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The split Grainyhead protein of Cnidaria GRH-1 Grainyhead Grainyhead is a transcription factor. It was first identified in Drosophila as the locus mutated in the embryonic lethal grainyhead mutant. (Bray & Kafatos, 1991) Subsequently, the gene was identified in a range of triploblastic animals, and most recently in four diploblastic phyla. (Traylor- Knowles et al., 2010) It plays a presumably homologous role in establishing and maintaining epithelial integrity in Drosophila, C. elegans, and Mus. (Harden, 2005) GRH-2 GRH in Wound Repair Harden (2005) Origin of the Epithelium DIPLOBLASTIC ANIMALS TRIPLOBLASTIC ANIMALS Deuterostomia Chordata Hemichordata Echinodermata Acoelomorpha Calcispongia Silicispongiae Porifera Ctenophora Placozoa Cnidaria Ecdysozoa Lophotrochozoa Arthropoda Onychophora Nematoda Annelida Mollusca Platyhelminthes PROTOSTOMIA ? ? eumetazoan ancestor ancestor of eumetazoans + calcisponges GRH role in epithelial integrity is at least this old. Grainyhead in diploblastic animals • Traylor-Knowles et al. (2010) identified putative GRH genes in representatives of four diploblastic animal phyla: Cnidaria, Ctenophora, Placozoa, and Porifera. phylum Cnidaria Nematostella vectensis phylum Porifera Amphimedon queenslandica phylum Placozoa Trichoplax adhaerens phylum Ctenophora Vallicula multiformis A neighbor-joining analysis of the amino acid sequences in the conserved motifs supported the conclusion that these were bona fide GRH sequences in the diploblastic animals. Conserved & family-specific motifs in GRH/LSF Traylor-Knowles et al. (2010) analyzed the motif architecture of GRH proteins and the related LSF proteins from 8 metazoan phyla using MEME [Bailey & Elkan (1994) Multiple Em for Motif Elicitation; Em = expectation maximization]. 1 2 3 4 5 6 9 10 11 12 13 15 18 19 LSF Grh Nine motifs were shared between GRH and LSF proteins. Two were unique to LSF. Three were unique to GRH. Based on the motif architecture, the basal metazoan sequences appeared to be bona fide GRH proteins Timothy L. Bailey and Charles Elkan, "Fitting a mixture model by expectation maximization to discover motifs in biopolymers", Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park, California, 1994. Vertebrata (Homo) Cephalochordata (Branchiostoma) Urochordata (Ciona) Insecta (Drosophila) Crustacea (Daphnia) Annelida (Capitella) Mollusca (Lottia) Cnidaria (Nematostella) Placozoa (Trichoplax) Ctenophora (Vallicula) Poriferan (Amphimedon) GRH function in humans Grainyhead proteins bind to DNA as dimers, where they activate the expression of target genes involved in epithelial integrity. In the human protein GRHL2 the centrally conserved motifs constitute the DNA binding domain. the amino-terminal motif represents the activation domain. the carboxy-terminal motif represents the dimerization domain. Activation DNA binding Dimerization Split GRH locus in Nematostella In Nematostella, the ancestral GRH protein appears to have become split between two separate loci. GRH-1 harbors the conserved motifs that constitute the DNA binding domain. GRH-2 harbors the dimerization domain. Given this altered protein structure, is the ancestral function still preserved (perhaps requiring cooperation among the GRH-1 and GRH-2 loci)? Activation DNA binding GRH-1 