Axel Bethke

Bethke, Axel et al. (2009) International Worm Meeting "cis-regulatory elements in promoters of the let-7 family."

Wiese, Mary, Antebi, Adam, Bethke, Axel

[International Worm Meeting, 2009]

microRNAs play a critical role in development and disease, yet their transcriptional regulation is not well understood. Only a handful of transcription factors are known to directly activate their transcription. Among them, the nuclear hormone receptor DAF-12 regulates transcription of let-7 family homologs in a ligand-dependent manner, to influence developmental progression in the heterochronic circuit. Our studies show that DAF-12 binds to specific cis-regulatory response elements (REs) present within the microRNA promoters. However, genetic experiments also reveal that DAF-12 is not the only factor involved. To better understand the transcriptional networks regulating microRNA expression, we have undertaken a genetic and biochemical analysis of the cis- and trans-acting factors governing their transcriptional control. In promoters of the let-7 family members, daf-12 activated REs appear separate or as dimers in inverted or direct repeats with various base pairs spacing between each element. To investigate the individual influence of each daf-12 RE and the sequence directly surrounding it, we generated repeats of 100 to 150bp long fragments surrounding the REs and fused these repeats to a minimal promoter and a YFP reporter. We found isolated REs that show complete daf-12 and ligand dose dependence, while other REs are only moderately influenced by daf-12. Moreover, different REs typically have non-overlapping tissue specificity, and activate transcription only in a subset of tissues compared to the full length promoter. We are now using this approach to investigate nuclear hormone receptor-mediated transcriptional regulation of separate REs isolated from the full-length promoter. We are also screening for other transcription factors that bind to REs in direct proximity to daf-12 dependent REs to influence stage and tissue specific expression. These studies should shed light on transcription factor-microRNA networks that regulate temporal and spatial aspects of development.

Axel Bethke et al. (2007) International Worm Meeting "Direct and ligand dependent activation of miRs by DAF-12."

Adam Antebi, David Mangelsdorf, Zhu Wang, Axel Bethke

[International Worm Meeting, 2007]

The let-7-family of miRNAs are involved in the temporal regulation of C. elegans development. Their transcriptional regulation is poorly understood despite their important role. Recently, the nuclear hormone receptor DAF-12 has been hinted to transcriptionally influence members of this miRNA family. DAF-12 regulates the developmental fate of C. elegans by either promoting reproductive growth and transcriptional activation in the presence of its ligand "dafachronic acid", or promoting dauer diapause and transcriptional repression in the absense of its ligand. In this work we show the direct and ligand induced activation of several let-7-family promotors by DAF-12. Luciferase assays in human cell lines showed strong DAF-12 and ligand dependent activation of some let-7-family members but not others. We have focused on one promotor and identified several DAF-12 response elements (RE), which directly mediate DAF-12 binding in vitro by gel shift assay. Mutation of these response elements in full length promotor constructs altered ligand dependent activation in cell culture. In worms, repeats of 100bp or 150bp long promotor blocks surrounding these REs have a tissue selective expression pattern, overlapping with that seen in reporter constructs with the full length promotor. Whereas several tissues expressed YFP reporters without obvious DAF-12 dependence, other tissues exhibited a strict dependence on DAF-12 and ligand. These constructs with isolated DAF-12 REs for in vivo expression also allow identification of coactivators and other transcription factors responsible for tissue selective expression. We are also investigating the mechanism of DAF-12 signalling phenotypes by using the tissue selective expression profile of these reporter constructs.

Axel Bethke et al. (2003) International Worm Meeting "Transcriptional complexes of the C. elegans dauer pathways"

Nanji Park, Axel Bethke, Andreas Ludewig, Adam Antebi

[International Worm Meeting, 2003]

