Cardin, Derrick L. et al. (2013) International Worm Meeting "Activity and functional interactions of the leucine-rich protein PAN-1 during larval development."

Gissendanner, Chris R., Cardin, Derrick L.

[International Worm Meeting, 2013]

PAN-1 is a novel nematode-specific leucine-rich (LRR) protein that exhibits complexity in both structure and function. The pan-1 gene encodes three protein isoforms: a predicted type I transmembrane protein with extracellular LRRs and two cytoplasmic proteins, one with a smaller number of LRRs and one lacking LRRs. PAN-1 participates in multiple developmental processes. It associates with P-granules in the germline (Gao et al, 2012) and is also required for larval development. For the latter, pan-1 is necessary for early larval development and the L4 to adult transition. pan-1 promotes growth of the germline, somatic gonad, and vulva during later larval stages and is required for progression of the L4 to adult molting cycle. To better understand the function of PAN-1 during larval development we have initiated a series of studies to address the cellular activities of this protein. Expression of the transmembrane isoform of PAN-1 lacking a cytoplasmic domain is dominant-negative, indicating that PAN-1 may dimerize and transduce an intracellular signal. Using these dominant-negative effects as an assay, we are performing conditional expression of different cytoplasmic domain-deleted constructs to identify critical regions of the intracellular domain. We are also characterizing interactions between pan-1 and other genes. We have found that lin-29 loss of function suppresses the molting progression and vulva development phenotypes in pan-1 RNAi animals. We are further characterizing this interaction to determine how pan-1 intersects with lin-29 in molting regulation. In addition, loss of function of lron-12, which encodes a secreted LRR protein, enhances the pan-1 RNAi phenotype. lron-12 mutants exhibit a larval growth phenotype indicating that pan-1 and lron-12 function together to regulate larval development. We propose that PAN-1 participates in a novel nematode-specific signaling pathway regulating life cycle progression.

Ge Gao et al. (2008) Development & Evolution Meeting "A P-granule Associated Novel Protein And Its Relationship To The C.elegans GLHs"

Ge Gao, Karen Bennett

[Development & Evolution Meeting, 2008]

P-granules are germline-specific protein and RNA complexes found surrounding the nuclei of C. elegans germ cells and germ cell precursors. GLH (germline RNA helicase) proteins are components of the P-granules and are necessary for fertility in C. elegans. PAN-1, a P-granule Associated Novel protein, was identified as a GLH-binding partner by yeast two hybrid assays. This binding was also confirmed by pull down assays after in vitro co-expression of His-tagged PAN-1 and GST-tagged GLH-1 in baculovirus. When pan-1 is eliminated by RNA interference (RNAi), C. elegans larvae arrest between the L1 and L2 stages and can survive eight days at 20oC (equal to several generations in wild type worms). After backcrossing to eliminate potential non-specific mutations, a pan-1(gk142) deletion strain exhibits the same "forever-young" phenotype as seen by RNAi. mRNA analysis and protein expression studies show that PAN-1 is not germline specific but is germline enhanced. To further study PAN-1 germline function, we made use of the rrf-1 strain. rrf-1 encodes an RNA directed RNA polymerase required for RNAi in C. elegans somatic tissue. By doing pan-1 RNAi into the rrf-1 strain, the pan-1 larval arrest phenotype is rescued and PAN-1 protein is expressed in the somatic tissue while it is eliminated in the germline. Use of pan-1(RNAi) in the rrf-1 strain gives us the ability to study pan-1 germline function. Our data show that GLH-1 protein levels are somewhat down regulated in the rrf-1 strain after pan-1(RNAi) compared to the rrf-1 strain alone, suggesting that PAN-1 could function as a regulatory protein that affects C. elegans germline development. pan-1(RNAi) into the rrf-1 strain also results in fewer progeny compared to the rrf-1 stain alone or to wild type worms. Experiments are ongoing to investigate whether PAN-1 co localizes with P-granule proteins such as PGL-1 and GLH-1. If PAN-1 regulates the GLHs, we predict it might form a regulatory complex with two other GLH binding partners, CSN-5 and KGB-1.

Li, Meng et al. (2019) International Worm Meeting "PAN-1 interacts with MYRF to regulate synaptic rewiring in DDs."

