Peng Wang et al. (2002) East Coast Worm Meeting "Finding Players in the CeTwist Pathway"

Ann K Corsi, Peng Wang

[East Coast Worm Meeting, 2002]

We are interested in understanding the function of the CeTwist protein in the mesoderm as a model for cell-fate specification during development. Previous characterization of a null allele in the CeTwist gene, hlh-8 (nr2061), showed that this protein plays a role in non-striated muscle development (1). Animals with a null mutation in hlh-8 do not properly form sex muscles, which leads to an egg-laying defective phenotype (1). A second semidominant allele hlh-8 (n2170) has also been characterized (2). These animals are also egg-laying defective but the cellular defects suggest an earlier blockage in sex muscle development when compared with hlh-8 (nr2061) animals. A suppressor screen is currently underway to identify mutations that can compensate for defects caused by the n2170 allele. We expect this screen will identify proteins that physically interact with CeTwist or are downstream of CeTwist. CeTwist is a member of the basic helix-loop-helix (bHLH) family of transcription factors. CeTwist can function as a heterodimer with CeE/DA, the product of the hlh-2 gene, to turn on egl-15 and ceh-24 (1,3). We have recently identified arg-1 (apx-1 related gene) as another CeTwist target. In order to reveal a complete pathway of CeTwist function in C. elegans mesoderm development, it will be necessary to ultimately identify all downstream target genes of hlh-8. Using DNA microarrays, it is possible to observe global changes in gene expression of nearly every mRNA during development (4). Since hlh-8 is expressed in at most 2% of cells at any given time during development, it may be difficult to obtain meaningful data using DNA microarrays by comparing mRNA from hlh-8 null animals to mRNA from wild type animals. To circumvent this difficulty, we will overexpress both hlh-8 and hlh-2 from heat shock promoters and compare mRNA isolated from this strain to mRNA isolated from isogenic wild type animals. The three known targets genes of CeTwist: egl-15, ceh-24, and arg-1 are overexpressed when both hlh-8 and hlh-2 are overexpressed from the heat shock promoter, and we predict unknown targets will also be overexpressed. To avoid measuring a secondary cascade of mRNA expression, the time of heat shock treatment will be optimized by studying the kinetics of mRNA induction of egl-15, ceh-24, and arg-1 by RT-PCR after heat shock. We are currently building the appropriate strains for this study.

Peng Wang et al. (2009) International Worm Meeting "O-GlcNAc modification and proteotoxicity in Caenorhabditis elegans."

Peng Wang, Brooke D. Lazarus, Michael W. Krause, John A. Hanover, Michele E. Forsythe, Dona C. Love

[International Worm Meeting, 2009]

O-linked N-acetylglucosamine (O-GlcNAc) addition is a type of post-translational modification that occurs on hundreds of proteins, including nuclear pore proteins, transcription factors, proteasome components and neuronal proteins. O-GlcNAc can be added onto and removed from serine or threonine residue by two evolutionally conserved enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Disruption of the O-GlcNAc cycling is involved in several human diseases, including diabetes mellitus and neurodegenerative diseases. One common signature of neurodegenerative diseases is the formation of protein aggregates, with toxic effects that could lead to neuronal degeneration and death. Human proteins that form protein aggregates in different neurodegenerative diseases have been expressed in the neurons or muscles of C. elegans to construct transgenic models. These human proteins include the following: different isoforms of Tau proteins (Alzheimers disease), beta-Amyloid (Alzheimers disease), Huntingtin (N-terminal)-polyglutamine fusion protein (Huntingtons disease), and yellow fluorescent protein (YFP)-polyglutamine fusion protein (PolyQ diseases). To investigate the effects of O-GlcNAc modification on proteotoxicity in a whole model organism, we crossed those Caenorhabditis elegans transgenic models into ogt-1 or oga-1 null mutant. The phenotypes of the transgenic animals were scored in wild type, ogt-1 mutant, or oga-1 mutant background. We found that the ogt-1 mutation alleviated and the oga-1 mutation worsened the phenotypes that are caused by the toxicity of those human proteins. In addition, in a Huntingtons disease model, we observed the same rescued phenotypes in ogt-1; oga-1 double mutant as in ogt-1 single mutant, indicating that alterations of the phenotypes depend on O-GlcNAc modification. The level of transcript of the transgenes, measured by SYBR green real-time PCR, did not correlate with the alteration of phenotypes that we observed in different genetic backgrounds. Thus, regulation of some protein quality control systems by O-GlcNAc modification might be the underlying mechanism. There are two main protein degradation pathways: proteasome and autophagy. Our working hypothesis is that both degradation pathways might be up-regulated in ogt-1 mutant and down-regulated in oga-1 mutant.

