Rottiers, Veerle et al. (2009) International Worm Meeting "Transketolase is a new target of SBP-1 and is required for lipid homeostasis in C. elegans."

Hart, Anne C, Rottiers, Veerle, Walker, Amy K, Naar, Anders M, Watts, Jennifer L

[International Worm Meeting, 2009]

Several prevalent diseases such as obesity and type II diabetes are linked to aberrant lipid homeostasis. The C. elegans SREBP/SBP-1 transcription factor is, like its mammalian homologs, critical for fatty acid and lipid homeostasis. Depletion of SBP-1 or its co-activator MDT-15 results in clear, sterile, lethal and slow growth phenotypes and a reduction in the fatty acid oleic acid, a precursor for triacylglycerides (TAGs) and poly-unsaturated fatty acids. Dietary supplementation with oleic acid markedly improved the phenotypes of sbp-1 and mdt-15 knockdown animals, suggesting a central role for oleic acid in SBP-1-function and lipid homeostasis. To identify novel conserved regulators of lipid homeostasis we performed an oleic acid auxotrophy screen. We screened the Ahringer RNAi library clones that cause clear, sterile, lethal, and slow growth phenotypes (1,300 genes) in search of clones that are, like sbp-1 and mdt-15, rescued upon dietary addition of oleic acid. We identified 6 genes in the screen. Three genes are required for mitochondrial function: sco-1 (C01F1.2) and mitochondrial S9 and S17 ((F09G8.3; C05D11.10) confirming the role of mitochondria in lipid homeostasis (McKay et al, 2003). We also found cul-1, pyr-1 and tkt-1(F01F10.2) in our screen. We focused on tkt-1, the homolog of human transketolase (TKT) since TKT mouse knockouts are lean. C. elegans tkt-1 is also necessary for normal lipid homeostasis since tkt-1 depleted animals contain less TAGs by GC-MS and show reduced Sudan Black staining. TKT is an enzyme of the Pentose Phosphate Pathway and is necessary for the production of NADPH. A large number of SREBP/SBP-1 targets need NADPH as a cofactor and mammalian SREBP activates transcription of NADPH generating enzymes. This activation seems to be conserved in C. elegans since 3 NADPH producing enzymes (G6PDH, PGD and cytosolic IDH) were identified in our microarray expression studies of sbp-1 depleted animals. tkt-1 seems to be a new, direct target of SBP-1 since Ptkt-1::gfp expression is dependent on SBP-1 and tkt-1 was identified in our microarray analysis of sbp-1(RNAi) animals. We are currently testing if mammalian TKTs are also activated by SREBP in cultured cells. Our oleic acid auxotrophy screen has identified genes necessary for lipid homeostasis and found a new target of SBP-1. We suggest a molecular mechanism for lipid depletion in TKT depleted animals, possibly across species, through SBP-1 and the requirement for NADPH.

Veerle Rottiers et al. (2002) European Worm Meeting "Gonadal and hormonal signals that regulate life span in C. elegans"

Diana McCulloch, Veerle Rottiers, Kiyoji Nishiwaki, David Gems, Adam Antebi

[European Worm Meeting, 2002]

Life span of C. elegans is influenced by the Insulin-like signaling pathway and by signals from the gonad. The nuclear hormone receptor (NHR) DAF-12 plays a role in both of these pathways since mutants enhance the longevity of daf-2(e1370) (the insulin-like receptor) and abolish the life span extension of germline laser ablated animals. daf-9 acts downstream of insulin-like inputs but upstream of daf-12. It encodes a cytochrome P450 suggesting a function in the production of a ligand for DAF-12. A sterol may be the daf-9 substrate and daf-12 ligand because cholesterol deprivation phenocopies mutant defects. The molecular identities of daf-12 and daf-9 indicate an endocrine mechanism regulating life span in C. elegans.

Veerle Rottiers et al. (2007) International Worm Meeting "RNAi screens for molecules involved in nuclear receptor signal transduction."