Xia, Shili, Qi, Yingchuan, Li, Meng, Shao, Dongyu

[International Worm Meeting, 2019]

In C. elegans, six GABAergic neurons (named DD neurons) rewire during development by reversing their axonal and dendritic domains without other obvious morphological transitions. This unique event provides an attractive system in which we can identify the molecular requirements of circuit remodeling. We have previously identified the conserved gene myrf-1 and its paralog myrf-2 as key positive regulators of DD neuron rewiring. Using immunoprecipitation of GFP::MYRF-1 from C. elegans tissues followed by quantitative mass spectrometry analyses, we have identified PAN-1 (P-granule Associated Novel protein), a novel leucine rich repeat (LRR) domain-containing protein, as an interactor of MYRF in vivo. pan-1 null mutants exhibit blocked synaptic rewiring in DD neurons. The membrane-associated isoform of PAN-1B rescues synaptic rewiring cell-autonomously. Over-expression of MYRF restores the rewiring defect of pan-1(0) mutants, indicating that PAN-1 acts upstream of MYRF.

Ge Gao et al. (2004) Mid-west Worm Meeting "Loss of pan-1 causes a Peter Pan-like phenotype in C. elegans"

Ge Gao, Karen Bennett

[Mid-west Worm Meeting, 2004]

P-granules are complexes of proteins and RNA found surrounding the nuclei of C. elegans germ cells and germ cell precursors. GLH (germline RNA helicase) proteins are components of the germline specific P-granules, which are necessary for fertility in C. elegans . PAN-1, a P-granule associated novel protein, was identified as a GLH-binding protein in yeast two hybrid assays. PAN-1 contains some conserved amino acids of N-terminal F-box motifs, as well as sixteen leucine-rich repeats and a weak FOG-2 homology (FTH) motif, each found in F-box proteins. F-box proteins, in the SCF (SKP-1, Cullin, F-box) complex, utilize ubiquitin-mediated substrate degradation. When pan-1 is eliminated by RNA interference (RNAi), the larvae arrest between the L1 and L2 stages and can survive eight days at 20 0 C. A pan-1(gk142) deletion strain exhibits the same 'forever-young' phenotype. mRNA analysis and protein expression show that PAN-1 is not germline specific but is germline enhanced. Experiments are ongoing to separate potential germline and somatic functions of PAN-1. If PAN-1 belongs to the family of F-box proteins, it may be implicated in regulating GLH protein levels, as are two other GLH binding proteins, CSN-5 and KGB-1.

Ge Gao et al. (2006) Development & Evolution Meeting "Loss Of pan-1 Causes a Peter Pan-like Phenotype in C. elegans"

Karen Bennett, Ge Gao

[Development & Evolution Meeting, 2006]

P-granules are complexes of proteins and RNA found surrounding the nuclei of C. elegans germ cells and germ cell precursors. GLH (germline RNA helicase) proteins are components of the germline specific P-granules, which are necessary for fertility in C. elegans. PAN-1, a P-granule Associated Novel protein, was identified as a GLH-binding protein in yeast two hybrid assays. PAN-1 contains some conserved amino acids of N-terminal F-box motifs, as well as sixteen leucine-rich repeats and a weak FOG-2 homology (FTH) motif, each found in F-box proteins. F-box proteins, in the SCF (SKP-1, Cullin, F-box) complex, utilize ubiquitin-mediated substrate degradation. When pan-1 is eliminated by RNA interference (RNAi), C. elegans larvae arrest between the L1 and L2 stages and can survive eight days at 20ºC, which is equal to several generations in wild type worms. After backcrossing to eliminate potential non-specific mutations, a pan-1(gk142) deletion strain exhibits the same "forever-young" phenotype as seen by RNAi. mRNA analysis and protein expression studies show that PAN-1 is not germline specific but is germline enhanced. Experiments are ongoing to confirm the PAN-1 binding to GLHs in the baculovirus system, as well as potential germline functions of PAN-1. If PAN-1 belongs to the very large (>400) family of C. elegans F-box proteins, it may be implicated in regulating GLH protein levels, as are two other GLH binding proteins, CSN-5 and KGB-1.

Leyva-Diaz, Eduardo et al. (2017) International Worm Meeting "Identification of transcription factors regulating pan-neuronal gene expression."