Zhang, Peng et al. (2017) International Worm Meeting "Phosphorylation by MPK-1/ERK regulates the SKN-1/Nrf1 proteasomal stress response."

Walhout, Albertha J.M., Zhang, Peng, Blackwell, T. Keith, Na, Huimin, Hourihan, John, Zhang, Fang, Isik, Meltem

[International Worm Meeting, 2017]

Protein degradation by the ubiquitin-proteasome system is vital to cellular homeostasis and survival, and mechanisms that promote healthy aging. The proteasome is also a critical therapeutic target in cancers that rely on the endoplasmic reticulum (ER) secretory system. An isoform of SKN-1 (SKN-1a; Nrf1 in mammals) regulates essentially all proteasome subunit genes, and mediates a pathway that maintains proteasome levels. Under conditions of proteasome inhibition, this recovery pathway generates additional proteasomes. In this little-understood pathway, SKN-1a/Nrf1 is processed through a complex mechanism that involves localization to and extrusion from the ER, and cleavage. Here, we performed a genome-scale RNAi screen in C. elegans to identify genes required to detect proteasome dysfunction, and activate SKN-1a/Nrf1. We show that a critical regulator in the proteasome recovery pathway is the kinase MPK-1/ERK, which responds to growth signals. We find that this pathway is regulated distinctly from the PMK-1/p38-dependent oxidative stress response that is mediated by a different SKN-1 isoform. MPK-1/ERK1 is activated by proteasomal stress, and inhibition of this kinase blocks the proteasome recovery pathway, and sensitizes C. elegans and human tumor cells to proteasome inhibition. In human tumor cells, phosphorylation by ERK on a single residue is required for Nrf1/SKN-1 to localize to nuclei and activate proteasome genes. Our data indicate that SKN-1a/ERK is directly regulated by MPK-1/ERK in the proteasomal recovery/synthesis pathway, suggesting that it is responsive to growth factor signaling. They also identify ERK/MPK-1 and possibly other hits from our screen as potential therapeutic targets for blocking compensatory proteasome synthesis in cancers that depend upon the proteasome.

Peng Huang et al. (2002) East Coast Worm Meeting "FGF Regulation of Fluid Balance in C. elegans"

Michael J Stern, Peng Huang

[East Coast Worm Meeting, 2002]

clr-1 encodes a receptor tyrosine phosphatase (RTP) that negatively regulates an EGL-15 FGFR signaling pathway in C. elegans. The pseudocoelomic cavity of clr-1 mutants fills with clear fluid, leading to the characteristic Clear (Clr) phenotype. The Clr phenotype suggests that the balance of EGL-15 signaling is essential for fluid homeostasis in worms. Animals that completely lose EGL-15 function arrest early in larval development, and less severe reduction of EGL-15 function results in a Scrawny (Scr) phenotype. To understand how FGFR signaling controls fluid homeostasis in C. elegans, we are investigating the cellular basis of these phenotypic defects. A number of approaches have been taken to investigate the cellular site of EGL-15 and CLR-1 function. Although these approaches have not given completely consistent results, several lines of experimentation suggest that CLR-1 acts in the excretory cell to regulate EGL-15 signaling. First, a clr-1::gfp reporter is expressed robustly in the excretory cell as well as a number of other cells. Second, mosaic analysis of clr-1 indicates that clr-1 activity is required in the AB.plp sublineage (C.Z. Borland, unpublished data); this lineage gives rise to a group of cells that include the excretory cell. Consistent with these results, laser ablation of the excretory cell has been shown to result in a similar Clr phenotype (F.K. Nelson and D.L. Riddle, 1984). These data suggest that CLR-1 is normally expressed and functions in the excretory cell. To explore this further, we have used tissue-specific promoters to identify cells in which CLR-1 or EGL-15 can be expressed to rescue their respective mutant phenotypes. Promoters (vha-1p and itr-1p) that drive expression in the excretory cell can drive full clr-1 rescue activity, while a body wall muscle promoter (unc-54p) cannot. Surprisingly, expression of CLR-1 by neuronal promoters (snb-1p and unc-14p) can also rescue the Clr phenotype, suggesting a potential role of CLR-1 in neurons. It is unlikely that CLR-1 acts in neurons within the AB.plp lineage; most neurons derived from the AB.plp lineage have bilaterally symmetrical cells derived from the AB.prp lineage, which presumably carry out the exact same biological function. As a complementary approach, we have dissected the endogenous egl-15 promoter by deletion analysis to look for important regulatory elements. A 200bp sequence (E15) was found to be essential for full-scale egl-15 promoter activity. Moreover, the E15 enhancer fragment is able to drive egl-15 rescue activity when fused to a minimal pes-10 promoter. Therefore, this E15 element is both necessary and sufficient for complete egl-15 promoter activity. Intriguingly, a GFP reporter under the control of two copies of this enhancer element (2E15::pes-10::GFP) is expressed mainly in hypodermal cells. Surprisingly, neither the lin-26 nor the jam-1 hypodermal promoters can drive clr-1 or egl-15 rescue activity, but these negative results may have failed for other reasons. Although our data do not pinpoint a single site of action of EGL-15 function, it is striking that the three potential sites of rescue, neurons, hypodermal cells, and the excretory cell, all contribute to the tissue domain surrounding the excretory canal outside of the basal lamina. Additional studies are in progress to understand FGF-regulated fluid flux in worms.

