Pop, C et al. (2005) Cell "The nematode death machine in 3D."

Pop, C, Salvesen, GS

[Cell, 2005]

Regulated apoptosis is part of the development of the nematode Caenorhabditis elegans. In a recent paper in Nature, Yan et al. (2005) describe the in vitro reconstitution of the core components of the worm apoptotic pathway. Based on a structural analysis of the complex between the death activator CED-4 and the antiapoptotic protein CED-9, the authors explain the regulation of activity of CED-4. Intriguingly, CED-4 comprises a AAA+ type ATPase domain yet does not seem to need ATP hydrolysis for activity.

Lin, Rueyling et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "WRM-1 functions as a scaffold for phosphorylation of POP- 1 by LIT-1 kinase"

Robertson, Scott, Huang, Shuyi, Lin, Rueyling

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

During C. elegans embryogenesis, Wnt and MAP kinase signaling pathways specify the endoderm precursor E. We showed previously that whereas Wnt increases the level of ?-catenin/SYS-1, MAP kinase reduces the level of TCF/POP-1, resulting in a robust increase in the SYS-1-to-POP ratio and transcriptional activation of endoderm genes in E. The ?-catenin SYS-1 was shown to bind to the conserved N-terminus of POP-1, functioning as an essential coactivator for POP-1 to mediate Wnt signaling. A second ?-catenin protein, WRM-1, is also required for E specification. WRM-1 does not, however, bind to the N-terminal domain of POP-1, nor does it function as a coactivator of POP-1. Instead, WRM-1 has been shown to be an obligate component of the LIT-1 kinase complex, together phosphorylating POP-1 which promo tes its nuclear export, resulting in lowered nuclear POP-1 levels in signal-receiving cells. While the requirement of WRM-1 for LIT-1 activity has been clearly established, the molecular mechanism by which WRM-1 regulates LIT-1 kinase activity remains unclear. We show here that WRM-1 functions as a scaffold protein that brings POP-1 and LIT-1 into close proximity. Unlike SYS-1 binding to the N-terminal domain, WRM-1 binds to the C-terminal domain of POP-1. POP-1 deleted of the C-terminal domain does not bind to WRM-1, nor does it exhibit LIT-1-mediated phosphorylation in tissue culture cells. In vivo, POP-1 delta C is symmetric between A-P nuclei, and has a reduced activity, both in repression and activation. We show that binding between WRM-1 and POP-1 is regulated: binding decreased when LIT-1 kinase was coexpressed in mammalian tissue culture cells, with the decrease preferentially in hyperphosphorylated POP-1. An epitope at the C-terminal domain is differentially p resented in different populations of POP-1, suggesting a possible mode of regulation in vivo.

Miao-Chia Lo et al. (2002) West Coast Worm Meeting "Nuclear POP-1 asymmetry between A-P sisters is regulated by a 14-3-3 mediated nuclear export mechanism"

Miao-Chia Lo, Rueyling Lin

[West Coast Worm Meeting, 2002]

We have shown previously that the HMG domain protein POP-1 is asymmetrically localized in the nuclei of all sisters born of A-P divisions using a monoclonal antibody to the POP-1 protein. In most cases examined, the POP-1 asymmetry is required for A-P fate differences between sisters. We and others have shown that the A-P fate difference and POP-1 asymmetry between MS and E sisters require a Wnt/MAPK signaling pathway. How the Wnt/MAPK signaling results in a lower level of nuclear POP-1 and POP-1 activity in the posterior sister is not clear. Using a functional POP-1-GFP reporter expressed from an exogenous promoter, Maduro and Rothman have shown that the coding region of POP-1 is sufficient to confer the asymmetric pattern observed by POP-1 antibody staining. Moreover, the overall level of POP-1 in A-P sisters is equal and the apparent difference in the nuclear POP-1 is a result of differential nuclear-cytoplasmic distribution. However, the mechanism by which POP-1 is transported between cytoplasm and nuclei as well as how the differential distribution is achieved between A-P sisters remain unclear.

Roehrig, Casey et al. (2009) International Worm Meeting "POPping the Patterning Question."