Stefanakis, Nikolaos, Hobert, Oliver, Leyva-Diaz, Eduardo, Carrera, Ines

[International Worm Meeting, 2017]

While neuronal cell types in a nervous system display an astounding degree of diversity in their molecular composition, all neuron types share a panel of common features defined by the expression of pan-neuronal genes. Though a great deal is known about how the expression of neuron-type specific identity features is controlled in the nervous system, there is a lack of knowledge into which factors are controlling pan-neuronal gene expression programs. Through an extensive analysis of the cis-regulatory control region of a battery of pan-neuronal genes, we addressed several distinct scenarios for how pan-neuronal gene expression could be achieved. We found that pan-neuronal gene expression is controlled through a surprisingly complex array of redundantly acting cis-regulatory modules that direct expression to broad, overlapping domains through the nervous system. These redundantly acting cis-regulatory modules are responsive to a host of distinct trans-acting factors. We found that terminal selector factors and HOX genes as some examples of these factors, however the identity of most of them remains unknown. We are currently conducting multiple genetic screens to identify these trans-acting factors that bind to those cis-regulatory elements and control pan-neuronal gene expression. Our first mutants are presently being cloned and tested for involvement in the regulation of different pan-neuronal genes. In addition, we are performing a small-scale cis-regulatory analysis of a conserved vertebrate pan-neuronal gene to test whether this redundant modular model is conserved in vertebrates. Identifying factors involved in pan-neuronal gene regulation will provide insights into how the fully differentiated neuronal fate is developed, and might reveal key regulators of neuronal identity maintenance during normal development and neurodegeneration.

Ilya Ruvinsky et al. (2005) International Worm Meeting "A cis-regulatory motif that allows identification of pan-neuronal genes in C. elegans"

Gary Ruvkun, Uwe Ohler, Christopher Burge, Ilya Ruvinsky

[International Worm Meeting, 2005]

Pan-neuronal genes are expressed in most or all types of neurons and thus define generic neuronal features and provide a window into the developmental origin of the nervous system. We computationally predicted a ten-nucleotide cis-regulatory motif shared by many pan-neuronal genes and demonstrated that it is required for broad neuronal expression. Our results suggest that global and cell-type specific controls act in concert to establish pan-neuronal gene expression. Using the newly discovered motif and genome-level gene expression data, we systematically identified a set of 234 candidate pan-neuronal genes, the largest collection of such genes in any organism. The known involvement of many of these genes in neurogenesis and physiology of the nervous system supports the utility of this set for future targeted analyses.

Stefanakis, Nikolaos et al. (2015) International Worm Meeting "Redundant modular control of pan-neuronal gene expression in C. elegans."

Hobert, Oliver, Carrera, Ines, Stefanakis, Nikolaos, Leyva-Diaz, Eduardo

[International Worm Meeting, 2015]

While neuronal cell types in a nervous system display an astounding degree of diversity in their molecular composition, all neuron types share a panel of common features defined by the expression of pan-neuronal genes. Though a great deal is known about how the expression of neuron-type specific identity features is controlled in the nervous system, there is a striking dearth of insight into how pan-neuronal gene expression programs are controlled. We define here a set of 23 conserved genes which are expressed throughout the entire nervous system of C. elegans, most of them encoding for genes involved in synaptic vesicle biology and neuropeptide processing. Through an extensive analysis of the cis-regulatory control region of this pan-neuronal gene battery, we address several distinct scenarios for how pan-neuronal gene expression could be achieved. We found that pan-neuronal gene expression is controlled through a surprisingly complex array of redundantly acting cis-regulatory modules that direct expression to broad, overlapping domains throughout the nervous system. These redundantly acting cis-regulatory modules are responsive to a host of distinct trans-acting factors. We found that terminal selector factors and HOX genes as some examples of these factors, however the identity of most of them remains unknown. We are currently conducting multiple genetic screens to identify these trans-acting factors that bind to those elements and control pan-neuronal gene expression. Neuronal gene expression programs therefore fall into two fundamentally distinct classes. Neuron type-specific genes are generally controlled by discrete and non-redundantly acting regulatory inputs, while pan-neuronal gene expression is controlled by diverse, redundant molecular mechanisms. These two fundamentally distinct strategies of controlling gene expression in the nervous system may be a reflection of the evolutionary history of neuronal cell types.