Peng, Felicia et al. (2021) International Worm Meeting "The Role of mRNA Decay in Embryonic Cell Fate Specification"

Peng, Felicia, Murray, John

[International Worm Meeting, 2021]

During development, cells undergo dynamic changes in gene expression that are required for appropriate cell fate specification. Although the regulation of mRNA degradation contributes to developmental gene expression, its influence on these expression patterns is not well understood relative to that of transcriptional regulation. Like transcriptional regulation, however, the regulation of mRNA decay has the potential to be highly complex. Transcript stability is largely regulated by the binding of protein or RNA factors to cis-regulatory elements within the 3' untranslated region (3' UTR) of transcripts. Considering the great diversity of RNA-binding proteins and miRNAs in eukaryotes, along with differential usage of 3' UTR isoforms, a complex mRNA degradation code may contribute to precise gene expression patterns during development. One relatively well-characterized phenomenon of developmentally regulated mRNA decay occurs during early embryogenesis, in which the widespread degradation of maternal mRNAs is required for the control of development to switch from maternally to zygotically encoded products. Increasing evidence is also emerging for the importance of zygotic mRNA degradation in cell fate decisions, though these studies tend to occur on a gene-by-gene basis or in the context of cell culture systems. Thus the extent of developmentally regulated zygotic mRNA decay remains unclear. We hypothesize that the regulation of zygotic mRNA degradation contributes to appropriate cell fate decisions throughout embryogenesis. To explore this, we are examining mRNA decay rates during embryonic development in Caenorhabditis elegans. By sequencing embryonic cells treated with a transcription inhibition time course, we have measured mRNA half-lives globally in C. elegans embryos. We found that half-lives can vary by more than an order of magnitude from gene to gene, and differences in mRNA stability correlate with functional classes of genes. Furthermore, analysis of the half-life distributions of tissue- and developmental stage-enriched genes suggests that mRNA decay is differentially regulated across different tissues and stages. Future work should extend these results to measure differential degradation of the same mRNAs in different cell types and stages. Our results highlight that zygotic mRNA degradation is a highly regulated process with tissue- and stage-specific dynamics during development.

Chan, Shih-Peng et al. (2011) International Worm Meeting "RACK-1 acts in let-7-mediated heterochronic development of Caenorhabditis elegans."

Chan, Shih-Peng, Slack, Frank

[International Worm Meeting, 2011]

In Caenorhabditis elegans, the let-7 miRNA regulates the exit of cell cycle and terminal differentiation at the L4-to-adult switch. Dysfunction of let-7 results in irregular hypodermal and vulval development of C. elegans and is also a feature of human lung cancer. Here we show that Receptor of Activated C Kinase (RACK-1) modulates let-7-mediated regulation in C. elegans. The RACK-1 protein co-immunoprecipitated with the nematode Argonaute protein, ALG-1. Reduction of rack-1 gene expression by RNAi suppressed the aberrant vulva and hypodermis development phenotypes of let-7(n2853) mutant animals and promoted adult-specific gene expression. Our results indicate a relationship between RACK-1 and let-7 in regulation of terminal differentiation that may help understand the mechanism of translational control by miRNAs.