Hunter, Craig, Roehrig, Casey

[International Worm Meeting, 2009]

In C. elegans, many cell fate decisions rely on a variant of the canonical Wnt signaling pathway in which post-translational modifications of the TCF transcription factor, POP-1, modulate its interactions with cofactors that regulate its activity. These interactions allow POP-1 to both activate and repress a number of different genes to determine the fates of cells throughout the animal. For example, in the EMS lineage, POP-1 has been implicated in both the activation and repression of endodermal genes including end-3 (Maduro et al. 2005). In the C lineage, POP-1 has been shown to play a role in distinguishing between skin and muscle cell fates. Loss of pop-1 causes the C lineage to generate only muscle, whereas loss of its regulator, the kinase lit-1, causes C to produce hypodermis (Fukushige and Krause 2005). The role that POP-1 plays in this process, however, remains unclear. Does it bind to and repress muscle gene expression, or promote the transcription of epidermal genes, or both? To better understand POP-1''s role in this decision, we are investigating the binding of POP-1 to the promoters of transcription factors required for skin and muscle cell fates. We have performed chromatin immunoprecipitation of embryos carrying a GFP-POP-1 fusion protein that is expressed either in the EMS lineage or the C and D lineages. Semi-quantitative PCR analysis suggests that POP-1 binds to the promoters of transcription factors involved in skin and muscle specification in a lineage-specific fashion to coordinate cell fate determination. Further analysis and identification of binding sites are underway to clarify POP-1''s function in cells destined to take on different fates and to determine how POP-1 interacts with other factors required for these cell fate decisions.

KM Mickey et al. (1999) International C. elegans Meeting "Specifying cell fates in the C lineage"

KM Mickey, JR Priess

[International C. elegans Meeting, 1999]

The daughters of the C blastomere, Ca and Cp, have nearly identical lineage patterns. However, Ca produces two neurons and has high levels of the POP-1 protein, while Cp does not make any neurons and has low levels of POP-1. We wondered if pop-1 plays a role in the Ca/Cp difference since pop-1(+) is required to specify many other a/p differences in the early embryo. Using a neuronal GFP marker (provided by D. Pilgrim), we find that neither Ca nor Cp produces neurons in embryos lacking POP-1 ( pop-1RNAi ). In embryos with high levels of POP-1 (such as mom-2(wingless);apr-1(apc)RNAi double mutants), both Ca and Cp can produce neurons. Epistasis analysis indicates that pop-1(+) activity is required for Cp to make neurons in the mom-2;apr-1 double. These results indicate that high levels of POP-1 are required for the ability of Ca to make neurons and that the asymmetry of POP-1 between Ca and Cp is important for making these sisters different. When Ca and Cp divide, they each produce an anterior daughter that has high levels of POP-1 and makes hypodermis, and a posterior daughter that has low levels of POP-1 and makes muscle. Does pop-1(+) play a role in this a/p difference? When Caa or Cpa, the hypodermal precursors, are isolated in pop-1(RNAi) embryos, they each make muscle and appear to lack hypodermis, suggesting that pop-1(+) is required. We noticed that when we lineaged Cpa in an intact pop-1(RNAi) embryo, it produced hypodermal cells. This surprising result suggests a possible cell-cell interaction influencing cell fate decisions in the pop-1 mutant embryos. We currently are investigating the biological relevance of such an interaction. We are interested in identifying factors that may act with POP-1 to specify cell fates in the daughters and granddaughters of the C blastomere. We have isolated a mutant, zu352 , that is defective in specifying the fate of Cp. In zu352 mutant embryos, Cp makes intestine while Ca produces hypodermis and muscle. We are interested in how the cell fates of Ca and Cp in zu352 are specified, and if pop-1(+) plays a role in this Ca/Cp difference.

