Kamila Sekiewicz et al. (2003) International Worm Meeting "Cross-species comparisons of signal transduction pathways."

Chi-hua Chiu, Garth Patterson, Kamila Sekiewicz

[International Worm Meeting, 2003]

C. elegans senses environmental cues such as pheromone, temperature and food, and develops accordingly into a reproductive adult or a dauer larva. The decision as to which developmental path is followed is controlled in part by a TGF signaling pathway.The components of the TGF signaling pathway in C. elegans include a ligand encoded by daf-7, two receptors encoded by daf-1 and daf-4 and transcription factors encoded by daf-8, daf-14 and daf-3. Another identified component of the pathway is DAF-5, which is antagonized by the TGF pathway and which was cloned in our lab (see abstract by Da Graca et al. this program).C. briggsae is a nematode worm morphologically and developmentally almost identical to C. elegans. The two species have diverged 20-100 MYA. When comparing the amino acid sequence of C. elegans DAF-5 and its C. briggsae ortholog, we have noticed a surprisingly low identity of only about 40%. To determine whether this low percent identity is a property of DAF-5 alone, or perhaps a group of TGF/dauer genes, we looked at the sequence conservation between C. elegans and C. briggsae in the following groups of genes: TGF dauer pathway, TGF body size pathway, insulin mediated pathway, other dauer genes, and a group of randomly selected genes.We have found that the weak sequence conservation between C. elegans and C. briggase genes was not specific to DAF-5 alone, and all the genes in the TGF dauer pathway share this property with the average of 41% identity. In contrast to this group, we have found that all of the genes in the TGF body size pathway are very highly conserved, with the exception of DAF-4, which acts in both the body size and the dauer TGF pathways. Excluding DAF-4 from the TGF body size group, the average percent identity is 84.6. The genes in the insulin mediated pathway and other dauer genes show a greater range of values, and their average percent identities are 72.2 and 79.2 respectively. The group of randomly selected genes gives an average of 77.5% identity. All possible pairwise comparisons of these groups show that only the genes in the TGF dauer pathway are significantly different from all the other groups.To further determine the signs of positive, negative, or neutral selection acting on individual genes in the TGF dauer pathway, we are taking a closer look at the conservation of individual functional domains of these genes.

Kamila A Sekiewicz et al. (2002) East Coast Worm Meeting "Identifying new genes involved in TGFbeta signaling in C. elegans"

Kamila A Sekiewicz, Garth I Patterson

[East Coast Worm Meeting, 2002]

All animals, including humans are affected by various outside stimuli and respond accordingly to those stimuli. Caenorhabditis elegans senses environmental cues (pheromone, food and temperature) and develops accordingly into a reproductive adult or a dauer larva. The decision as to which developmental path is followed is controlled by a TGFbeta signaling pathway. The components of the TGFbeta pathway in C. elegans include a ligand encoded by daf-7 and two receptors encoded by daf-1 and daf-4. daf-8, daf-14 and daf-3 code for transcription factors called Smads. (Daf stands for dauer formation.) Mutations in daf-7, daf-1, daf-4, daf-8 and daf-14 will result in a dauer-constitutive (Daf-c) animal (one which will go into dauer even under conditions which do not induce dauer development in wild type). Mutations in daf-3 result in a dauer-defective (Daf-d) animal, which suppresses the Daf-c phenotype. Other mutations have also been identified that are Daf-d and/or suppress the Daf-c phenotype of TGFbeta pathway mutants. They include daf-5, daf-12 and scd-1. scd stands for suppressor of constitutive dauer. The above model of the dauer pathway describes the members of the pathway and the path of the TGFbeta signal. The mechanism by which the TGFbeta pathway regulates transcription of dauer specific or non-dauer genes is still not understood. Our genetic analysis suggests that there are other, still unidentified factors in the TGFbeta pathway (such as transcription factors or co-factors) that regulate transcription of these genes. Identification of these factors might help to explain in more detail the transcriptional events. Our search for the new genes concentrated downstream of ther TGFbeta pathway. Screens were performed yielding eighty suppressors of the daf-7 (Daf-c) mutation. I tested thirteen suppressors for penetrance and complementation of four other known Daf-c suppressors - daf-3, daf-5, daf-12 and scd-1. Our tests indicate that we have isolated alleles of these known suppressors, as well as four alleles that do not complement any of these mutants. We will test these alleles for complementation with the other two known suppressors: scd-2 and scd-3. I have also started to map selected suppressors using a snip-SNP method, which utilizes presence of SNPs (single nucleotide polymorphisms) in Bristol N2 and Hawaii CB4856 strains.

