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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 mutation activates the DNA damage response.(A–C) Acridine orange staining showing apoptosis in the 50 hpf flo retina that is rescued by the tp53 morpholino (mo) knockdown. (D) Histological cross section showing rescue of flo retinal architecture defects by tp53 knockdown [compare (F) with Figure 1G and 1H]. (E) Intestinal defects persist in flo/tp53 double mutants. Arrow, thin intestinal wall; Arrowhead, apoptotic cells in the intestinal lumen. (F) Western blot showing elevated levels of phospho-Chk2 (Serine 33) in the intestine of flo larvae, compared with sibling wild type larvae, but not slim jim larvae (I). (G) Western blot showing comparable levels of phospho-Chk1 (Ser 345) in flo and sibling wild type larvae, before and after γ-radiation (30 Gy) and treatment with hydroxyurea (HU). (H) Western blot showing enhanced phospho-Chk2 activation in the intestine of flo and wild type larvae following γ-radiation (30 Gy). (I) γH2AX is not detected in the flo or wild type intestine (75 hpf), but is detected at this stage following γ-irradiation (30 Gy) or hydroxyurea treatment (HU).
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pgen-1000240-g006: The flo mutation activates the DNA damage response.(A–C) Acridine orange staining showing apoptosis in the 50 hpf flo retina that is rescued by the tp53 morpholino (mo) knockdown. (D) Histological cross section showing rescue of flo retinal architecture defects by tp53 knockdown [compare (F) with Figure 1G and 1H]. (E) Intestinal defects persist in flo/tp53 double mutants. Arrow, thin intestinal wall; Arrowhead, apoptotic cells in the intestinal lumen. (F) Western blot showing elevated levels of phospho-Chk2 (Serine 33) in the intestine of flo larvae, compared with sibling wild type larvae, but not slim jim larvae (I). (G) Western blot showing comparable levels of phospho-Chk1 (Ser 345) in flo and sibling wild type larvae, before and after γ-radiation (30 Gy) and treatment with hydroxyurea (HU). (H) Western blot showing enhanced phospho-Chk2 activation in the intestine of flo and wild type larvae following γ-radiation (30 Gy). (I) γH2AX is not detected in the flo or wild type intestine (75 hpf), but is detected at this stage following γ-irradiation (30 Gy) or hydroxyurea treatment (HU).

Mentions: To gain a better understanding of how NPC disruption caused apoptosis and cell cycle arrest in flo intestine and retina, we assayed expression of tp53 and p21 in 50 hpf and 75 hpf flo mutants via real-time quantitative PCR. These experiments showed increased expression of tp53, p21 and also mdm2, a negative regulator of p53 whose expression is induced in response to p53 activation (Figure S6). To further assess the role of tp53 in flo mutants, we injected antisense morpholinos known to target zebrafish tp53 mRNA translation [27],[38],[39] into newly fertilized embryos derived from matings of heterozygous flo/+ fish. The tp53 knockdowns restored normal p21 and tp53 expression in flo larvae. We also generated larvae that were homozygous for both the flo mutation and a previously described tp53 mutation that inhibits p53-dependent radiation induced apoptosis [40]. The homozygous tp53 mutation as well as the tp53 knockdowns rescued retinal apoptosis in 50 hpf flo mutants (n = 25 flo/tp53 double mutants analyzed and >200 tp53 morpholino injected larvae analyzed; Figure 6A–6C). Retinal size and architecture was restored or greatly improved by the tp53 knockdown and mutation in most flo larvae (n>50, 5 dpf larvae examined; Figure 6D), but neither rescued intestinal morphology, differentiation or apoptosis (Figure 6E; n = 25 flo/tp53 double mutants analyzed at 4 dpf). These data show that disruption of NPCs activates the DNA damage response in a tissue-specific manner.


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 mutation activates the DNA damage response.(A–C) Acridine orange staining showing apoptosis in the 50 hpf flo retina that is rescued by the tp53 morpholino (mo) knockdown. (D) Histological cross section showing rescue of flo retinal architecture defects by tp53 knockdown [compare (F) with Figure 1G and 1H]. (E) Intestinal defects persist in flo/tp53 double mutants. Arrow, thin intestinal wall; Arrowhead, apoptotic cells in the intestinal lumen. (F) Western blot showing elevated levels of phospho-Chk2 (Serine 33) in the intestine of flo larvae, compared with sibling wild type larvae, but not slim jim larvae (I). (G) Western blot showing comparable levels of phospho-Chk1 (Ser 345) in flo and sibling wild type larvae, before and after γ-radiation (30 Gy) and treatment with hydroxyurea (HU). (H) Western blot showing enhanced phospho-Chk2 activation in the intestine of flo and wild type larvae following γ-radiation (30 Gy). (I) γH2AX is not detected in the flo or wild type intestine (75 hpf), but is detected at this stage following γ-irradiation (30 Gy) or hydroxyurea treatment (HU).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000240-g006: The flo mutation activates the DNA damage response.(A–C) Acridine orange staining showing apoptosis in the 50 hpf flo retina that is rescued by the tp53 morpholino (mo) knockdown. (D) Histological cross section showing rescue of flo retinal architecture defects by tp53 knockdown [compare (F) with Figure 1G and 1H]. (E) Intestinal defects persist in flo/tp53 double mutants. Arrow, thin intestinal wall; Arrowhead, apoptotic cells in the intestinal lumen. (F) Western blot showing elevated levels of phospho-Chk2 (Serine 33) in the intestine of flo larvae, compared with sibling wild type larvae, but not slim jim larvae (I). (G) Western blot showing comparable levels of phospho-Chk1 (Ser 345) in flo and sibling wild type larvae, before and after γ-radiation (30 Gy) and treatment with hydroxyurea (HU). (H) Western blot showing enhanced phospho-Chk2 activation in the intestine of flo and wild type larvae following γ-radiation (30 Gy). (I) γH2AX is not detected in the flo or wild type intestine (75 hpf), but is detected at this stage following γ-irradiation (30 Gy) or hydroxyurea treatment (HU).
Mentions: To gain a better understanding of how NPC disruption caused apoptosis and cell cycle arrest in flo intestine and retina, we assayed expression of tp53 and p21 in 50 hpf and 75 hpf flo mutants via real-time quantitative PCR. These experiments showed increased expression of tp53, p21 and also mdm2, a negative regulator of p53 whose expression is induced in response to p53 activation (Figure S6). To further assess the role of tp53 in flo mutants, we injected antisense morpholinos known to target zebrafish tp53 mRNA translation [27],[38],[39] into newly fertilized embryos derived from matings of heterozygous flo/+ fish. The tp53 knockdowns restored normal p21 and tp53 expression in flo larvae. We also generated larvae that were homozygous for both the flo mutation and a previously described tp53 mutation that inhibits p53-dependent radiation induced apoptosis [40]. The homozygous tp53 mutation as well as the tp53 knockdowns rescued retinal apoptosis in 50 hpf flo mutants (n = 25 flo/tp53 double mutants analyzed and >200 tp53 morpholino injected larvae analyzed; Figure 6A–6C). Retinal size and architecture was restored or greatly improved by the tp53 knockdown and mutation in most flo larvae (n>50, 5 dpf larvae examined; Figure 6D), but neither rescued intestinal morphology, differentiation or apoptosis (Figure 6E; n = 25 flo/tp53 double mutants analyzed at 4 dpf). These data show that disruption of NPCs activates the DNA damage response in a tissue-specific manner.

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