Kenneth Thompson et al. (2008) Development & Evolution Meeting "Identification of Transcriptional Targets and Characterization of Binding Sites for the C. elegans TCF /LEF Protein POP-1"

Kenneth Thompson, David Eisenmann

[Development & Evolution Meeting, 2008]

In C. elegans, Wnt signaling functions in endoderm induction, Q neuroblast migration, P12 and vulva precursor cell fate specification, and T cell and somatic gonad precursor cell polarity. Key components of this pathway are members of the beta-catenin (BAR-1 and SYS-1) and TCF/LEF (POP-1) families of proteins. Two of the C. elegans beta-catenin homologs, BAR-1 and SYS-1, interact with POP-1 to activate gene expression. POP-1 has been shown to bind DNA via a high mobility group DNA binding domain in a sequence specific manner. To date, only a few POP-1 target genes have been identified. Thus, the identification of POP-1 binding sites within the C. elegans genome will potentially aid in the identification of POP-1 target genes. In order to identify POP-1 target genes, I am using a selection and amplification method with the DNA-binding domain of POP-1 to identify genomic binding sequences. Fragments bound by POP-1 utilizing this technique are subjected to alignment and sequence comparison with the C. elegans genome to identify putative gene targets. To date, my results using this method have yielded 280 fragments (118 from the third round and 162 from the fourth round) with 20 of these fragments repeated in both rounds. Bioinformatic analysis of these fragments identified a common sequence motif of TTTTTGA, which shows similarity to known POP-1 binding sites. In support of this motif, EMSA analysis has shown that unlike other members of the TCF/LEF family, POP-1 may equally prefer a C or a T in the first position of the binding site. Previous work has identified POP-1 binding sites that resemble those of other TCF/LEF family members. However, the DNA-binding domain of POP-1 shows several amino acids changes where DNA contact is made compared to other TCF/LEF family members. We hypothesize that POP-1 may have affinity for sites dissimilar to known TCF/LEF family member binding sites. To identify novel POP-1 binding sites, I am carrying out a bacterial one-hybrid screen using a bait/prey interaction to activate transcription of a reporter gene. In this procedure, I will use a randomized 18-mer oligonucleotide library as the binding site prey and the DNA binding domain of POP-1 as the bait. Control experiments with a known POP-1 binding site in the prey vector and the DNA-binding domain of POP-1 in the bait vector have verified the use of this system. The construction of the prey reporter library is underway and further results will be reported.

Yang XD et al. (2011) Development "Distinct and mutually inhibitory binding by two divergent -catenins coordinates TCF levels and activity in C. elegans."

Xu W, Yang XD, Sawa H, Lin R, Robertson S, Huang S, Lo MC, Mizumoto K

[Development, 2011]

Wnt target gene activation in C. elegans requires simultaneous elevation of -catenin/SYS-1 and reduction of TCF/POP-1 nuclear levels within the same signal-responsive cell. SYS-1 binds to the conserved N-terminal -catenin-binding domain (CBD) of POP-1 and functions as a transcriptional co-activator. Phosphorylation of POP-1 by LIT-1, the C. elegans Nemo-like kinase homolog, promotes POP-1 nuclear export and is the main mechanism by which POP-1 nuclear levels are lowered. We present a mechanism whereby SYS-1 and POP-1 nuclear levels are regulated in opposite directions, despite the fact that the two proteins physically interact. We show that the C terminus of POP-1 is essential for LIT-1 phosphorylation and is specifically bound by the diverged -catenin WRM-1. WRM-1 does not bind to the CBD of POP-1, nor does SYS-1 bind to the C-terminal domain. Furthermore, binding of WRM-1 to the POP-1 C terminus is mutually inhibitory with SYS-1 binding at the CBD. Computer modeling provides a structural explanation for the specificity in WRM-1 and SYS-1 binding to POP-1. Finally, WRM-1 exhibits two independent and distinct molecular functions that are novel for -catenins: WRM-1 serves both as the substrate-binding subunit and an obligate regulatory subunit for the LIT-1 kinase. Mutual inhibitory binding would result in two populations of POP-1: one bound by WRM-1 that is LIT-1 phosphorylated and exported from the nucleus, and another, bound by SYS-1, that remains in the nucleus and transcriptionally activates Wnt target genes. These studies could provide novel insights into cancers arising from aberrant Wnt activation.

