Over the past few years, we have identified and characterized a number of genes encoding novel regulators of centrosome size and centriole duplication. All such szy genes were identified in a screen for factors that genetically interact with the kinase ZYG-1, a conserved upstream regulator of centrosome duplication. Analysis of individual szy genes has led to the identification of several molecular pathways that control centriole duplication by controlling the expression levels of centriole assembly factors. Over the past two years we have mainly continued to work on two pathways of particular interest, one of which operates at a post-transcriptional level and a second that operates at a transcriptional level. The first pathway involves the activity of protein phosphatase I (PP1) in negatively regulating centrosome duplication. Loss of either the PP1- isoform GSP-1 or one of two highly conserved PP1 regulators (named I-2 and SDS-22) suppresses the centriole assembly defect of a zyg-1 hypomorphic mutation. This suggests that PP1 normally opposes the activity of ZYG-1, and accordingly we find a moderate increase in the level of ZYG-1 at centrosomes in embryos compromised for PP1 activity. Furthermore we find that down regulation of PP1 activity results in a three- to five-fold increase in the total cellular levels of ZYG-1 indicating that PP1 functions to limit expression of ZYG-1. As zyg-1 mRNA levels are unaffected by inhibition of PP1 activity, PP1 appears to act post-transcriptionally. Additionally we have found that PP1 does not act to regulate translation of zyg-1 through the 3-utr. In sum, our results indicate that PP1 acts post-translationally to control the stability of ZYG-1. Interestingly, we find that in a zyg-1(+) background, strong down regulation of either I-2, SDS-22, or PP1 results in the overproduction of centrioles, the formation of multipolar spindles and ultimately lethality. Using structured illumination microscopy (SIM) we have confirmed the centriole over-duplication defect and have found that more than one daughter forms next to each mother centriole. Currently we are trying to identify the molecular target(s) of PP1. The most obvious candidate is ZYG-1, which contains a near-consensus PP1-docking motif. We have taken several approaches to determine if PP1 and ZYG-1 physically interact. These include co-immunoprecipitation (co-IP) experiments to determine if ZYG-1 and GSP-1 reside in a complex in vivo and mutating zyg-1 to ablate the docking motif and determine if this affects ZYG-1 protein levels. So far we have not been able to detect an interaction between ZYG-1 and PP1, I-2, or SDS-22. However it is possible that co-IP experiments failed to detect an interaction because it is transient in nature. To address this we have used CRISPR-CAS9 genome editing to fuse PP1 to the biotin ligase birA and ZYG-1 to the biotin-acceptor tag. We have co-expressed these two fusion proteins in the same strain and find that ZYG-1 is reproducibly biotinylated suggesting that the two proteins physically interact in vivo. We are currently testing the specificity of this interaction. The second pathway of interest regulates centriole duplication at a transcriptional level. This is an important yet overlooked area of investigation as most studies have focused on post-transcriptional mechanisms of regulation. We have found that in the worm, the heterodimeric transcription factor E2F-DP1 represses centrosome duplication. Specifically we find that loss of E2F or DP1 activity can suppress a zyg-1 hypomorphic allele. Accordingly, conserved binding sites for this transcription factor are found in the promoter regions of most of the centriole duplication genes including zyg-1 and sas-6, and recent ChiP-on-Chip analysis demonstrates that these sites are bound in vivo by E2F-DP1 (Kudron et al. 2013 Genome Biol. 14: R5). We have performed qRT-PCR analysis of transcript levels in wild-type and dpl-1 mutants and find that E2F-DP1 is likely to play a positive role in regulating transcription of centriole duplication genes. Consistent with this, deletion of E2F-binding site in the promoter of ZYG-1 abolishes expression. Despite our finding that E2F-DP1 plays a positive role in regulating expression of centriole duplication genes including SAS-6, we find that loss of E2F-DP1 specifically results in an increase in the level of SAS-6 protein. This seemingly contradictory result can be explained if E2F-DP1 also positively regulates the expression of a gene that down regulates SAS-6 protein levels. Our data thus favors a model whereby E2F-DP1 through its role in transcription sets the balance between positive and negative regulators of centriole duplication