Twisha Chakravarty, Joshua Wollam, Veerle Rottiers, Raha Nabbi, Anne Schadach, Brittany E. Ford, Adam Antebi, Kanwar Singh

[International Worm Meeting, 2007]

Nuclear hormone receptors are transcription factors regulated by lipophilic hormones. They direct gene expression through association of co-activator or co-repressor complexes. The nuclear hormone receptor DAF-12 regulates C. elegans dauer diapause, developmental timing, stress resistance and lifespan. So far only a co-repressor, din-1, and two genes involved in hormone biosynthesis, daf-9/CYP450 and daf-36/Rieske oxygenase, have been described suggesting that several more molecules, such as co-activators, remain to be identified. daf-9 and daf-36 mutants, as well as ligand binding mutants of daf-12, share the same phenotypic profile, including constitutive dauer formation (Daf-c), migration defects in the gonad (Mig) and sensitivity to cholesterol deprivation. However, the daf-36 null phenotype is much weaker than the daf-9 null phenotype, suggesting a branched pathway. We have performed a genome-wide RNAi screen looking for enhancement of daf-36 phenotypes. As expected, we found genes involved in hormone biosynthesis (e.g. a CYP450, a short chain dehydrogenase, a cytochrome P450 reductase) and transport (e.g. the Niemann-Pick C homolog ncr-1). Another class of genes acts upstream of hormone synthesis and includes insulin signaling components (e.g. akt-1). Interestingly, we identified several transcription factors or potential co-regulators (e.g. a nuclear hormone receptor, lin-29/Zinc finger protein, lin-42/period), as well as various chromatin factors. We are currently analyzing these genes for genetic interactions with each other and their epistatic relation to daf-12. Finally, a number of mitochondrial genes enhanced the daf-36 Daf-c phenotype suggesting an input of mitochondrial function into dauer formation. Further analysis should yield novel insights into nuclear hormone receptor signaling, as well as the physiological outputs on development and life span.

Veerle Rottiers et al. (2003) International Worm Meeting "The role of daf-36, a putative terminal oxygenase in the regulation of dauer formation in C. elegans"

Adam Antebi, Kiyoji Nishiwaki, Veerle Rottiers

[International Worm Meeting, 2003]

Under unfavourable conditions, C. elegans forms an alternative third stage larvae, the dauer larvae. Signals from the environment are transduced through insulin, TGF and cGMP signaling pathways to the nuclear hormone receptor DAF-12. daf-12 then selects developmental programs for reproductive growth or dauer formation. The CYP450 daf-9 acts downstream of insulin-like inputs but upstream of daf-12. daf-9 is thought to be involved in the production of a (steroid like) hormone binding to daf-12. Steroid hormones are typically built in a series of enzymatic steps. We wanted to identify more components in this putative biosynthetic pathway. Therefore we characterised the mutant daf-36(k114), which shares a number of features with daf-9. First, the mutant displays both gonadal and Daf-c phenotypes. Second, epistasis analysis showed that daf-36 acts downstream or in parallel to TGF and Insulin-like signaling but upstream of daf-12. Finally, upon cholesterol starvation, the gonadal migration phenotype of daf-36 is strongly enhanced. We cloned daf-36 and found it to be a putative terminal oxygenase subunit of a ring-hydroxylating dioxygenase. The most homologous described gene is kshA, a subunit of 3-ketosteroid-9-hydroxylase in Rhodococcus. This homology suggests a function for daf-36 in modification or breakdown of a steroid hormone. We are currently analysing the enzymatic activity of DAF-36 in Rhodococcus. A daf-36::gfp construct reveals strong expression in the instestine. By contrast daf-9 is expressed in 2 head neurons, hypodermis and spermatheca suggesting that different hormonal metabolic steps take place in different tissues. We are currently analysing the daf-36 expression in different genetic backgrounds. With the identification of daf-36, we describe a new gene involved in the endocrine signaling in C. elegans that regulates dauer formation.

Veerle Rottiers et al. (2001) International C. elegans Meeting "Role of DAF-9 CYP450 in the regulation of fat storage and life span in C. elegans."

Veerle Rottiers, Adam Antebi

[International C. elegans Meeting, 2001]