Shih-peng Chan et al. (2005) International Worm Meeting "Identification of ALG-1/ALG-2 containing miRNP complexes in C. elegans"

Shih-Peng Chan, Frank J Slack

[International Worm Meeting, 2005]

The let-7 microRNA, one of the founding members of the miRNA family, controls the timing of terminal differentiation decisions in C. elegans. The let-7 miRNA acts as a post-transcriptional repressor of lin-41, let-60/RAS, hbl-1/lin-57 and other genes containing 3' UTR sequences with imperfect complementary to let-7. In humans, let-7 regulates the RAS oncogene and defective expression of let-7 is associated with lung cancer. The C. elegans Argonaute proteins ALG-1 and ALG-2 have been implicated in let-7 function through genetic studies and demonstrated to be components of a let-7 containing ribonucleoprotein complex by biochemical assays. ALG-1 and ALG-2 are homologs of RDE-1, which is required for the initiation of the RNAi pathway. In order to investigate the roles of ALG-1 and ALG-2 in the let-7 miRNA pathway, we aim to purify ALG-1/ALG-2 containing complexes from GFP::ALG-1 and GFP::ALG-2 transgenic strains. We have successfully integrated GFP::ALG-1 and GFP::ALG-2 fusion constructs into the C. elegans genome and found that they recapitulate endogenous ALG-1/ALG-2 expression, i.e. basal levels of expression throughout the body with elevated expression in the pharynx, seam cells, vulva and germline. ALG-1/ALG-2 expression begins during early embryonic stages and persists into the adult. As let-7 miRNA expression begins during the early L3 stage, the broad temporal pattern of ALG-1/ALG-2 expression suggests that the Argonaute proteins may be involved in developmental processes that involve miRNAs other than let-7. We are in the process of identifying the proteins that copurify with ALG-1/ALG-2 containing complexes. Current experimental work is aimed at understanding the mechanism of gene regulation by the let-7 miRNA and the ALG-1/ALG-2 related miRNA pathway in C. elegans. Our results may provide valuable information about the roles of let-7 miRNAs in human development and cancer.

Peng Wang et al. (2006) Development & Evolution Meeting "Investigating the roles of CeTwist and CeE/DA in C. elegans mesoderm development"

Ann Corsi, Peng Wang, Mary Philogene, Jie Zhao

[Development & Evolution Meeting, 2006]

A fundamental question of developmental biology is how pluripotent cells accomplish cell-fate specification and differentiation. Many transcription factors are key regulators in these events. The basic helix-loop-helix transcription factor Twist has been shown to play an important role in mesoderm development in a variety of organisms including humans. In humans, mutations in the TWIST gene result in an autosomal dominant disorder called Saethre-Chotzen syndrome (SCS). A major phenotype of SCS is the premature fusion of cranial sutures, a condition called craniosynostosis. Mutations in several human genes cause other syndromes characterized by craniosynostosis, and homologs of these genes in C. elegans are all downstream of CeTwist. Our laboratory has been studying CeTwist to understand its role in mesoderm development in the nematode. A major goal is to understand how CeTwist, coded for by hlh-8, and its heterodimeric partner, the more broadly-expressed CeE/DA, coded for by hlh-2, are turning on target genes in the mesoderm. We have taken several approaches to investigate this problem. In the first approach, we have overexpressed CeTwist and CeE/DA to identify upregulated genes by Affymetrix oligonucleotide microarrays. A subset of the genes identified in the microarrays has been studied using transcriptional GFP reporters. These studies have led to the discovery of 9 novel target genes of CeTwist and CeE/DA. The second approach, involves a careful examination of the promoter region of the target gene, arg-1. Through deletion analysis and site-directed mutagenesis, we have determined that the arg-1 promoter contains three elements that are uniquely involved in regulating its gene expression. The final approach has been to examine a temperature-sensitive loss-of-function mutation in hlh-2. The hlh-2 mutant animals have an altered pattern of arg-1::gfp expression that is inconsistent with regulation of this gene in the mesoderm exclusively by CeTwist/CeE/DA heterodimers. Additionally, rescue experiments of hlh-8 null mutants using tethered CeTwist molecules suggest that homodimers may play a role that is distinct from the heterodimers in the mesoderm. A search for target genes regulated by CeTwist homodimers is currently underway.

