Download The split Grainyhead protein of Cnidaria Grainyhead

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