The C. elegans dauer pathways are a remarkable example of how environmental signals impact development and life span. In particular, how identified transcription factors mediate the physiological output of insulin/IGF, TGF and cGMP dauer-signaling pathways is not well understood. By examining transcriptional complexes of the dauer pathways, including those of DAF-12/nuclear receptor DAF-3/smad and DAF-16/forkhead, we hope to better understand gene regulatory networks. DAF-12 acts near the end of the signalling cascades inducing dauer formation and is regulated by DAF-9, a cytochrome P450 likely involved in a hormone biosynthesis. DAF-12 transcriptional complexes should contain interacting proteins like coactivators and corepressors, whose activity on target genes are hormonally regulated. Yeast two hybrid screens identified DIN-1, a homolog of the mammalian SHARP corepressor, as a DAF-12 interacting protein. This interaction was verified by GST-pulldown experiments with radiolabelled DIN-1 and GST-tagged DAF-12. We are now trying immunoprecipitation experiments to look at complex formation. One limitation is that DAF-12 and DIN-1 are very low abundance proteins. To identify further interacting factors, we will use co-immunoprecipitation and MALDI-fingerprinting. PTR-23 was also identified as a DAF-12 interactor. We are now doing GST-pulldown experiments with PTR-23. Interestingly, ptr-23 RNAi shows moulting defects, as do some daf-12 mutants (e.g. Daf-c alleles). It remains to be determined how these genes are functionally connected. Since hormone is a central regulatory component of these complexes, we are also beginning experiments to identify the DAF-12 hormone. By comparing steroid profiles from daf-9 mutant and wild type worms, we hope to identify relevant differences that lead us to a measurable biological activity. For example, the activity of different sterols will be tested on daf-9 mutants for rescue of the mutant phenotypes.

Axel Bethke et al. (2007) International Worm Meeting "Dauer signaling through UTR-mediated repression?"

Nicole Fielenbach, Adam Antebi, David Mangelsdorf, Christopher T. Hensley, Zhu Wang, Axel Bethke

[International Worm Meeting, 2007]

Post-embryonic animal development is often contingent on environmental cues, such as the availability of nutrients and resources. The C. elegans nuclear hormone receptor DAF-12 integrates environmental information to mediate the decision between reproductive development or the dauer diapause, a long lived stress resistant stage. DAF-12 also works in the heterochronic pathway in several tissues. In particular, daf-12 mutants perturb L3 and later stage programs in the seam cells, a phenotype shared with mutants of the let-7-family of microRNAs, mir-48, mir-84, and mir-241. Individual mutants exhibit impenetrant phenotypes, whereas daf-12;miR doubles leads to an enhancement. Notably, we have shown that DAF-12 directly activates let-7-family miR transcription in response to its identified ligand, "dafachronic acid" (see abstract by Bethke et al). These observations together imply that DAF-12 activates the miRs during reproductive development, and represses them during dauer formation, connecting environmental input to the heterochronic pathway. Interestingly, these microRNAs also influence dauer formation itself. The miR triple deletion has a Daf-c phenotype, which is readily suppressed by daf-12 loss of function. This observation suggests that in addition to being transcriptional targets, miRs could also feedback regulate daf-12 through its 3 UTR. In addition, a number of other dauer regulators contain potential let-7-family miRNA binding sites, suggesting they too could be direct targets. In this work we look at two predicted 3 UTR targets for repression, DAF-12 and the FOXO transcription factor DAF-16. To test this hypothesis, we quantified the degree of repression: a YFP construct with the daf-12 promotor and unc-54-3''UTR showed high expression levels. By contrast, the expression level of the chromophore is strongly repressed when the unc-54-3''UTR is exchanged for either the daf-12 or the daf-16 3''UTR, indicating a tight repression of translation or decrease in post transcriptional mRNA stability. We are currently conducting genetic experiments to find out the exact identity of the suppressing miRNAs and their major upstream activators, as well as Q-PCR experiments to look at mRNA stability of the miR-targets.

Axel Bethke et al. (2002) European Worm Meeting "Identifying transcriptional targets of the DAF-12 nuclear hormone receptor"

Axel Bethke, Adam Antebi

[European Worm Meeting, 2002]

The daf-12 gene encodes a nuclear hormone receptor that integrates inputs from insulin/IGF, TGF- and cGMP signalling pathways and influences dauer formation, the heterochronic circuit and life span. Therefore, the set of daf-12 target genes should be highly interesting. Shostak and Yamamoto (IWM, 2001) defined several putative DAF-12 DNA target sequences in the lit-1 promotor by in vitro and in vivo experiments. We are searching for additional physiological candidate genes in identified signalling pathways. Surprisingly, one is daf-9, a cytochrome P450 related to vertebrate steroidogenic hydroxylases. DAF-9 is thought to metabolize a DAF-12 hormone and acts by genetic epistasis experiments upstream of DAF-12. In contrast to this, we find that hypodermal daf-9::gfp expression does not occur in daf-12 null mutants, suggesting feedback regulation by DAF-12 (See abstract by Gerisch et al). Another candidate target is let-7, a heterochronic gene coding for a small temporal RNA (stRNA) that regulates the larval to adult switch. Slack and Johnson (IWM, 2001) showed that a let-7::gfp construct is temporally upregulated by the L4 stage in the same cell type as daf-12::gfp. In addition, daf-12 alleles with delayed heterochronic phenotypes, such as rh61, also result in delayed or inhibited let-7 expression (personal com. Slack et al). To investigate whether these candidates are directly regulated by DAF-12 in vivo, we are beginning chromatin immunoprecipitation (X-ChIP) experiments to define DNA regions to which the nuclear receptor binds. We will also dovetail these experiments with gel mobility shift assays using protein from nuclear extracts to narrow down the region of receptor binding and to identify corresponding response elements.