In C. elegans , dauer arrest and fat storage are controlled by TGF-beta and Insulin-like signaling pathways. The nuclear hormone receptor daf-12 acts downstream of both of these pathways, mediating the choice between dauer arrest and reproductive growth. daf-12 also plays a role in aging since mutants enhance the longevity of daf-2(e1370) (the insulin-like receptor) and abolish the life span extension of germline laser ablated animals. daf-9 acts downstream of TGF-beta and insulin-like inputs but upstream of daf-12 for dauer formation. It encodes a cytochrome P450 suggesting a function in the production of a ligand for daf-12 . We investigated the role of daf-9 in fat storage and in the gonad signaling pathway affecting life span. Under dauer inducing conditions, wild type animals shift metabolism towards fat storage. L2 predauers (L2d) and dauers of TGF-beta (e.g. daf-7 ) and insulin-like (e.g. daf-2 ) pathway mutants are dark and stain with Sudan black. This phenotype is blocked by Daf-d mutants daf-3 and daf-16 , respectively. We find that daf-9 L2d larvae store fat independently of daf-3 and daf-16 and dependent on daf-12 . These results place daf-9 downstream or parallel to daf-16 and daf-3 but upstream of daf-12 . daf-9 L3 dauers on the other hand, are somewhat lighter indicating that fat is mobilized during partial recovery from diapause. We performed laser ablations of somatic gonad and germline precursors in daf-9(rh50) mutant animals. rh50 is a hypomorphic allele of daf-9 . Germline ablation did not lengthen life span and somatic gonad ablation did not further shorten life span. We conclude that daf-9(+) is necessary for life span extension. Since daf-9 is expressed in the spermatheca it might define the life extending signal from the somatic gonad. Finally we want to identify more genes in the putative hormone signaling pathway. We are currently analyzing one candidate that displays gonadal and Daf-c phenotypes similar to daf-9 .

Veerle Rottiers et al. (2005) International Worm Meeting "A multistep hormone biosynthetic pathway that regulates C. elegans development and dauer formation."

Birgit Gerisch, Veerle Rottiers, Adam Antebi, Kailly Harrell, Axel Bethke, Raha Nabbi, Anne Schadach, Harald Hutter

[International Worm Meeting, 2005]

During larval development, C. elegans evaluates environmental cues and must choose between reproductive growth or arrest at the dauer diapause. Insulin/IGF and TGF- signalling integrate neurosensory cues, and subsequently produce downstream secondary endocrines to coordinate tissues throughout the body. One such endocrine is proposed to be an unidentified lipophilic hormone that regulates the nuclear hormone receptor daf-12. Molecular genetic evidence suggests that in presence of this hormone, worms undergo reproductive development, whereas in its absence they enter the dauer diapause. The cytochrome P450 daf-9, is a major player in hormone production, but typically, such hormones are built by several enzymes. Accordingly, we previously reported that a novel Rieske-like oxygenase, daf-36, is also involved. daf-36 shares both dauer constitutive (Daf-c) and gonadal migration defect (Mig) phenotypes with daf-9 as well as daf-12 ligand binding domain mutants. However, daf-36 null mutant defects are weaker than daf-9 mutants, and both genes have non-overlapping expression patterns. This suggests a complex biosynthesis pathway involving several genes with distributed production sites, but the extent of this pathway is unknown. Here, we report a more detailed analysis of the hormone biosynthesis pathway. Using RNAi-enhancer screens of daf-36, we screened for Daf-c and Mig phenotypes, which are signatures of the hormone pathway. So far we have identified a dehydrogenase, another CYP450 and a nuclear hormone receptor. We are currently characterizing the phenotypes and epistasis patterns of these loci. A further prediction of the hormone hypothesis is that lipid extracts of worms should contain the daf-12 ligand. Indeed, we found that lipid extracts of wild type L3 worms can rescue the daf-9 and daf-36 mutant phenotypes, whereas each mutant cannot be rescued by its own extract. These results confirm the lipophilic nature of the hormone and demonstrate that these genes are involved in hormone production. We are currently performing cross-feeding experiments with mutant extracts to see whether the genes can be ordered relative to one another. This should allow us to dissect the biosynthetic pathway and facilitate the biochemical identification of the daf-12 hormone. In sum, these studies provide the first evidence for a multistep hormone biosynthetic pathway in C. elegans.

Veerle Rottiers et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Identification of Regulators Governing Lipid Homeostasis in C. elegans"

