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.

Alexander, K. et al. (2017) International Worm Meeting "Investigating Molecular Mechanisms Underlying Cholinergic Synapse Remodeling in the C. elegans Motor Circuit."

Doitsidou, M., Oliver, D., Alexander, K., Francis, M.M., Benard, C., Lambert, C., Philbrook, A., Thackeray, A.

[International Worm Meeting, 2017]

The nervous system comprises complex neural circuits that require precise patterns of connectivity. To accomplish this, there is often considerable remodeling of connectivity during development, typically an initial over proliferation of synapses that is followed by activity-dependent refinement or remodeling. We have only a limited molecular understanding of how this is achieved. To address this question, we have been investigating synaptic remodeling of GABAergic Dorsal D class (DD) motor neurons. Initially, DD motor neurons form neuromuscular synapses ventrally and receive synaptic inputs dorsally. By late larval stages, the polarity of these connections is reversed. DD motor neurons receive synaptic inputs from cholinergic motor neurons in the ventral nerve cord and form neuromuscular synapses to dorsal body wall muscles. Prior work has focused on the remodeling of presynaptic components in DD neurons and identified several genes involved. My work is focused on the remodeling of postsynaptic components using a specific marker (ACR-12::GFP) to label synaptic inputs to DD neurons. Interestingly, only a subset of genes implicated in presynaptic remodeling also affect postsynaptic remodeling. For example, disruption of the heterochronic gene hbl-1 similarly affects both processes [1]. In contrast, the caspase ced-3, involved in the removal of pre-synaptic components during remodeling [2], is not required for removal of dorsal ACR-12::GFP clusters. We are carrying out a forward genetic screen to identify novel genes involved in the remodeling process. To date, we have obtained two candidate mutants in which dorsal ACR-12 receptor clusters persist into adulthood. Through our ongoing characterization of these candidates and additional mutants identified from the screen, we aim to uncover conserved molecular mechanisms underlying synaptic refinement and remodeling. 1. Thompson-Peer et al., Neuron (2012) 2. Meng et al., Cell Reports (2015).

Sagulo, Seth et al. (2015) International Worm Meeting "Investigating the Role of Host Cell Factor 1 and Ribosomal S6 Kinase in Regulation of Longevity."

Sagulo, Seth, Lee, Siu Sylvia

[International Worm Meeting, 2015]

The Lee lab has identified Host Cell Factor 1 (HCF-1) as a transcriptional corepressor of DAF-16/FOXO, a major downstream effector of the highly conserved insulin/insulin-like (IIS) signaling pathway. C. elegans that are mutant in hcf-1 have an extended lifespan that is dependent on daf-16. Host Cell Factor 1 is a nuclear expressed protein that acts as a scaffolding protein as it does not have a clear DNA binding domain. Thus it likely plays a role in various protein-protein interactions, some of which may be important for regulating lifespan. Since many of the proteins that interact with HCF-1 have not yet been identified immunoprecipitation-mass spectrometry (IP-MS) was performed in collaboration with the Ruvkun lab and Ming Zhu, Meng-Qiu Dong of the Dong lab. This was done using a strain of C. elegans expressing HCF-1 tagged with GFP. Several chromatin related factors including the histone deacetylase SIN-3 which has been previously shown to interact with HCF-1 in addition to many metabolism proteins were found to interact with HCF-1. Surprisingly, the downstream kinase of the highly conserved target of rapamycin (TOR) pathway, ribosomal S6 kinase (RSKS-1/S6K1) was also identified which suggests it may play a role in the regulation of HCF-1 through phosphorylation. Interestingly, previously published data shows that rsks-1 mutants have an extended lifespan that is dependent on daf-16 which is similar to hcf-1 mutants. To support the IP-MS data, the genetic relationship of rsks-1 and hcf-1 was tested. The rsks-1;hcf-1 double mutant does not show an additive extension of lifespan compared to either of the rsks-1 or hcf-1 single mutants providing evidence that HCF-1 and RSKS-1 may be working through the same pathway to regulate longevity in C. elegans. Since HCF-1 and RSKS-1 are both highly conserved proteins understanding the novel biochemical and genetic connection between them will be relevant to aging in mammals.

Koelle MR (1998) East Coast Worm Meeting "G protein regulators in C. elegans: a survey of their expression patterns and functions"

Koelle MR

[East Coast Worm Meeting, 1998]

RGS proteins (regulators of G protein signaling) inhibit G protein signaling by binding directly to G protein alpha subunits and converting them from their active GTP-bound form to their inactive GDP-bound form. We have previously described the C. elegans RGS protein EGL-10 protein, which is expressed in most or all C. elegans neurons and inhibits signaling by the G protein GOA-1 to regulate several behaviors, including egg laying and locomotion. We found that both the mammalian and C. elegans genomes encode large families of proteins that share sequence similarity with EGL-10 in the ~120 amino acid "RGS domain", the part of these proteins that interacts with G protein alpha subunits. To help understand why there might be so many G protein regulators, we have analyzed the expression patterns and functions of all 11 RGS genes identified so far by the C. elegans genome sequencing project. We used GFP reporter transgenes to examine the expression patterns of the RGS genes. Our results suggest that about half of the RGS genes in C. elegans are expressed quite widely, and have expression patterns that overlap extensively. For example, egl-10, C05B5.7, C41G11.3, and the RGS genes on cosmids C16C2 and F45B8 each appear to be expressed in most or all neurons, and B0336.4 appears to be expressed in the neurons as well as most other cells in the animal. Because these genes do not all have the same functions (for example, egl-10 has a mutant phenotype, and thus is not completely redundant with the other RGS genes expressed in the same cells), we suggest that different RGS proteins may achieve specific functions by acting specifically on different G protein targets. Five RGS genes in C. elegans appear to be expressed in patterns that are highly spatially and/or temporally restricted. These RGS proteins may achieve specific functions in part by virtue of this fact. For example, GFP reporters for C29H12.3 are expressed only in seven amphid neurons and the two phasmid neurons. This suggests a specific role for this RGS gene in down-regulating or desensitizing responses to the chemosensory or mechanosensory stimuli detected by these cells through G protein signaling pathways. We have begun to examine the functions of the RGS genes by analyzing animals that overexpress them from multicopy transgenes. Overexpression of egl-10 in this manner gives a phenotype similar to that obtained by deleting the gene for its known G protein target, GOA-1. We hope that overexpression of the other RGS genes will similarly result in gain-of-function phenotypes for these genes. As described in abstracts for this meeting by Meng-Qiu Dong and Georgia Patikoglou, two additional RGS genes besides egl-10, when overexpressed, also stimulate egg laying, presumably by also inhibiting the G protein GOA-1. These RGS genes, C05B5.7 and F16H9.1, appear to be expressed in patterns that overlap the GOA-1 expression pattern in the nervous system, consistent with this idea. Another RGS gene, on cosmid C16C2, also appears to be widely expressed in the nervous system, but causes an opposite phenotype when overexpressed. This RGS gene could encode an inhibitor of EGL-30, a G protein that activates egg laying. We are further testing these ideas by examining loss-of-function mutations in the RGS genes.