Peng, Felix et al. (2015) International Worm Meeting "Analysis of foci created by the telomere binding protein POT-1 in C. elegans."

Maddox, Paul, Ahmed, Shawn, Shtessel, Ludmila, Peng, Felix

[International Worm Meeting, 2015]

Telomeres are tracts of tandem DNA repeats that protect the ends of linear chromosomes from replicative erosion and from being identified and repaired as double-strand breaks. Shelterin is a complex of proteins found to directly associate with telomeres and functions to distinguish telomeres from double-strand breaks. The shelterin component POT1 binds to single stranded telomeric DNA, which is present at the displacement loop created by strand invasion of the 3' overhang into the telomeric duplex at the chromosome terminus. POT1 homologues have recently been identified and characterised in C. elegans. A transgene expressing pot-1::mCherry reveals strong punctate foci within the germline corresponding to discrete telomeric termini. Here, we examine the dynamics of POT1 foci in the germline and during early development within living embryos using time-lapse imaging. We interrogate the behaviour of POT1 foci in the context of fused chromosomal ends harbouring telomeric repeats of varying lengths. We also analyse POT1::mCherry foci in genetic backgrounds where telomerase activity is abolished, leading to shortened telomeres, and in mutants causing telomere elongation, as occurs with mutant pot-1. Our results suggest that POT1 creates large, discrete structures within the nucleus and we are testing the hypothesis that dynamics of POT1 foci during early development could be relevant to telomere biology.

Peng Wang et al. (2005) International Worm Meeting "Identification and characterization of the downstream target genes of CeTwist and its partner CeE/DA"

Peng Wang, Ann Corsi

[International Worm Meeting, 2005]

CeTwist, encoded by the hlh-8 gene, is a member of the basic helix-loop-helix (bHLH) family of transcription factors and appears to function as a heterodimer with CeE/DA, another bHLH protein encoded by the hlh-2 gene (1). Identification of new downstream target genes of CeTwist and CeE/DA will shed more light on the functions of CeTwist in C. elegans mesoderm development and may provide information about a class of human diseases that cause craniosynostosis, or premature fusion of skull sutures. Several human genes, whose mutations are believed to cause human craniosynostotic disease, have homologues in CeTwist pathway in C. elegans. Based on the striking parallel relationship, we predict the new CeTwist target genes which are found in our work might explain genetic defects in other related syndromes. To accomplish this goal, Affymetrix oligonucleotide microarray experiments were performed to detect global gene expression changes between animals with over-expressed CeTwist and CeE/DA from the heat shock promoter and otherwise isogenic wild type animals. Microarray data was analyzed with Microarray Suite 5.0 (Affymetrix) and Genespring 6.0, and 193 genes including 161 up-regulated genes and 23 down-regulated genes were identified. 47 potential target genes were chosen to be studied initially based on the analysis of the potential CeTwist control elements in their upstream sequences and their functional predictions reported in Wormbase. Transcriptional GFP reporters for all the 47 genes were constructed and their temporal and spatial expression patterns were examined. 12 candidate genes showed coincident expression with hlh-8. We are further testing these 12 genes by examining whether the GFP reporters of these genes show ectopic or strong up-regulation upon over-expression of CeTwist and CeE/DA and whether the GFP reporters are not expressed in an hlh-8 null mutant (2). So far, 7 genes have been tested with these two methods and 5 of them are identified as the most promising genes. Site directed mutagenesis of their potential promoter control elements of CeTwist and functional characterization of these genes by RNAi will start soon. 1. Harfe, B. D., Gomes, A. V., Kenyon, C., Liu, J., Krause, M. and Fire, A. (1998). Analysis of a Caenorhabditis elegans Twist homolog identifies conserved and divergent aspects of mesodermal patterning. Genes Dev 12, 2623-2635. 2. Corsi, A. K., Kostas, S. A., Fire, A. and Krause, M. (2000). Caenorhabditis elegans twist plays an essential role in non-striated muscle development. Development 127, 2041-51.