07/0948, TFF postdoctoral fellowship 700046, NIH grant CA020535.

Thurtle-Schmidt, Deborah et al. (2019) International Worm Meeting "Dissection of the NHR-25 transcriptional landscape reveals distinct roles for the regulatory element and transcription factor in target gene regulation."

Ward, Jordan, Thurtle-Schmidt, Deborah, Asahina, Masako, Sinks, Alexander, Yamamoto, Keith

[International Worm Meeting, 2019]

Transcription factors bind to genomic regulatory elements to direct precise gene networks, which are necessary for establishment and maintenance of cellular identity. Regulating only the appropriate target gene is integral to maintaining the cell's transcriptional network, yet how a transcription factor regulates only the appropriate target gene is not well understood. Due to its compact yet complex metazoan genome, C. elegans is an ideal model to investigate how transcription factors identify and regulate their target genes. To elucidate mechanisms of transcription factor regulation, we have profiled the genome-wide binding of an endogenously tagged transcription factor, NHR-25 in L1s. This analysis revealed one-quarter as many peaks as compared to the low-copy NHR-25 modENCODE integrant, suggesting that transcription factor binding is highly dependent on expression level. Motif analysis of the NHR-25 enriched regions identified the NR5A motif, confirming that NHR-25 likely binds this conserved motif in vivo. Additionally, twenty total motifs were enriched at NHR-25 peaks, including EOR-1 and an unknown AT-rich motif, implying that regulatory elements bind combinations of transcription factors to direct gene expression. To identify NHR-25 responsive genes and correlate target genes to NHR-25 enriched regulatory elements, we profiled gene expression in worms with mutant or knocked down nhr-25. Interestingly, many NHR-25 occupied regions were not adjacent to NHR-25 responsive genes. To directly correlate target genes of identified regulatory elements, we deleted NHR-25 enriched regions using CRISPR/Cas9 and profiled gene expression by RNA-seq. For a single deleted regulatory element, we identified two nearby genes with altered expression. Interestingly, only one of these adjacent genes showed NHR-25 dependent expression. Our data suggests that a single regulatory element can confer distinct patterns of regulation on multiple target genes through differential actions of transcription factor complexes assembled on the element. Through the deletion and mutation of complementary cis and trans genome regulators, this study reveals basic mechanisms as to how transcription factors complexes identify and regulate their target genes.

Niwa, Ryusuke et al. (2009) International Worm Meeting "The nuclear hormone receptor nhr-25 is a heterochronic gene that has dual roles in both promoting and inhibiting C. elegans adult programs."

Niwa, Ryusuke, Hasegawa, Hiroshi, Slack, Frank, Hada, Kazumasa, Kanaho, Yasunori, Asahina, Masako

[International Worm Meeting, 2009]

Developmental timing in C. elegans is controlled by heterochronic genes, mutations in which cause changes in the relative timing of developmental events. During the fourth larval stage to adult transition, the lateral hypodermal seam cells exit the cell cycle, fuse and secrete an adult specific cuticle called lateral alae. The timing of this switch is under the control of a growing set of genes including let-7, a founder member of microRNAs (miRNAs). The let-7 family miRNAs and their targets are evolutionally conserved in many animals, suggesting that similar genetic pathways control developmental timing across phylogeny. Recently we have reported that the expression in seam cells of apl-1, a homolog of Alzheimer''s amyloid precursor protein gene, is regulated by the let-7 family miRNAs and their target heterochronic genes, such as hbl-1 and lin-41 (Niwa et al. Dev. Biol. 315: 418-425, 2008). We proposed that the expression analysis of apl-1 in seam cells is a new approach to elucidate the let-7-dependent terminal differentiation pathway in C. elegans. Here we report that the apl-1 expression analysis reveals the nuclear hormone receptor gene nhr-25 as a novel heterochronic gene. During the course of an RNAi-based loss-of-function screen, we found that nhr-25 positively regulated apl-1 expression in seam cells. Similar to hbl-1 and lin-41, nhr-25 controlled not only the temporal apl-1 expression but also other let-7-dependent developmental timing functions. In addition to the let-7- and nhr-25-directing the molting process (Heyes et al. Development 133: 4631-4641, 2006), we showed that nhr-25 also controlled adult-specific collagen gene col-19 expression, seam cell division, seam cell fusion and alae formation. Loss of nhr-25 function leaded to the precocious expression of the adult-specific collagen gene col-19 in the larval stage. However, interestingly, loss-of-nhr-25 function resulted in retarded phenotypes in seam cell division, seam cell fusion and alae formation. These results suggest that nhr-25 is a novel heterochronic gene that has dual roles in both promoting and inhibiting C. elegans adult programs.

