Velayudhan, Satheeja Santhi et al. (2022) MicroPubl Biol

Velayudhan, Satheeja Santhi, Ellis, Ronald E

[MicroPubl Biol, 2022]

To learn if orthologous mutations are temperature-sensitive in related species, we studied four C. briggsae mutations orthologous to alleles of important C. elegans genes. Both Cel-glp-4(bn2) and Cbr-glp-4(v473) are temperature-sensitive, causing sterility at 25C. By contrast, Cel-fog-1 ( q253) is strongly ts , but its ortholog Cbr-fog-1(v442) causes a loss-of-function at all temperatures. Finally, the C. elegans glp-1 alleles bn18 and e2141 are ts sterile. However, their C. briggsae orthologs, Cbr-glp-1(v429) and Cbr-glp-1(v438) respectively, are wild-type at all temperatures. Thus, a ts mutation in one species provides clues about how to design ts alleles in another, but all theoretical outcomes are possible.

Velayudhan, Satheeja Santhi et al. (2019) International Worm Meeting "Control of C. briggsae germline development by TRA-1-interacting co-factors."

Ellis, Ronald E, Shen , Yongquan, Velayudhan, Satheeja Santhi

[International Worm Meeting, 2019]

Chromatin remodelers work with transcriptional regulators to control gene expression during development. Many studies have focused on the role of transcription factors play in evolutionary change, but much less is known about the role of chromatin remodelers. Gli proteins form a conserved group of Zn-finger transcription factors that regulate many important developmental processes. In Caenorhabditis nematodes, TRA-1 is the sole Gli protein, and acts as the terminal regulator of the sex-determination pathway. Both the activator and repressor functions of this Gli protein can be studied in the germ line, where they regulate the sperm/oocyte decision. TRA-1 activity is influenced by several chromatin regulators that may work as co-factors. In C. briggsae, it interacts genetically with TRR-1 (part of the TIP-60 HAT complex), WDR-5.1 and the NURF complex to promote spermatogenesis. We are studying these epigenetic interactions in the germ line to learn how each co-factor works with TRA-1 to control its target fog-3. To do this, we are (1) setting up ChIP assays to identify sites bound by each regulator, and (2) purifying TRA-1 complexes to identify interacting factors and protein modifications. To begin, we used gene editing to make these control strains: Cbr-glp-1(v429) III, Cbr-glp-1(v433) III, Cbr-glp-1(v438) III, Cbr-glp-1(v439) III and Cbr-fog-1(v442) I, (Three of these mutations are orthologous to temperature-sensitive alleles from C. elegans, but only one of them is temperature-sensitive in C. briggsae). Next, we produced alleles of cbr-tra-1 with either N-proximal or C-proximal OLLAS tags that retain wildtype function. We are now beginning the ChIP studies. WDR-5.1 is a member of an H3K4 trimethylation (H3K4me3) complex, so we are doing ChIP assays using WDR-5.1 specific Anti-Histone H3 (tri-methyl-K4) antibodies. We will use OLLAS epitope-tagged TRA-1, TRR-1 and NURF-1.1 for similar ChIP analyses, and the OLLAS tagged TRA-1 for purification and analysis. Eventually, we plan to expand to C. elegans and C. tropicalis to identify evolutionary changes.

Velayudhan, Satheeja Santhi et al. (2021) International Worm Meeting "Control of C. briggsae germline development by TRA-1-interacting co-factors"

Ellis, Ronald, Velayudhan, Satheeja Santhi, Shen , Yongquan

[International Worm Meeting, 2021]

Chromatin remodelers work with transcriptional regulators to control gene expression during development. Many studies have focused on the role transcription factors play in evolutionary change, but much less is known about the role of chromatin remodelers. Gli proteins form a conserved group of Zn-finger transcription factors that regulate many important developmental processes. In Caenorhabditis nematodes, TRA-1 is the sole Gli protein, and acts as the terminal regulator of the sex-determination pathway. Both the activator and repressor functions of this Gli protein can be studied in the germ line, where they regulate the sperm/oocyte decision. TRA-1 activity is influenced by several chromatin regulators that may work as co-factors. In C. briggsae, it interacts genetically with TRR-1 (part of the TIP-60 HAT complex), WDR-5 and the NURF complex to promote spermatogenesis. We are studying epigenetic interactions in the germ line to learn how each co-factor works with TRA-1 to control its target fog-3. We made these strains to allow us to alter germ cell fates: Cbr-glp-4 (v473ts) I and Cbr-trr-1(v104) II. The glp-4 strain is temperature sensitive for germline development, just as the orthologous mutation is in C. elegans, and trr-1 mutants have decreased Tip-60 activity causing 70% of the worms become female. Next, we produced alleles of Cbr-tra-1 with either N-proximal (Cbr-tra-1-N-OLLAS (V455) III) or C-proximal (Cbr-tra-1-c-OLLAS (V424) III) OLLAS tags that retain wildtype function. We performed anti-H3K4me3 and anti-OLLAS ChIP-qPCR to amplify the TRA-1 binding site from the fog-3 promoter at different developmental stages, focusing on the L1 stage (long before spermatogenesis), L3 stage (initiation of spermatogenesis) and L4 stage (completion of spermatogenesis). The H3K4me3 CHIP signal gradually decreased with age, so it appears to act in early stages of germline development and not in the later stages. Perhaps one WDR-5 function is to promote early germline development by marking genes for potential transcription. Preliminary results with OLLAS CHIP revealed higher levels of TRA-1 at fog-3 when we used the N-terminal OLLAS strain (which should detect both TRA-1 activator and repressor) than the C-terminal strain (which should only detect activator).