Morris Maduro et al. (2008) Development & Evolution Meeting "Your Pop's a Mom: An E to MS transformation in C. briggsae pop-1(RNAi) embryos"

Morris Maduro, Serena Cervantes, Katy Lin, Gina Broitman-Maduro, Wendy Hung

[Development & Evolution Meeting, 2008]

In C. elegans, the bZIP/homeodomain transcription factor SKN-1 and the Wnt effector TCF/POP-1 are central to the maternal specification of the endomesoderm prior to gastrulation. The 8-cell stage blastomere MS is primarily a mesodermal precursor, giving rise to cells of the pharynx and body muscle among others, while its sister E clonally generates the entire endoderm (gut). In C. elegans, loss of SKN-1 results in the absence of MS-derived tissues all of the time, and loss of gut most of the time, while loss of POP-1 results in a mis-specification of MS as an E-like cell, resulting in ectopic gut (the Pop phenotype). We have found that in C. briggsae, RNAi of the skn-1 ortholog results in a stronger E defect but no apparent defect in production of GLP-1-independent pharynx, one of the tissues made by MS. Laser ablations confirm that MS still makes pharynx muscle in Cb-skn-1(RNAi) embryos. Second, RNAi of pop-1 results in a highly penetrant loss of gut, and both MS and E make pharynx cells - an apparent Mom phenotype. This is the opposite of the pop-1 knockdown phenotype seen in C. elegans, in which both MS and E make gut. RNAi of the divergent beta-catenin gene Cb-sys-1 gives a much more penetrant phenotype, approximately 50% gutless, suggesting that the positive contribution of POP-1 to endoderm in C. briggsae requires interaction of Wnt-signaled POP-1 with SYS-1, just as in C. elegans. By in situ hybridization, we find that the difference in pop-1(-) phenotypes correlates with changes in how the endogenous endoderm-specifying end genes are regulated in the two species: Whereas end-1 and end-3 in C. elegans are de-repressed in MS in pop-1(RNAi), mRNAs for the C. briggsae end genes (Cb-end-1, Cb-end-3.1 and Cb-end-3.2) are no longer detectable in Cb-pop-1(RNAi). One hypothesis is that a change in cis-regulation is responsible for the difference in pop-1(-) readout. To test this, we introduced a Cb-end-3.2::GFP reporter into C. elegans. While the reporter is activated in the early E lineage in a manner similar to Ce-end-1/3 reporters, pop-1(RNAi) results in de-repression of Cb-end-3.2::GFP in the MS lineage. With the caveat that we may not have included all regulatory sites, this result suggests that the POP-1 regulation differences cannot be collectively explained solely by promoter changes. As Cb-POP-1 can complement maternal loss of Ce-pop-1 in C. elegans, we hypothesize that degeneracy of the SKN-1 pathway may account for the differences we are seeing in C. briggsae. Curiously, C. remanei pop-1(RNAi) results in an apparent ectopic endoderm phenotype, further suggesting that these changes have evolved on a relatively short timescale (tens of millions of years), as C. remanei and C. briggsae are considered to be more closely related. Our results suggest that both Wnt-dependent and Wnt-independent cell fate specification pathways in Caenorhabditis embryos have an unexpected degree of flexibility.

Yang, X. et al. (2011) International Worm Meeting "Distinct and mutually inhibitory binding by two b-catenins, SYS-1 and WRM-1, coordinates TCF/POP-1 nuclear levels and activity."

Lin, R., Huang, S., Yang, X., Xu, W., Robertson, S., Lo, M.

[International Worm Meeting, 2011]