Axel Bethke et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "LIN-29/ZnF co-activates DAF-12/NHR to drive miR-84 expression, placing miR-84 towards the end of the heterochronic pathway"

Nicole Fielenbach, Axel Bethke, Adam Antebi, Linyan Meng

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

The nuclear hormone receptor DAF-12 acts as the switch between continuous development or arrest at the dauer diapause during the L2 stage. It also works in the heterochronic circuit, promoting L3-stage specific developmental programs. Another heterochronic factor, the zinc finger protein LIN-29 acts during L4 to promote terminal differentiation of various tissues like gonad or hypodermis, a function referred to as the larval-to-adult switch. Despite this terminal position, here we present evidence that LIN-29 is involved in transcriptional activation of the let-7 sister mir-84, which was thought to act at an earlier step. Previously, we had shown DAF-12 and ligand dependent activation of mir-84 promoter constructs in human cell culture and in worms. We wondered whether any of the heterochronic factors could serve as DAF-12 co-activators to augment transcription. Remarkably, we found that co-transfection of LIN-29 increased DAF-12 dependent transcription two-fold, but had no such activity alone, suggesting LIN-29 may be a DAF-12 co-activator. Accordingly, point mutation of DAF-12 response elements strongly decreased DAF-12 and ligand dependent activation as well as the co-activator effect, The DNA-motive that LIN-29 binds to is unknown, but EMSA with an oligo containing two functional DAF-12 sites show no LIN-29 binding, making it unlikely that LIN-29 can bind directly to DAF-12 response elements. Taken together, the experiments suggest that LIN-29 works through DAF-12 to exert these transcriptional effects. The mir-84 promoter also shows DAF-12 and LIN-29 dependence in vivo. In particular, the lin-29 null mutants exhibited decreased pharyngeal mir-84p::GFP expression, even below the level observed in a daf-12 null. Moreover, a lin-29, daf-12 double null nearly lost all GFP expression. These in vivo data are consistent with the notion that LIN-29 acts through DAF-12 and its response elements, but also hint at a DAF-12 independent function of LIN-29 on the mir-84 promoter. The placement of LIN-29 upstream of mir-84 inverts its conventional placement as the terminal factor in the heterochronic pathway. Interestingly, Hayes et al showed that mir-84 over expression can suppress lin-29 for supernumerary molting heterochronic phenotypes. This, together with the data presented here, suggests that LIN-29 could drive terminal differentiation in the hypodermis through activating mir-84 expression, placing miR-84 downstream of LIN-29 to the very end of the heterochronic signaling cascade. We are currently testing this hypothesis.

Gudibanda, Pooja et al. (2015) International Worm Meeting "Small molecule mediated nuclear receptor DAF-12 signaling regulates development and lifespan in C. elegans."

Gudibanda, Pooja, Bethke, Axel, Mahanti, Parag, Schroeder, Frank, Ludewig, Andreas

[International Worm Meeting, 2015]

Nuclear receptors (NRs) are ligand-modulated transcription factors that interact with corepressor and coactivator proteins to control development, homeostasis and metabolism. In C. elegans, the vitamin-D and liver-X receptor homolog, DAF-12, functions as a central regulator of development and lifespan. For example, DAF-12 is required for the more than 50% lifespan increase observed when germline stem cells (GSCs) are ablated from the gonad. Three chemically distinct, endogenous steroidal ligands of DAF-12, the dafachronic acids (DAs), have recently been identified, which are required for reproductive development and DAF-12 dependent lifespan increase. DAF-12 has been shown to upregulate members of the let-7 microRNA (miRNA) family; however, the mechanisms by which the DAs regulate these different phenotypes are unclear.To investigate how binding of different ligands regulates interaction of DAF-12 with co-regulators and its DNA response elements, we have developed a cell-free, bead-based AlphaScreen assay. Using this assay, we have confirmed that a previously described corepressor, DIN-1, interacts with DAF-12 in its unliganded state. We have further identified several coactivator candidates that may interact with DAF-12 upon ligand binding. We hypothesize that binding of different ligands controls interaction with different coactivators and corepressors, and that different DNA response elements in let-7 family miRNA promoters favor specific DAF-12-coregulator interactions. To validate results from the AlphaScreen, in an HEK293T cell-based system, we employed luciferase assays which showed that DA-mediated DAF-12 activation under the let-7 family of promoters mir-241p and mir-84p is repressed by DIN-1. We propose a model in which promoter-guided DAF-12 dimerization mediates interaction with different ligands and coregulators, facilitating different transcriptional outcomes associated with the different functions of DAF-12 in the regulation of development and lifespan. .

