Thurtle, Deborah et al. (2015) International Worm Meeting "Insulation of Nuclear Receptor Gene Regulation by Boundary Effectors."

Thurtle, Deborah, Yamamoto, Keith

[International Worm Meeting, 2015]

Cellular identity is established and maintained through proper genetic regulation by transcriptional regulatory factors. Transcriptional regulatory factors bind to regulatory elements to modulate the expression of cognate target genes. Regulating only the appropriate target gene is integral to the fidelity of the cell's transcriptional regulatory network, yet how a transcriptional regulatory factor regulates only the appropriate target gene is not well understood. We are exploiting the compact genome of C. elegans, which likely necessitates very efficient mechanisms to insulate a transcriptional regulatory factor's influence due to the close proximity of response elements to multiple promoters, to examine the mechanism of transcriptional regulatory factor insulation. We have performed RNA-Seq of wildtype (N2), nhr-25(RNAi) and an nhr-25 hypomorphic mutant, ku217, to determine the nhr-25 dependent transcriptome in L4 animals. Analysis of this RNA-Seq data in combination with the NHR-25 ChIP-Seq dataset from modencode (Araya et al. 2014) has revealed several pairs of divergently transcribed genes in which one gene is regulated by NHR-25 and the other gene is not. We are developing a boundary reporter assay using fluorescent reporters, and will utilize this assay to screen for boundary effectors. .

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.

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).

Stefan Taubert et al. (2004) West Coast Worm Meeting "Biochemical Characterization of the C. elegans Nuclear Hormone Receptor NHR-49"

Keith R Yamamoto, Stefan Taubert, Marc Van Gilst

[West Coast Worm Meeting, 2004]

Nuclear Hormone receptors (NHRs) are a family of ligand-regulated transcription factors involved in diverse metazoan functions, such as sexual differentiation, organ development, and regulation of metabolism. The C. elegans genome contains over 270 NHR genes, approximately five times as many as the human genome. For most of these no function has been described yet. We focus on characterizing NHR-49, which regulates fat metabolism, and lifespan (Marc van Gilst and Keith R. Yamamoto, unpublished). Interestingly, this mirrors the functions of human Peroxisome-proliferator-activated-receptor a (PPARalpha), while the sequence of NHR-49 suggests similarity to another human receptor, Hepatocye Nuclear Receptor 4alpha (HNF4alpha). Since HNF4alpha dimerizes via its ligand-binding-domain (LBD), we utilized yeast two-hybrid based technology to assess whether the LBDs of NHR-49 and 12 related HNF4alpha-like NHRs, can also homodimerize. While several of these LBDs readily dimerized (including NHR-49), some others did not (such as DAF-12). Since the homodimerization of NHR-49's LBD is predicted to occur via two conserved salt-bridges (E5/K55 and E50/R93), we mutated the E50 and K55 residues to alanines. We will present the effect of these mutations on LBD homodimerization and activation of a transcriptional reporter in yeast. In an attempt to identify ligands of NHR-49 we treated worms with clofibrate, a synthetic PPARa ligand used to treat hyperlipidemia. This treatment caused a strong reduction of worm body fat levels, and was accompanied by lethality at higher concentrations. However, similar effects were observed in a strain harboring an NHR-49 deletion, suggesting that the effects of clofibrate are independent of NHR-49. We are currently characterizing changes in the transcriptional program in response to clofibrate treatment.

Ratnappan, Ramesh et al. (2013) International Worm Meeting "Molecular Outsourcing: Reproductive Signals Deploy NHR-49/PPARa to Reorganize Lipid Homeostasis and Alter Lifespan."

Ratnappan, Ramesh, Ghazi, Arjumand, Gill, Hasreet, Ward, Jordan, Holden, Kyle, RG Amrit, Francis, Yamamoto, Keith

[International Worm Meeting, 2013]