Dimerization GRH-2 Test for Functional Conservation Use in situ hybridization to see if GRH1 and/or GRH2 transcripts are expressed in epithelia, with and without wounding. Develop antibodies to GRH-1 and GRH-2 proteins. See if they co-localize within the cell. Use antibodies to GRH-1 and GRH-2 in a ChIP-seq assay to see if both proteins are associated with the same DNA targets. Comparative studies on other Cnidaria to see if split GRH of Nematostella is not an DNA binding anomaly. Dimerization GRH-1 GRH-2 Functional Conservation? Does the Nematostella GRH-1 bind DNA targets? Does it upregulate transcription of target genes? Does it form a dimer to do so? Does it form a complex with GRH-2 in order to form a dimer? Is it involved in epithelial integrity and wound healing? Is this split in the GRH locus widespread among Cnidaria? GRH-1 GRH-2 Cnidarian genomic model systems Nematostella Edwardsiella Pocillopora Acropora Hydra The sea walnut (the comb-jelly Mnemiopsis leidyii) comb jelly parasites The Lined Sea Anemone (Edwardsiella lineata; or “Ed”) larva comb jelly parasites larva parasite New life cycle stage. New body plan. Novel developmental program. Novel developmental regulatory gene cascade. Novel gene expression pattern. polyp host absent host present larva Sequence all expressed genes. polyp parasite host absent host present larva Cnidarian genomic model systems Nematostella Edwardsiella Available data from Edwardsiella lineata: mRNA was isolated from whole animals in five developmental stages: parasite planula larva adult polyp parasite to planula transition planula to polyp 188 million read pairs sequenced on an Illumina Genome Analyzer IIX. The raw reads were assembled into ~95,000 contigs with an average length of 1,002 nucleotides (Lubinski & Granger; unpublished) A BLAST interface is available at EdBase (Lubinski & Granger; unpublished) GRH-1 BLAST matches to Nematostella GRH-1 among contigs in Edbase Nv-GRH1 4 FGLFNRYTFILEAPTSIVQRRGDDTLTYLNKGQFYAIDFEGNFDPPSTEEDIIRVKSVVH F 10642 63 YTFILEAPTSIVQRRGDDTLTYLNKGQFYAIDFEGN DPP +EEDI RVKSVVH 648 FSAQESYTFILEAPTSIVQRRGDDTLTYLNKGQFYAIDFEGNVDPPCSEEDIARVKSVVH 469 64 LVFRDEKDPRAELEHWNYWHSQQPNPQQRAFDIDRKSCQNIDENITDQAYNAAGFTWSPH 123 Nv-GRH1 LVFRDEKDPRAELEHW+YWHSQQPNPQQRAFDIDRKSCQNIDEN++D +YNAAGFTWSPH 10642 468 LVFRDEKDPRAELEHWHYWHSQQPNPQQRAFDIDRKSCQNIDENVSDMSYNAAGFTWSPH 289 Nv-GRH1 124 LNAKIVIRINCLSTDFSPQKGVKGIPLHLQIDTYEDVDNPDAEPVHRAFCQIKVFRDKGA 183 LNAKIVIRINCLSTDFSPQKGVKGIPLHLQIDTYEDVDNPDAEPVHRAFCQIKVFRDKGA 10642 Query 288 184 LNAKIVIRINCLSTDFSPQKGVKGIPLHLQIDTYEDVDNPDAEPVHRAFCQIKVFRDKGA ERKNKDESKSAERR ERKNKDES+SAERR 197 109 GRH-2 BLAST matches to Nematostella GRH-2 among contigs in Edbase NvGRH-2 1 Edbase 151 NvGRH-2 73 Edbase 3 NvGRH-2 133 Edbase 183 MVVQRMQSRLLSFDVNRYSTPLLRQLQEKARKRTSMQAFTETAADALDELD MVV+ MQ+ +SFD+++YSTPLL+QL+EKARKRT+ AFTET ADALD+L+ MVVKIMQAGCISFDLDKYSTPLLKQLREKARKRTATVAFTET-ADALDDLE 51 QETEKAYNAVFMEELTVESFKEAVSLRYGTPIKSVRKIEIQTNSGSIEKVDDNTIHGFDE +TEKAYNA+FMEELTV++FKEAVSLRYG P++S++K+EI+T+SGSIE++ + I GF + NKTEKAYNAIFMEELTVDNFKEAVSLRYGAPVESIKKLEIRTHSGSIEELGNEAIGGFID EDTFTIVLNYNKVEGTCDILLSS EDTF I L+Y+ EGTC+ILL + EDTFIIQLDYDNAEGTCNILLCT 155 251 Motif Conservation Comparison of Nv and El GRH proteins using MEME LSF GRH-1 GRH-2 132 182 Phylogenetic Analysis Not done yet Conclusions Future Directions Acknowledgements