Amy Walker, Fajun Yang, Anders Naar, Veerle Rottiers, Anne Hart, Karen Jiang

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Cholesterol and fatty acids have important functional roles in metazoans, such as modulating membrane fluidity, serving as signaling molecules, and providing energy storage in the form of triacylglycerides. Abnormal cholesterol and fat levels have been linked to prevalent diseases, including atherosclerosis, obesity, type 2 diabetes and hypertension underscoring the importance of understanding fully how cholesterol and lipid homeostasis are regulated and maintained. The mammalian SREBP transcription factors are critical regulators of adipocyte differentiation and cholesterol and fatty acid homeostasis. We have previously shown in vitro that SREBP activates gene expression by recruiting CBP/p300 acetyltransferases and the ARC/Mediator co-activators and that this mechanism of activation is conserved in C. elegans. Depletion of SBP-1/SREBP and MDT-15/ARC105 results in a clear phenotype, sterility, lethality and slow growth. Triacylglycerides and poly-unsaturated acids are strongly decreased and stearic acid is increased when sbp-1 and mdt-15 are knocked down by RNAi. Moreover, the expression of fat-6 and fat-7, the homologs of the stearoyl-CoA desaturase genes, is dramatically decreased upon depletion of both sbp-1 and mdt-15. fat-6 and fat-7 are involved in synthesis of oleic acid from stearic acid and, indeed, dietary supplementation with oleic acid markedly improved the phenotypes of sbp-1, mdt-15 and fat-6/fat-7 RNAi knockdown animals. This suggests a central role of oleic acid and stearoyl CoA desaturases in SBP-1 function and lipid homeostasis. To identify novel conserved regulators of fatty acid desaturation and lipid homeostasis we are performing 2 large scale RNAi screens. The first is an Oleic Acid Auxotrophy screen: based on our data suggesting a critical role of oleic acid regulation in C. elegans physiology we are currently screening through all the RNAi clones that cause clear, sterile, lethal and slow growth phenotypes in the C. elegans genome (~1,300 genes) in search of clones that are rescued upon dietary addition of oleic acid. The second, genome-wide screen, derives from our finding that the fat-7 stearoyl CoA desaturase is a key target of central lipid regulators including SBP-1. In this screen, we will identify genes whose loss enhances or reduces the expression of a FAT-7 promotor::GFP fusion reporter. We expect to identify key fat regulators that are conserved across species and may be relevant in human diseases.

Veerle Rottiers et al. (2007) International Worm Meeting "The influence of D4-dafachronic acid, a DAF-12 ligand, on lifespan, development and stress resistance in C. elegans."

Dongling Li, Daniel L. Motola, David J. Mangelsdorf, Adam Antebi, Carolyn L. Cummins, Joshua Wollam, Birgit Gerisch, Veerle Rottiers

[International Worm Meeting, 2007]

The nuclear hormone receptor DAF-12 regulates C. elegans developmental timing, dauer formation and lifespan. Recently, the endogenous ligands of DAF-12 were shown to be 3-keto bile-acid like steroids, termed dafachronic acids. These ligands rescue the larval defects of hormone-deficient mutants, such as daf-9/cytochrome P450 and daf-36/Rieske oxygenase, and activate DAF-12. Supplementation of <font face=symbol>D</font>4-dafachronic acid enables us to directly test its effect on lifespan and stress resistance and allows us to pinpoint when the ligand is necessary for each of the processes regulated by DAF-12. We tested the effect of <font face=symbol>D</font>4-dafachronic acid on lifespan and found that it shortened the lifespan of long-lived daf-9 mutants, indicating that the ligand is pro-aging in response to signals from the dauer pathways. However, the ligand extended the lifespan of germ-line ablated daf-9 and daf-36 mutants, showing that it is anti-aging in the germ-line longevity pathway. Thus, dafachronic acid regulates C. elegans lifespan according to signaling state. We have also tested the effect of the DAF-12 ligand on stress resistance and found that it abolishes the resistance to heat and oxidative stress of daf-9 mutants, consistent with its life shortening role in response to dauer signaling. We are currently investigating the role of dafachronic acid in response to other types of stress such as pathogen exposure. These results provide the first evidence that bile acid-like steroids modulate stress resistance and aging in animals.

Dach, Neal A. et al. (2009) International Worm Meeting "Genome-wide RNAi screen for regulators of lipid homeostasis in C. elegans."

Naar, Anders M., Rottiers, Veerle, Hart, Anne C., Dach, Neal A.

[International Worm Meeting, 2009]