Aging is an inherently entropic but remarkably plastic phenomenon. The rate of aging can be altered by modifications to an energy-intensive process such as reproduction. In worms, removal of the Germline-Stem Cells (GSCs) results in increased fat accumulation and the activation of a transcriptional network in intestinal cells that extends lifespan. Two key regulators in this network are DAF-16/FOXO and TCER-1/TCERG1. In a screen designed to identify nuclear hormone receptors (NHRs) that impact DAF-16/FOXO and TCER-1/TCERG1 function, one of the NHRs we isolated was NHR-49/PPARa, a conserved and crucial modulator of energy and fat metabolism. Independently, we also identified nhr-49 as a gene jointly upregulated by DAF-16/FOXO and TCER-1/TCERG1 through RNA-Seq transcriptomics. Predictably, nhr-49 mutation suppressed the longevity of GSC(-) worms, but did not appear to change the overall elevated fat content. A GFP-tagged translational reporter showed expression in the cytoplasm and nuclei of all somatic cells and rescued nhr-49(-) phenotypes. NHR-49/PPARa overexpression increased the lifespan of nhr-49 mutants substantially without a concomitant loss of fertility. NHR-49/PPARa regulated the expression of fatty-acid desaturases, specific mitochondrial b-oxidation pathway genes and acted in a feedback loop to increase DAF-16/FOXO and TCER-1/TCERG1 activity. At least part of these transcriptional changes may be brought about in co-operation with NHR-71, another NHR identified in our screen that interacted with both NHR-49/PPARa and TCER-1/TCERG1 in yeast two-hybrid experiments. Our results suggest that upon GSC loss, NHR-49/PPARa is recruited to fundamentally remodel the lipid metabolic profile of the worm. This reorganization not only allows the mobilization of fat that would otherwise have been transported to the oocytes, but also induces transcriptional and signaling events that direct the allotment of cellular resources towards somatic maintenance and longevity.

S Taubert et al. (2004) European Worm Meeting "CHARACTERIZATION OF THE C. ELEGANS NUCLEAR HORMONE RECEPTOR NHR-49"

S Taubert, K Yamamoto, M Van Gilst

[European Worm Meeting, 2004]

Nuclear Hormone receptors (NHRs) are a family of ligand-regulated transcription factors involved in diverse metazoan functions, such as sexual differentiation, organ development, and regulation of metabolism. The C. elegans genome contains over 270 NHR genes, approximately five times as many as the human genome. For most of these no function has been described yet. We focus on characterizing NHR-49, which regulates fat metabolism, and lifespan (Marc van Gilst and Keith R. Yamamoto, unpublished). Interestingly, this mirrors the functions of human Peroxisome-proliferator-activated-receptor a (PPARa), while the sequence of NHR-49 suggests similarity to another human receptor, Hepatocye Nuclear Receptor 4a (HNF4a). Since HNF4a dimerizes via its ligand-binding-domain (LBD), we utilized yeast two-hybrid based technology to assess whether the LBDs of NHR-49 and 12 related HNF4a-like NHRs, can also homodimerize. While several of these LBDs readily dimerized (including NHR-49), some others did not (such as DAF-12). Since the homodimerization of NHR-49's LBD is predicted to occur via two conserved salt-bridges (E5/K55 and E50/R93), we mutated the E50 and K55 residues to alanines. We will present the effect of these mutations on LBD homodimerization and activation of a transcriptional reporter in yeast. In an attempt to identify ligands of NHR-49 we treated worms with clofibrate, a synthetic PPARa ligand used to treat hyperlipidemia. This treatment caused a strong reduction of worm body fat levels, and was accompanied by lethality at higher concentrations. However, similar effects were observed in a strain harboring an NHR-49 deletion, suggesting that the effects of clofibrate are independent of NHR-49. We are currently characterizing changes in the transcriptional program in response to clofibrate treatment.

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.

Arda, H Efsun et al. (2009) International Worm Meeting "An Intricate Network of Nuclear Hormone Receptors Regulates Energy Balance in the Nematode C. elegans."

Doucette-Stamm, Lynn, Walhout, Albertha JM, Conine, Colin, Sequerra, Reynaldo, Taubert, Stefan, van Gilst, Marc, Yamamoto, Keith, Arda, H Efsun

[International Worm Meeting, 2009]

Obesity is a growing epidemic and results from an imbalanced energy homeostasis. Most metabolic genes are transcriptionally regulated, therefore deciphering the complex regulatory networks that govern energy homeostasis is crucial to understand the mechanisms underlying obesity. The nematode C. elegans is a powerful model organism to study metabolic transcriptional regulatory networks. Already ~400 genes have been identified that alter the body fat of the worm when inactivated, and an additional ~25 genes were shown to give transcriptional response upon fasting. In order to elucidate the transcriptional regulation of these "fat" and "fasting" genes, we mapped a transcription regulatory network using high-throughput, gene-centered yeast one-hybrid assays. This network contains numerous nuclear hormone receptor (NHR) type transcription factors, which are involved in fat metabolism throughout the Metazoa. We found that most of these previously uncharacterized NHRs partition into different modules based on the targets they share in the network. We show that several of them are necessary to maintain normal levels of body fat. Finally, we also found that many of the transcription factors identified in the fat network can interact with a mediator subunit, MDT-15, which is required for the regulation of fat metabolism both in C. elegans and mammalian systems. Taken together, our results provide an integrated network of transcriptional regulation of energy balance of C. elegans.