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Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress.

Davuluri G, Gong W, Yusuff S, Lorent K, Muthumani M, Dolan AC, Pack M - PLoS Genet. (2008)

Bottom Line: Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine.These in vivo data indicate a role for Elys in Mcm2-chromatin interactions.Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACT
The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

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The flo locus encodes zebrafish elys.(A) Schematic representation of the genomic region surrounding the flo locus. The names of the polymorphic markers with the corresponding number of recombinants are listed. (B) DNA sequence analysis showing the cytosine to thymidine transition encoding the premature stop codon in the elysti262c allele. (C) Schematic representation of the functional domains of the human (hs) and zebrafish (dr) Elys protein and the protein encoded by the elysti262c allele (flo-ELYS). (D–I) Acridine orange staining showing apoptotic cells in the retina and growth plate of the optic tectum of 48 hpf flo (E,H) and elys-morpholino injected (F,I) larvae but not wt (D,G). (J–R) Confocal projections through the posterior intestine of 96 hpf larvae showing wheat-germ agglutinin positive goblet cells in the epithelium of the posterior intestine of wt (J) but not flo (K) or elys-morpholino injected (L) larvae; secretory cells in wt (M) but not flo (N) or elys-morpholino (O) injected larvae; enterocytes in wt (P) but not flo (Q) or elys-morpholino (R) injected larvae. (S–T) Carboxy-peptidase A positive cells are abundant in the 5 dpf wt (S) but not in elys-morpholino injected (T) exocrine pancreas. (U) Histological cross section through the retina of a 4 dpf elys morpholino injected larva showing retinal disorganization that is comparable to the 4 dpf flo retina (Figure 1H).
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pgen-1000240-g002: The flo locus encodes zebrafish elys.(A) Schematic representation of the genomic region surrounding the flo locus. The names of the polymorphic markers with the corresponding number of recombinants are listed. (B) DNA sequence analysis showing the cytosine to thymidine transition encoding the premature stop codon in the elysti262c allele. (C) Schematic representation of the functional domains of the human (hs) and zebrafish (dr) Elys protein and the protein encoded by the elysti262c allele (flo-ELYS). (D–I) Acridine orange staining showing apoptotic cells in the retina and growth plate of the optic tectum of 48 hpf flo (E,H) and elys-morpholino injected (F,I) larvae but not wt (D,G). (J–R) Confocal projections through the posterior intestine of 96 hpf larvae showing wheat-germ agglutinin positive goblet cells in the epithelium of the posterior intestine of wt (J) but not flo (K) or elys-morpholino injected (L) larvae; secretory cells in wt (M) but not flo (N) or elys-morpholino (O) injected larvae; enterocytes in wt (P) but not flo (Q) or elys-morpholino (R) injected larvae. (S–T) Carboxy-peptidase A positive cells are abundant in the 5 dpf wt (S) but not in elys-morpholino injected (T) exocrine pancreas. (U) Histological cross section through the retina of a 4 dpf elys morpholino injected larva showing retinal disorganization that is comparable to the 4 dpf flo retina (Figure 1H).

Mentions: Bulk segregant analysis identified markers on zebrafish chromosome 17 that were linked to the flo locus (Figure 2 and Figure S1). Analyses of 2629 mutant larvae ultimately identified a zero recombinant marker within a predicted open reading frame that encoded a gene orthologous to mammalian Elys (also known as AT hook containing transcription factor 1; ATHCF1), a gene recently shown to be required for NPC assembly and nuclear integrity in worms [18]–[21]. Elys is expressed in a wide range of tissues and is essential for early mammalian development [16]. The longest open reading frame of zebrafish elys encodes a predicted protein that consists of 2527 amino acid residues. Computational analysis of the Elys protein revealed two WD-40 repeats and three nuclear export signals in the N-terminus plus a coiled-coil region (involved in protein-protein interactions) and several nuclear localization signals in the C-terminus (Figure 2C and Figure S2). The consensus sequence of the AT hook domain present in mammalian Elys was only partially conserved in the zebrafish ortholog. Sequence analyses of cDNA derived from flo larvae revealed a single base pair mutation encoding a cytidine to thymidine transition that generated a premature stop codon within the predicted zebrafish elys translation product (Figure 2B). This mutation is predicted to generate a truncated Elys protein that lacks 1209 amino acids containing the coiled-coil region and the nuclear localization signals. Protein truncation, coupled with reduced elys expression in flo larvae (Figure S3), most likely arising from termination codon associated mRNA decay [31], supports the idea that transcription of the elysti262c allele generates only a small amount of active Elys protein.


Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress.