Ward, Jordan D. et al. (2013) International Worm Meeting "Sumoylated NHR-25/NR5A regulates cell fate during C. elegans vulval development."

Asahina, Masako, Bernal, Teresita, Bojanala, Nagagireesh, Ashrafi, Kaveh, Ward, Jordan D., Yamamoto, Keith

[International Worm Meeting, 2013]

Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The exquisite cell and tissue specificity of gene regulation by a given TF appear to be achieved combinatorially, within multifactor regulatory complexes with distinct compositions and activities. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here we examine how a single regulatory input (sumoylation) differentially modulates the activity of the conserved C. elegans nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on three lysines. Genetic studies revealed that loss of smo-1 phenocopied NHR-25 overexpression, with respect to maintenance of the 3o cell fate in vulval precursor cells (VPCs) during development. Furthermore, in vivo overexpression using NHR-25 alleles that could not be sumoylated and SUMO-NHR-25 fusions indicated that NHR-25 sumoylation is critical for maintaining 3o cell fate. SUMO also regulated formation of an NHR-25 accumulation gradient in VPCs. Using an NHR-25::GFP translational reporter, we discovered that NHR-25 levels were uniform across VPCs at the beginning of development, but as cells began dividing a smo-1-dependent NHR-25 gradient formed with highest levels in 1o fated VPCs, lower levels in 2o fated VPCs, and the lowest levels in 3o fated VPCs. Our findings support a model where the ratio of unsumoylated to sumoylated NHR-25 regulates 3o cell fate determination and maintenance during vulval development. Supported by GAAV IAA500960906, MODBIOLIN 7FP-REGPOT-2012-2013-1, Terry Fox Foundation (#700046), CIHR (#234765) and the NIH (CA20535).

Ward, Jordan D. et al. (2011) International Worm Meeting "Sumoylation of C. elegans nuclear receptor NHR-25 promotes proper organogenesis."

Jindra, Marek, Asahina, Masako, Ward, Jordan D., Bojanala, Nagagireesh, Ashrafi, Kaveh, Yamamoto, Keith R., Bernal, Teresita

[International Worm Meeting, 2011]

