Clary, Lynn et al. (2011) International Worm Meeting "C. elegans TBX-2 is a SUMOylation dependent transcriptional repressor."

Ronan, Tom, Clary, Lynn, Okkema, Peter

[International Worm Meeting, 2011]

T-box transcription factors are crucial developmental regulators in all multicellular animals, and they have been implicated in a variety of human diseases and cancers. Despite their importance, few direct targets of T-box factors have been identified. C. elegans TBX-2 is a member of the Tbx2 sub-family of T-box factors, which in other species includes both transcriptional activators and repressors, and it is required for development of anterior pharyngeal muscles. We are interested in determining if TBX-2 is a transcriptional activator or repressor, and in identifying TBX-2 targets to characterize its molecular mechanism. To identify targets of TBX-2, we compared mRNA expression levels in wild-type and hypomorphic tbx-2(bx59) mutant embryos using Affymetrix microarrays. Of 19,885 genes examined, we found 980 mRNAs that were significantly up-regulated in tbx-2(bx59) and 175 mRNAs that were significantly down-regulated. We analyzed a subset of these differentially expressed genes and found the gene D2096.6 is directly repressed by TBX-2 at a variant T-box binding site in its promoter. Our previous genetic evidence suggests TBX-2 function is SUMOylation dependent, as reduction of the SUMO-conjugating enzyme UBC-9 produces pharyngeal phenotypes similar to loss of TBX-2. Consistent with these results we see that ubc-9(RNAi) causes over expression of D2096.6 similar to what we have observed in tbx-2 mutants. By comparing mRNA expression levels in wild-type and tbx-2(bx59) mutant embryos we have identified the first direct target of TBX-2 in C. elegans. Our results suggest TBX-2 functions as a transcriptional repressor whose function is dependent on SUMOylation. We are identifying additional TBX-2 targets among the differentially expressed genes using bioinformatic techniques.

Ronan, Tom et al. (2011) International Worm Meeting "Leveraging existing genomic and microarray data to find direct targets of the C. elegans transcription factor TBX-2."

Clary, Lynn, Ronan, Tom, Okkema, Peter G.

[International Worm Meeting, 2011]

Direct targets of transcription factors are time-consuming to find biologically and difficult to predict accurately solely using bioinformatics methods. Predictions can be significantly improved by utilizing the large repository of existing genomic and structural data in combination with select newly-generated gene expression data. Here we combine a single microarray experiment with classic transcription factor binding site prediction and novel uses of existing genomic and expression data sets to predict direct targets of the C. elegans transcription factor TBX-2. Binding data is not available for TBX-2, but is available for related T-box transcription factors. Alignments and structural data are used to distinguish among key characteristics and relevant regions of existing T-box binding motifs to produce a novel binding motif for predicting TBX-2 binding sites. This new motif is used to search five-prime cis-regulatory regions in five nematode genomes to identify potential direct targets. An alignment-independent definition of regulatory conservation is implemented to limit potential targets to binding sites conserved in multiple species. Genome-wide profiles of transcription factor binding characteristics, recently published by the modENCODE project, are used to narrow the field to targets most likely to be biologically significant. As TBX-2 is hypothesized to be a repressor, only potential targets which are up-regulated in a microarray comparing gene expression in wild-type and tbx-2(bx59) mutant embryos are considered. And finally, using an existing embryonic time-course mRNA expression data set, the subset of genes which anti-correlate with tbx-2 at various time-lags are used as evidence that they are directly targeted by TBX-2. This results in a small set of 18 biologically testable genes which are likely to be enriched for direct targets of the C. elegans transcription factor TBX-2, representing a 99% reduction over the microarray and a 91% reduction over bioinformatic predictions alone.

Huber, Paul et al. (2013) International Worm Meeting "Function of the C. elegans T-box factor TBX-2 depends on SUMOylation."

Packard, Adelaide, Okkema, Peter, Clary, Lynn, Huber, Paul, Crum, Tanya, Ronan, Tom

[International Worm Meeting, 2013]