Pungaliya, Chirag et al. (2009) International Worm Meeting "Systematic identification of steroids in Caenorhabditis elegans."

Pungaliya, Chirag, Wollam, Joshua, Antebi, Adam, Seim, Kristen, Schroeder, Frank, Malik, Rabia, Bethke, Axel

[International Worm Meeting, 2009]

Recently Motola et. al. identified the dafachronic acids, bile-acid like steroids that act as the endogenous ligands of the nuclear hormone receptor DAF-12. They directly bind to DAF-12 to influence interactions with its cofactors and thereby regulate transcriptional activity. It is possible that other endogenous steroids influence DAF-12 or act on other nuclear hormone receptors that modulate various C. elegans life history traits. We have begun a systematic analysis of C. elegans steroid metabolism based on chromatographic separation, bioassays and differential analysis by 2D NMR spectroscopy (DANS), comparing the wild-type metabolome to that of mutants with suspected defects in steroid metabolism. So far our investigations have revealed several steroids whose presence in C. elegans had not previously been recognized.

Bose, Neelanjan et al. (2011) International Worm Meeting "Revising the DAF-12 ligand biosynthetic pathway in C. elegans: A comprehensive NMR-based approach."

Antebi, Adam, Mahanti, Parag, Schroeder, Frank, Bose, Neelanjan, Hu, Patrick, Judkins, Joshua, Bethke, Axel, Wollam, Joshua

[International Worm Meeting, 2011]

In C. elegans, the nuclear hormone receptor DAF-12 plays a central role in regulating developmental progression from larva to adult. Two steroid hormones D4 and D7 -dafachronic acids have been predicted to function as ligands of DAF-12, promoting reproductive development and suppressing entry into larval diapause (dauer)1. Previous investigations1,2,3 predominantly based on indirect feeding experiments and phenotypic based proposed a putative biosynthetic pathway for D4 and D7 -dafachronic acids involving a cytochrome P450, DAF-9; a Rieske oxygenase, DAF-36 and a hydroxysterol dehydrogenase, HSD-1. Using NMR based comparative metabolomics, we show that the biosynthetic pathway for DAF-12 ligands requires substantial revision. For example, comparison of the metabolome of hsd-1 mutants with that of hormone deficient daf-9:daf-12 mutant worms revealed the presence of a novel endogenous DAF-12 ligand that is absent in wild-type worms. In contrast, hsd-1 mutants lacked a major wild-type ligand proposed to be upregulated in hsd-1. In addition we report changes in dafachronic acid biosynthesis in several other mutant strains, including daf-36, dhs-16 and strm-1. Separate ligand biosynthetic pathways for DAF-12 leading to structurally different ligands may represent a mechanism for controlling DAF-12 function4. The identification of new components and the association of the known compounds to specific mutant backgrounds will provide a better understanding of the convergence of signals from the upstream dauer network: Guanylyl Cyclase, TGF-b and insulin/IGF-1 pathways on to the central regulator DAF-12. 1. Motola DL, Cummins CL, Rottiers V, Sharma KK, Li T, Li Y, Suino-Powell K, Xu HE, Auchus RJ, Antebi A, Mangelsdorf DJ (2006). Cell 124, 1209. 2. Rottiers, V, Motola, DL, Gerisch, B, Cummins, CL, Nishiwaki, K, Mangelsdorf, DJ, and Antebi, A (2006). Dev. Cell.10, 473. 3. Patel DS, Fang LL, Svy DK, Ruvkun G, Li W. (2008). Development. 135, 2239. 4. Dumas KJ, Guo C, Wang X, Burkhart KB, Adams EJ, Alam H, Hu PJ. (2010). Dev Biol. 340, 605.