Tissue-specific regulatory programs governed by nuclear receptors (NRs) are a critical component of metazoan development and homeostasis with aberrations in these programs leading to pathophysiology. We are using the simplicity and powerful genetics of C. elegans to deconvolute how regulatory inputs differentially converge on NRs in specific cell and tissue-types. The single C. elegans NR5A family receptor, NHR-25, coordinates diverse tissue-specific developmental events such as molting, seam cell differentiation, fat metabolism, cell fate decisions and vulva formation. To uncover novel co-regulators of NHR-25 activity we performed a genome-wide yeast two-hybrid (Y2H) screen that uncovered the C. elegans SUMO1 homolog (SMO-1) as an NHR-25 interacting protein. SUMO Y2H interactions can reflect both covalent sumoylation and non-covalent interaction; to distinguish between these possibilities we performed mutational analysis. Both deletion of the SMO-1 C-terminal di-glycine repeat and mutations in the SMO-1 beta sheet, which abrogate covalent sumoylation and non-covalent binding, respectively, prevented NHR-25 binding in Y2H assays. in vitro biochemical assays confirmed the sumoylation of NHR-25. We have identified three lysines in NHR-25 responsible for the interaction, as well as non-covalent Sumo interacting motifs (SIM) located in the ligand binding domain of NHR-25. These data argue that NHR-25 initially binds SMO-1 non-covalently in order to promote its sumoylation, similar to thymine DNA glycosylase sumoylation. The C. elegans vulva is a paradigm of organogenesis since the progression from 22 epithelial precursor cells to the seven toroidal disks in the completed organ involve a combination of reproducible cell divisions, migrations, remodeling of adherens junctions, cell fusions and muscle attachments. Deletion of the smo-1 gene causes severe protruding vulva (Pvl) and weak multivulva (Muv) phenotypes. When NHR-25 activity was reduced in a smo-1 mutant, Muv induction in P3.p, P4.p and P8.p was enhanced. Interestingly, daughters of P(5-7).p vulval precursor cells (VPCs) were also affected and could not complete the full program of vulval cell divisions, thereby suppressing the smo-1 Pvl phenotype. Our work suggests that SMO-1 and NHR-25 function together during VPC cell division in a cell context-dependent manner. These data highlight how the sumoylation of nuclear receptors fine-tunes target gene regulation and thus ensures proper organ development. Supported by GACR 204/09/H058, 204/07/0948, NPVII 2B06129, TFF postdoctoral fellowship 700046, NIH grant CA020535.

Ward, Jordan D. et al. (2011) International Worm Meeting "Vulval organogenesis involves an interaction between SMO-1 and the nuclear receptor NHR-25"

Asahina, Masako, Ashrafi, Kaveh, Ward, Jordan D., Jindra, Marek, Bernal, Teresita, Yamamoto, Keith R., Bojanala, Nagagireesh

[International Worm Meeting, 2011]

Organogenesis at the cellular level involves a complex interplay of cell fate acquisition, cell cycle control, remodeling of adherens junctions, cell migrations and cell fusions. Thus, this process is tightly regulated temporarily as well as spatially. Tissue-specific regulatory programs governed by nuclear receptors (NRs) are a critical component of metazoan development and homeostasis with aberrations in these programs leading to pathophysiology. We are using the simplicity and powerful genetics of C. elegans to deconvolute how regulatory inputs differentially converge on NRs in specific cell and tissue-types. The single C. elegans NR5A family receptor, NHR-25, coordinates diverse tissue-specific developmental events such as molting, seam cell differentiation, fat metabolism, cell fate decisions and vulva formation. To uncover novel co-regulators of NHR-25 activity we performed a genome-wide yeast two-hybrid (Y2H) screen that uncovered the C. elegans SUMO homolog (SMO-1) as an NHR-25 interacting protein. Both deletion of the SMO-1 C-terminal di-glycine repeat and mutations in the SMO-1 beta sheet, which abrogate covalent sumoylation and non-covalent binding, respectively, prevented NHR-25 binding in Y2H assays. in vitro biochemical assays confirmed the sumoylation of NHR- 25. We have identified three lysines in NHR-25 responsible for the interaction, as well as non-covalent SUMO interacting motifs (SIM) located in the ligand binding domain of NHR-25. These data argue that NHR-25 initially binds SMO-1 non-covalently in order to promote its sumoylation, similar to thymine DNA glycosylase sumoylation. Deletion of the smo-1 gene in C. elegans causes severe protruding vulva (Pvl) and weak multivulva (Muv) phenotypes. When NHR-25 activity was reduced in a smo-1 mutant, Muv induction in P3.p, P4.p and P8.p was enhanced. Interestingly, daughters of P(5-7).p vulval precursor cells (VPCs) were also affected and could not complete the full program of vulval cell divisions, thereby suppressing the smo-1 Pvl phenotype. Our work suggests that SMO-1 and NHR-25 function together during VPC cell division in a cell context-dependent manner. The C. elegans vulva is a paradigm of organogenesis and these data highlight how the sumoylation of nuclear receptors fine-tunes target gene regulation and thus ensures proper organ development. Supported by GACR 204/09/H058, 204/07/0948, NPVII 2B06129, TFF postdoctoral fellowship 700046, NIH grant CA020535.