T-box transcription factors are critical developmental regulators in all multi-cellular animals, and altered T-box factor activity is associated with a variety of human congenital diseases and cancers. Despite the biological significance of T-box factors, their mechanism of action is not well understood. Here we examine whether SUMOylation affects the function of the sole C. elegans Tbx2 sub-family T-box factor TBX-2. TBX-2 is required for ABa-derived pharyngeal muscle, and tbx-2 null mutants arrest as L1 larva. We have previously shown that TBX-2 interacts directly with the E2 SUMO-conjugating enzyme UBC-9, and that loss of TBX-2 or UBC-9 produces identical defects in ABa-derived pharyngeal muscle development. We now show that TBX-2 is SUMOylated in mammalian cell assays, and that both UBC-9 interaction and SUMOylation depends on two SUMO consensus sites located in the T-box DNA binding domain and near the TBX-2 C-terminus, respectively. In co-transfection assays, a TBX-2:GAL4 fusion protein represses expression of a 5xGal4:tk:luciferase construct. However, this activity does not require SUMOylation, indicating SUMO is not generally required for TBX-2 repressor activity. In C. elegans, reducing SUMOylation enhances the phenotype of the temperature sensitive tbx-2(bx59) mutant and results in ectopic expression of a gene normally repressed by TBX-2, demonstrating that SUMOylation is important for TBX-2 function in vivo. Finally, we show mammalian orthologs of TBX-2, Tbx2 and Tbx3, can also be SUMOylated, suggesting SUMOylation is a conserved mechanism controlling T-box factor activity. We are currently examining how SUMOylation affects TBX-2 activity in vivo. We expect these studies will provide further insight into the mechanisms regulating T-box factor activity.

Guo S et al. (1995) International C. elegans Meeting "PAR-1: Asymmetrically Localized Kinase Required for Embryonic Polarity"

Kemphues KJ, Guo S

[International C. elegans Meeting, 1995]

The par-1 gene is required for many aspects of embryonic polarity including unequal cleavage and the asymmetric distribution of cytoplasmic components. We cloned the par-1 gene using a combination of YAC transformation rescue and antisense phenocopy. It encodes a putative serine/threonine kinase with strong similarity to kinases identified in yeasts and mammals. Two par-1 alleles have mutations in invariant kinase residues, suggesting that the kinase activity is crucial for PAR-1 function. PAR-1 protein is localized to the posterior periphery of the zygote. After the first embryonic division, PAR-1 is detected at the P1 periphery but is absent from the AB periphery. As the cell cycle proceeds, the peripheral PAR-1 becomes asymmetrically localized and partitioned into P2. Asymmetric localization of PAR-1 occurs again in P2 and P3, but does not occur in P4. The localization of PAR-1 shows a striking correlation with the distribution of P granules, and thus raises the possibility that PAR-1 is not only required for the early anterior-posterior polarity, but is also involved in later P lineage asymmetric divisions or germ-line development. To better understand interactions among par genes, we examined PAR-1 distribution in other par mutants. Interestingly, in par-2 mutants, PAR-1 is not detectable at the periphery of the zygote or P lineage blastomeres, but in par-3 mutants and in par-2 par-3 double mutants PAR-1 is present around the entire periphery of all cells in the early embryo. These results, together with other results from our lab (see abstracts by B. Etemad-Mogadam and Lynn Boyd), lead us to propose that PAR-1 is restricted to the posterior by PAR-3 activity and that PAR-2 is required to properly localize PAR-3.

Lynn Boyd (2001) International C. elegans Meeting "A simple lab using single worm PCR and dpy-5"

Lynn Boyd

[International C. elegans Meeting, 2001]

A simple lab has been designed for use in a sophomore genetics course. There are four primary objectives for this laboratory exercise: 1) students predict genotypes based on phenotype 2) students design primers for PCR 3) students use the PCR technique 4) students analyze results using gel electrophoresis The learning elements of the lab can be broken down into two main areas. First, it serves as a demonstration of the relationship between genotype and phenotype. Students observe the phenotype of worms and then make predictions about the genotype which they subsequently test. A second valuable learning element has been having the students design primers for PCR. Students must have a good understanding of both DNA structure and DNA polymerase activity to design primers successfully. This laboratory exercise makes use of a dpy-5 mutation that contains a deletion of 1009 bp (gracious thanks to Colin Thacker and Ann Rose for supplying the strain).. The mutation (e907) is semidominant. Therefore, students can identify all three genotypes in a population of worms containing the wild type and e907 alleles (+/+; +/e907; e907/e907). The investigation occurs over three lab periods. In the first lab, students become familiar with observing the Dpy phenotype and with picking worms. They also design primer pairs that will amplify both the wild type and e907 alleles. In the second lab, students use these primer pairs in single worm PCR reactions. They are asked to pick worms that represent each of the three genotypes and use these for PCR. In the third period, an agarose gel is run of the PCR reactions. Students determine the genotypes of the worms they chose and find out if their genotype predictions were correct. I have found that sophomore students are able to do this lab with a high percentage of success. If you would like more information or would like the lab handout, please contact Lynn Boyd at boydl@uah.edu.