and that a partial loss of E2F-DP1 tips the balance in favor of the positive regulators. This past year we completed our work on this project and published our findings (Miller et al., 2016 G3 6:709-720). In a related study, we have been characterizing yet another regulator of centriole duplication. The chromodomain helicase CHD-1 is known to positively and negatively regulate transcription. Among its known targets are genes encoding components of the centriole duplication pathway. Interestingly, CHD-1 co-precipitates with the ZYG-1 ortholog Plx4 in Xenopus egg extracts (Hatch et al. 2010 JCB 191: 721) suggesting the possibility that CHD-1 regulates centriole duplication. Consistent with this idea, we have found that RNAi of the worm chd-1 homolog suppresses the centriole duplication defect of the zyg-1(it25) mutant. We have also constructed a chd-1 null allele and find that it also suppresses zyg-1(it25). Curiously, mutation of the ATPase domain of the endogenous protein leads to suppression, while mutation of the DNA-binding domain does not. Recently we have found that loss of chd-1 affects expression of certain components of the centriole assembly pathway. Specifically, chd-1 mutants show an increase in the levels of SPD-2 and SAS-6 proteins. Interestingly, spd-2 mRNA levels are also elevated in the mutant while sas-6 mRNA levels are unaffected. Our results indicate that CHD-1 regulates centriole duplication via an effect on transcription. Finally, we have also launched a new project aimed at understanding how ZYG-1 might regulate the downstream components of the centriole duplication pathway. SAS-5 and SAS-6 are coiled-coil-domain-containing proteins that form the structural scaffold of the centriole. These proteins are known to form dimers and higher order oligomers that are important for their function. However, the potential role of ZYG-1 in regulating the oligomeric state of these proteins has not been investigated. We have expressed full-length recombinant proteins in E. coli and purified them to near homogeneity. We have found that ZYG-1 and SAS-5 physically interact in vitro and that ZYG-1 is capable of phosphorylating SAS-5 in vitro. We have mapped the phosphorylation sites and have begun using CRISPR-Cas9 genome editing to mutate these phosphorylations sites in the endogenous SAS-5 protein. We also are currently using analytical ultracentrifugation to study how oligomerization of SAS-5 and SAS-6 in vitro might be affected by ZYG-1. Together these experiments might allow us to better understand how these factors interact on a molecular level to build a nine-fold symmetric centriole.

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14
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2016
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U.S. National Inst Diabetes/Digst/Kidney
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Schwarz, Anna; Sankaralingam, Prabhu; O'Connell, Kevin F et al. (2018) Revisiting Centrioles in Nematodes-Historic Findings and Current Topics. Cells 7:
Peel, Nina; Iyer, Jyoti; Naik, Anar et al. (2017) Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication. PLoS Genet 13:e1006543
Miller, Jacqueline G; Liu, Yan; Williams, Christopher W et al. (2016) The E2F-DP1 Transcription Factor Complex Regulates Centriole Duplication in Caenorhabditis elegans. G3 (Bethesda) 6:709-20
O'Connell, Kevin F; Golden, Andy (2014) Confocal imaging of the microtubule cytoskeleton in C. elegans embryos and germ cells. Methods Mol Biol 1075:257-72
Peel, Nina; Dougherty, Michael; Goeres, Jacqueline et al. (2012) The C. elegans F-box proteins LIN-23 and SEL-10 antagonize centrosome duplication by regulating ZYG-1 levels. J Cell Sci 125:3535-44
Decker, Markus; Jaensch, Steffen; Pozniakovsky, Andrei et al. (2011) Limiting amounts of centrosome material set centrosome size in C. elegans embryos. Curr Biol 21:1259-67
Song, Mi Hye; Liu, Yan; Anderson, D Eric et al. (2011) Protein phosphatase 2A-SUR-6/B55 regulates centriole duplication in C. elegans by controlling the levels of centriole assembly factors. Dev Cell 20:563-71
Peters, Nathaniel; Perez, Dahlia E; Song, Mi Hye et al. (2010) Control of mitotic and meiotic centriole duplication by the Plk4-related kinase ZYG-1. J Cell Sci 123:795-805
Kumfer, Kraig T; Cook, Steven J; Squirrell, Jayne M et al. (2010) CGEF-1 and CHIN-1 regulate CDC-42 activity during asymmetric division in the Caenorhabditis elegans embryo. Mol Biol Cell 21:266-77
Song, Mi Hye; Aravind, L; Muller-Reichert, Thomas et al. (2008) The conserved protein SZY-20 opposes the Plk4-related kinase ZYG-1 to limit centrosome size. Dev Cell 15:901-12

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