Garth I Patterson et al. (2003) International Worm Meeting "Cracking the code: annotation of genes used for Kim microarray and other databases"

Tao Liu, Kamila Sekiewicz, Garth I Patterson

[International Worm Meeting, 2003]

Much pioneering genomic work, notably that from S. Kim's microarray facility and from J. Ahringer's RNAi library, relies on "GenePairs", a set of primers designed to amplify each C. elegans open reading frame. GenePairs were designed years ago, with an incompletely annotated genome, and many genes have changed since then. In particular, over one thousand GenePairs genes still had provisional names, which were quickly changed. Most analysis of these genes uses the GenePairs names to prevent ambiguity. In order to interpret some microarray data and to use some web resources, one needs to go from the current name of the gene to the GenePairs name or vice versa, which is not necessarily easy. For example, if you want to know what Wormbase has to say about Y59E9_116.A, which is an old GenePairs name for a gene, you might spend some time searching. A search of Wormbase turns up 13 matches, a few of which tell you that Y59E9_116.A is now called Y59E9AL.1, but most of which do not. In addition, since GenePairs were designed, many genes have been combined or split, usually because of cDNA sequencing. We have reannotated a list of genes from Stuart Kim's facility, so that we could easily "translate" from GenePairs to other databases. Data from the Stanford facility comes in tabular form. The table from Stanford has a column with a short description of the gene (from WormPD), a column with the genepairs name, and a column with alternate names. To that, we have added a column that has the current name of the gene, and several columns of brief functional annotations from Wormbase. In addition to the primary benefit of "translation", we have found several other benefits. First, the WormPD is no longer available to most C. elegans labs, which makes it hard to learn the rationale for the brief annotation. Second, some of the WormPD annotations are incorrect or incomplete. The Wormbase annotations are usually similar, sometimes better, sometimes worse, but with the database, genes that have differences in annotation can be identified and investigated. Third, the WormPD annotations are incompletesome genes have no description. Fourth, some GenePairs "genes" are now known to be more than one gene, which, of course, changes interpretation of results. Many, but unfortunately not all, of those are flagged in our database. In the hope that this database will be useful to others, we will present it (literally--you can pick up a CD) and describe its uses and limitations.

Ozdemir, Isa et al. (2021) International Worm Meeting "Regulation of transgenerational epigenetic H3K27me3 inheritance"

Steiner, Florian, Delaney, Kamila, Wenda, Joanna, Ozdemir, Isa

[International Worm Meeting, 2021]