We have shown that specification of endoderm requires a high b-catenin/SYS-1 to TCF/POP-1 ratio in the nucleus of E blastomere. Similar to vertebrate b-catenin binding to the N-terminal ~50 amino acids of TCF, SYS-1 binds to the N-terminal domain of POP-1 and functions as a coactivator in the activation of endoderm genes. Elevation of SYS-1 levels in E is dependent entirely on the Wnt signaling pathway, whereas lowering of nuclear POP-1 levels is regulated primarily by the MAP kinase pathway. The MAP kinase LIT-1, the C. elegans NLK homolog, phosphorylates POP-1, its only known substrate, promoting its nuclear export, the main mechanism for lowering nuclear POP-1 levels. We and others have shown previously that phosphorylation of POP-1 by LIT-1 requires a diverged b-catenin, WRM-1, although the precise molecular function of WRM-1 remains unclear. WRM-1 does not bind to the N-terminal domain of POP-1, function as a POP-1 coactivator, or function in cell adhesion. We demonstrate here two independent and distinct molecular functions for WRM-1: it serves as both the substrate-binding subunit and a regulatory subunit for the LIT-1 kinase. In addition, we present here a molecular mechanism by which a high nuclear level of SYS-1 can be maintained in E while its binding partner POP-1 is simultaneously exported out of nucleus. We show that WRM-1 binds to the C-terminus of POP-1, and that this binding is essential for all LIT-1-mediated POP-1 phosphorylation. The POP-1 C-terminus resembles the N-terminal SYS-1-binding domain in primary sequence. Computer-based structural analyses provide an explanation for the observed specificities in POP-1/WRM-1 and POP-1/SYS-1 binding. It has been shown that the POP-1/SYS-1 interaction primarily utilizes only one (extended strand) of the two TCF structural modules employed in the vertebrate TCF/b-catenin interaction. Structural modeling predicts that the POP-1/WRM-1 interaction primarily utilizes the other structural module (alpha helix). Finally, we show that the SYS-1/POP-1 (N-terminus) and WRM-1/POP-1 (C-terminus) interactions are mutually inhibitory, providing a molecular mechanism by which levels of the two interacting proteins, SYS-1 and POP-1, can be regulated in opposite directions in the E nucleus. Mutual inhibitory binding would result in two populations of POP-1: one bound by WRM-1 that is LIT-1 phosphorylated and exported from the E nucleus, and the other bound by SYS-1 that remains in the nucleus and transcriptionally activates Wnt target genes.

Calvo D et al. (2000) East Coast Worm Meeting "POP-1, A C. elegans TCF/LEF1 family member, possesses activation and repression domains and is a target of acetylation by CBP-1"

Calvo D, Shi Y

[East Coast Worm Meeting, 2000]

CBP-1 is the C. elegans homolog of mammalian histone acetyltransferase p300/CBP. Our lab has shown recently that CBP-1 is essential for the differentiation of endoderm, mesoderm and hypodermal tissues. In the endoderm differentiation CBP appears to antagonize the repressive activity of histone deacetylases, suggesting that the acetyltransferase activity of CBP plays an important role in endodermal gene activation. Studies in Drosophila and mammals have shown that, besides histones, p300/CBP also regulate the activities of a number of transcription factors through acetylation. These targets of p300/CBP include a Drosophila HMG-containing protein TCF that is related to the C. elegans POP-1protein. During C. elegans early embryogenesis, POP-1 plays an essential role in anterior-posterior cell fate decisions in C. elegans embryogenesis. Lack of POP-1 results in a mesodermal founder cell to adopt endodermal cell fate, suggesting that POP-1 functions to suppress genes important for endodermal fate in the mesodermal founder cell. To investigate the functional relationship between POP-1 and p300/CBP, we wish to determine whether p300/CBP can acetylate POP-1 in vitro and to analyze the consequence of POP-1 acetylation in C. elegans development. We have been able to show that p300 acetylates POP-1 efficiently in vitro and have identified the main lysine residue in POP-1 that is acetylated by p300. We have generated polyclonal antisera specific for this acetylation site and the results of these studies will be discussed. To investigate the molecular mechanisms that underlie the developmental function of POP-1, we also analyzed the transcriptional activity of POP-1. Consistent with its biological activity, we show that POP-1 can repress transcription. We identify a strong transcriptional repression domain that is necessary and sufficient for POP-1 to mediate repression in a GAL-4 fusion protein-based assay. We find multiple sequence motifs within the repression domain that each on its own can mediate repression. Further dissection of one such motif reveals amino acids that are important for the repression activity. POP-1 also possesses an activation domain, similar to its vertebrate homologs that have been shown to both activate and repress transcription.