Metabolic syndrome, which occurs in about 47 million Americans, is characterized by a group of risk factors including high cholesterol, hypertension, abdominal obesity, and insulin resistance. Metabolic syndrome is thought to largely result from lifestyle factors, such as excess energy intake and decreased exercise, however the underlying molecular mechanisms are still poorly understood. Defects in the regulation of fatty acid and cholesterol homeostasis may be involved in the development of metabolic syndrome. The SREBP family of transcription factors regulates genes responsible for fatty acid and cholesterol synthesis and uptake. SREBP activates gene expression by recruiting the chromatin-targeting CBP/p300 acetyltransferases and the ARC/Mediator complex which in turn recruits RNA Polymerase II. This mechanism of activation is conserved in the nematode C. elegans and depletion of the nematode SREBP ortholog sbp-1 or mdt-15 (ortholog of the ARC105 ARC/Mediator subunit) results in decreased polyunsaturated fatty acid and triacylglyceride levels as well as a buildup of stearic acid. The expression of fat-6 and fat-7, two stearoyl-CoA desaturases (SCDs) which convert stearic acid to oleic acid, is decreased in sbp-1 and mdt-15 mutants. Animals lacking fat-7, fat-6, sbp-1 and mdt-15 show similar phenotypes. Dietary supplementation with oleic acid significantly ameliorates their defects, indicating that the SCDs are physiologically important targets of SBP-1 function. To identify novel regulators of SCDs and lipid homeostasis in general, we have performed a genome-wide RNAi screen for modifiers of fat-7 expression. Employing the COPAS Biosorter, we have identified genes whose RNAi knockdown increases or reduces the expression of a fat-7 promotor::gfp fusion reporter construct. We find that RNAi of sbp-1 and mdt-15 reduces pfat-7::gfp expression, serving as positive controls for GFP expression reduction. RNAi of fat-6 and fat-7 results in up-regulation of the pfat-7::gfp reporter due to a compensatory mechanism, providing positive controls for GFP expression increase. Finally, we used unc-22 RNAi and L4440 empty vector as negative controls. We are now retesting and assessing the specificity of about 1,200 putative hits from the genome-wide screen. Our unconfirmed list of modifiers includes expected hits: sbp-1, nhr-49, cbp-1, fat-7, and C31E10.7, a putative cofactor for FAT-7, suggesting that functionally important genes will be identified. We expect many of the genes that emerge from this screen to expand our understanding of how animals maintain lipid homeostasis.

Jiang, Karen et al. (2009) International Worm Meeting "Conserved transcription factor networks link fatty acid and one carbon metabolism."

Rottiers, Veerle, Jiang, Karen, Watts, Jennifer, Hart, Anne, Naar, Anders, Walker, Amy

[International Worm Meeting, 2009]

Human metabolic disorders such as cardiovascular disease, obesity and metabolic syndrome are linked to changes in fatty acid or cholesterol biosynthesis; processes controlled by the sterol regulatory element binding protein (SREBP) family of transcription factors. To examine SREBP function in vivo, and to delineate SREBP transcriptional functions, we have examined SREBP (SBP-1) gene regulatory mechanisms in C. elegans, combined with complementary studies in mammalian cell lines. SREBP activates genes important for cholesterol, fatty acid and phospholipid biosynthetic pathways, in addition to ensuring production of co-factors such as Acetyl-CoA and NADPH. We have identified a novel set of SREBP-responsive genes in C. elegans, the methyl-group producing enzymes of the 1-carbon cycle (1CC). Multiple genes in this pathway, including sams-1(MAT1A) are responsive to SBP-1/SREBP in both C. elegans and mammalian cells. sams-1/MAT1A encodes a s-adenosyl methionine transferase, which provides the methyl donor (SAMe) for most methylation reactions. We hypothesize these genes are important components of SREBP function because methylation is necessary for phospholipid biogenesis, particularly the synthesis of phosphatidylcholine. Interference with the 1CC in C. elegans through RNAi or in sams-1(ok3033) disrupts lipid homeostasis, resulting in the accumulation of fat droplets in the intestine and body cavity. Importantly, this phenotype is highly reminiscent of lipid accumulation in fatty liver disease in humans and similar to the MAT1A KO in mice (Lu et al PNAS, 2001). Furthermore, we have found that phosphatidylcholine levels are low in sams-1(RNAi) animals suggesting that defects in phospholipid metabolism could contribute to the phenotypes of these animals. SInce sbp-1 is necessary for fat storage, it is surprising that a SBP-1 regulated gene causes increases in lipids. We have found that several SBP-1 target genes that also important for lipid metabolism (fatty acid desaturases fat-5, fat-6 and fat-7) are strongly upregulated in sams-1(RNAi) animals. In addition, sbp-1 and the FA desaturases are necessary for lipid accumulation in sams-1(RNAi) animals, suggesting that changes in 1C metabolism could impact key aspects of lipid biosynthesis through SBP-1. Alterations in 1C metabolism and SREBP function are associated with similar diseases, suggesting that co-regulation with lipid homeostasis may be a common impact point in metabolic disorders.