A crucial process in life is the ability of cells to pass on information to their descendants. This transmission can occur at several levels from genetics to epigenetics. Epigenetic inheritance refers to the heritable phenotypes affected by gene expression without altering the DNA sequence and occurs through various factors including histone modifications. Interruption of this transmission process can lead to severe developmental defects and fertility diseases. Methylation of histone H3 at position 27 (H3K27me3) is an epigenetic mark that is associated with heterochromatin and repressed gene expression. Several studies have demonstrated that the genomic patterns of H3K27me3 can be inherited from one generation to the next by depositing H3K27me3 histones and PRC21. However, the limits of this inheritance are difficult to test because PRC2 mutants are maternal-effect sterile in C. elegans. We have recently shown that the expression of an H3.3K27M mutant acts in a dominant-negative manner and alters genomic H3K27me3 patterns and distribution of PRC2 and results in infertility phenotypes in C. elegans2. We used this strain to follow the trans-generational inheritance of the H3.3K27M-induced phenotypes. Strikingly, we observed that the altered patterning of H3K27me3 and the fertility defects are heritable for multiple generations after the H3.3K27M mutation is lost. These findings were also validated in a tetracycline-inducible system where we can abruptly switch on and off the H3.3K27M expression. We performed a targeted RNAi screen in our tetracycline-inducible system and detected several enhancer and suppressor modifiers of H3.3K27M defects such as chromatin remodeling complexes, the nuclear RNAi pathway, and histone writer and reader complexes. Overall, our results revealed that the transmission of H3K27me3 is an epigenetically programmed event that can last for generations and that several biological pathways seem to be involved in the regulation of transmission of H3K27me3 patterns across generations. 1. L. J. Gaydos, W. Wang, S. Strome. H3K27me and PRC2 transmit a memory of repression across generations and during development. Science 345, 1515-1518 American Association for the Advancement of Science (AAAS), 2014. 2. Kamila Delaney, Maude Strobino, Joanna M. Wenda, Andrzej Pankowski, Florian A. Steiner. H3.3K27M-induced chromatin changes drive ectopic replication through misregulation of the JNK pathway in C. elegans. Nature Communications 10 Springer Science and Business Media LLC, 2019.

Li da Graca et al. (2003) International Worm Meeting "DAF-5, a putative transcriptional regulator, acts in the nervous system to control dauer development"

Li da Graca, Karen Zimmerman, Garth Patterson

[International Worm Meeting, 2003]

The reproductive/dauer decision is controlled by environmental cues such as food, pheromone and temperature. A TGF--related pathway promotes reproductive development over dauer. Mutations in daf-7 (TGF--like ligand), daf-1 and daf-4 (type I and type II receptor kinases), and daf-8 and daf-14 (Smad transcription factors) induce dauer at restrictive temperature regardless of environmental cues. The Daf-c phenotype of these mutants is suppressed by mutations in daf-3 (Smad) or daf-5. Epistasis analysis suggests that daf-5 and daf-3 are either antagonized by this TGF--related pathway or act in a parallel pathway. We found that daf-5 encodes a novel protein with weak similarity to chromatin remodeling proteins such as Sno/Ski and ACF-1. Vertebrate Sno has been recently shown to be capable of acting as an inhibitor of TGF- pathways by interacting with Smad proteins. DAF-5 is the best Sno/Ski homolog in C. elegans, but parasitic nematodes and Drosophila have apparent orthologs of Sno/Ski. Thus, either one group of nematodes has lost Sno/Ski, or daf-5 is a Sno/Ski that is so highly diverged as to be almost unrecognizable. Interestingly, DAF-5 has remarkably low conservation with its C. Briggsae ortholog (see Sekiewicz et al abstract). Our collaborators Muneesh Tewari et al (see abstract) find that DAF-5 binds to the DAF-3 (Smad) in a yeast-two-hybrid assay. Binding assays with individual domains of DAF-5 and DAF-3 suggest that the interaction of these proteins is similar to that of vertebrate Sno/Ski with Smad. Five out of twenty daf-5 alleles showed a mutation hotspot in a region that shows no significant homology to other proteins, but is well conserved in C. briggsae. GFP reporter constructs show strong daf-5 expression in neurons in head and tail ganglia and anterior pharynx, with weaker expression in several other cell types. We used tissue-specific expression constructs to show that daf-5 functions in the nervous system to non-autonomously control dauer morphogenesis. Work from the Riddle and Thomas labs shows that TGF-beta receptors also functions in the nervous system, suggesting that daf-3/daf-5 and the core TGF- pathway could act in the same cells. We currently are identifying the neurons that express daf-5 and will perform experiments to find the cells required for daf-5 function. We are seeking clues to construct a neuronal network and interpreting how this network integrates environmental cues and executes larval developmental programming.