Hui-Ju Yang et al. (2004) East Asia Worm Meeting "Characterization of TLK-1 function during mitosis"

Yu-Chin Albert Pan, Yi-Chun Wu, Chai-Wei Lee, Hui-Ju Yang, Chi-Ying Huang, Wei-Sin Chou, Chen-Kung Chou, Ju-Ling Liu

[East Asia Worm Meeting, 2004]

Aurora/Ipl1p-related kinases are a conserved family of proteins that have multiple functions during cell cycle progression [1]. A small-pool expression screening for substrates of Aurora-A kinase identified Tlk2, tousled-like kinase 2. To understand the function of Tlk2, we studied its C. elegans homolog tlk-1 . We performed RNA interference (RNAi) to inhibit the expression of the tlk-1 gene in C. elegans . The tlk-1 (RNAi) mutants arrested at about 50-cell stage showing the phenotype of aneupolidy. To understand the cause of aneuploidy, we utilized the strains carrying tubulin-GFP, histon-GFP or both transgenes to monitor the dynamics of chromosomes, centrosomes and spindle formation during cell cycle in living embryos. Time-lapse microscopic analyses revealed defects in DNA condensation and segregation in tlk-1 (RNAi) embryos.TLK-1 protein was expressed in a cell-cycle-dependent manner [2]. It is detected in interphase and prophase nuclei but not in mitotic cells from metaphase through anaphase. As TLK-1 harbors two D-box motifs, which are anaphase-promoting complex (APC)-dependent proteolysis signals, we investigated if these motifs are important for the dramatic disappearance of TLK-1 at metaphase. Mutations in D-box motifs caused the persistent presence of TLK-1 at metaphase, indicating that D box motifs are required for the degradation of TLK-1 at metaphase. The C. elegans mat-1 , mat-2 and emb-30 encodes subunits of APC. When any of these genes is mutated, TLK-1 protein is abnormally present at metaphase. In addition, we found the APC activator, CDH-1 but not CDC-20, is responsible for the cell-cycle-dependent degradation of TLK-1. We are currently investigating the physiological significance of APC-mediated TLK-1 degradation. Interestingly, human Tlk2 can partially rescued the lethality of tlk-1 (RNAi) embryos, suggesting the function of tlk-1 is conserved in evolution. 1. Bischoff, J. R. and G. D. Plowman. Trends Cell Biol. 9.11 (1999): 454-59.2. Han, Z. et al. Curr.Biol. 13.22 (2003): 1921-29.

Juan Wang et al. (2004) West Coast Worm Meeting "Characterization of a mutant affecting ADPKD Homologue Gene Expression and Male Mating"

Juan Wang, Maureen M Barr

[West Coast Worm Meeting, 2004]

lov-1 and pkd-2 are the homologues of the human autosomal dominant polycystic kidney disease (ADPKD) genes PKD1 and PKD2. lov-1 and pkd-2 are required for male mating behavior and function as male sensory genes. These two genes are exclusively expressed in male specific neurons: four head CEMs, ray RnBs rarely (in R2B and R6B) and hook HOB from late L4 through adulthood. How lov-1 and pkd-2 are temporally and spatially regulated is largely unknown. egl-46 is required for pkd-2 expression in the HOB neuron, but not the CEMs or RnBs (Yu et al. 2003). daf-19 , an RFX transcription factor, is required for expression of several cilium structure genes (Swoboda et al. 2000) as well as pkd-2 (Yu et al. 2003). We are interested in understanding the mechanisms underlying lov-1 and pkd-2 transcriptional regulation, male sensory neuronal development, and male mating behavior. n4132 was obtained in a screen for ceh-30 suppressors (See 2004 East Coast Worm Meeting abstract 248 Varner et al. ) and found to prevent pkd-2 expression. Our further characterization shows that the n4132 mutant lacks expression of both lov-1 and pkd-2 but not cilia structure genes such as osm-5 and osm-6 . In contrast to daf-19 mutants, we propose that n4132 disrupts transcriptional regulation of certain male sensory genes but not general cilia structure genes. I will describe the mapping, cloning, and molecular characterization of this new gene. References Swoboda P. et al. (2000). Molecular Cell 5: 411-421. Yu, H. et al. (2003). Development 130(21): 5217-5227.