Davuluri G, Gong W, Yusuff S, Lorent K, Muthumani M, Dolan AC, Pack M - PLoS Genet. (2008)

The flo locus encodes zebrafish elys.(A) Schematic representation of the genomic region surrounding the flo locus. The names of the polymorphic markers with the corresponding number of recombinants are listed. (B) DNA sequence analysis showing the cytosine to thymidine transition encoding the premature stop codon in the elysti262c allele. (C) Schematic representation of the functional domains of the human (hs) and zebrafish (dr) Elys protein and the protein encoded by the elysti262c allele (flo-ELYS). (D–I) Acridine orange staining showing apoptotic cells in the retina and growth plate of the optic tectum of 48 hpf flo (E,H) and elys-morpholino injected (F,I) larvae but not wt (D,G). (J–R) Confocal projections through the posterior intestine of 96 hpf larvae showing wheat-germ agglutinin positive goblet cells in the epithelium of the posterior intestine of wt (J) but not flo (K) or elys-morpholino injected (L) larvae; secretory cells in wt (M) but not flo (N) or elys-morpholino (O) injected larvae; enterocytes in wt (P) but not flo (Q) or elys-morpholino (R) injected larvae. (S–T) Carboxy-peptidase A positive cells are abundant in the 5 dpf wt (S) but not in elys-morpholino injected (T) exocrine pancreas. (U) Histological cross section through the retina of a 4 dpf elys morpholino injected larva showing retinal disorganization that is comparable to the 4 dpf flo retina (Figure 1H).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2570612&req=5

pgen-1000240-g002: The flo locus encodes zebrafish elys.(A) Schematic representation of the genomic region surrounding the flo locus. The names of the polymorphic markers with the corresponding number of recombinants are listed. (B) DNA sequence analysis showing the cytosine to thymidine transition encoding the premature stop codon in the elysti262c allele. (C) Schematic representation of the functional domains of the human (hs) and zebrafish (dr) Elys protein and the protein encoded by the elysti262c allele (flo-ELYS). (D–I) Acridine orange staining showing apoptotic cells in the retina and growth plate of the optic tectum of 48 hpf flo (E,H) and elys-morpholino injected (F,I) larvae but not wt (D,G). (J–R) Confocal projections through the posterior intestine of 96 hpf larvae showing wheat-germ agglutinin positive goblet cells in the epithelium of the posterior intestine of wt (J) but not flo (K) or elys-morpholino injected (L) larvae; secretory cells in wt (M) but not flo (N) or elys-morpholino (O) injected larvae; enterocytes in wt (P) but not flo (Q) or elys-morpholino (R) injected larvae. (S–T) Carboxy-peptidase A positive cells are abundant in the 5 dpf wt (S) but not in elys-morpholino injected (T) exocrine pancreas. (U) Histological cross section through the retina of a 4 dpf elys morpholino injected larva showing retinal disorganization that is comparable to the 4 dpf flo retina (Figure 1H).
Mentions: Bulk segregant analysis identified markers on zebrafish chromosome 17 that were linked to the flo locus (Figure 2 and Figure S1). Analyses of 2629 mutant larvae ultimately identified a zero recombinant marker within a predicted open reading frame that encoded a gene orthologous to mammalian Elys (also known as AT hook containing transcription factor 1; ATHCF1), a gene recently shown to be required for NPC assembly and nuclear integrity in worms [18]–[21]. Elys is expressed in a wide range of tissues and is essential for early mammalian development [16]. The longest open reading frame of zebrafish elys encodes a predicted protein that consists of 2527 amino acid residues. Computational analysis of the Elys protein revealed two WD-40 repeats and three nuclear export signals in the N-terminus plus a coiled-coil region (involved in protein-protein interactions) and several nuclear localization signals in the C-terminus (Figure 2C and Figure S2). The consensus sequence of the AT hook domain present in mammalian Elys was only partially conserved in the zebrafish ortholog. Sequence analyses of cDNA derived from flo larvae revealed a single base pair mutation encoding a cytidine to thymidine transition that generated a premature stop codon within the predicted zebrafish elys translation product (Figure 2B). This mutation is predicted to generate a truncated Elys protein that lacks 1209 amino acids containing the coiled-coil region and the nuclear localization signals. Protein truncation, coupled with reduced elys expression in flo larvae (Figure S3), most likely arising from termination codon associated mRNA decay [31], supports the idea that transcription of the elysti262c allele generates only a small amount of active Elys protein.

Bottom Line: Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine.These in vivo data indicate a role for Elys in Mcm2-chromatin interactions.Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACT
The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

Show MeSH
Related in: MedlinePlus