PUBLICATION

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

Authors

Davuluri, G., Gong, W., Yusuff, S., Lorent, K., Muthumani, M., Dolan, A.C., and Pack, M.

ID

ZDB-PUB-081105-5

Date

2008

Source

PLoS Genetics 4(10): e1000240 (Journal)

Registered Authors

Dolan, Amy, Lorent, Kristin, Pack, Michael

Keywords

Larvae, Gastrointestinal tract, DNA replication, Apoptosis, Zebrafish, Chromatin, Morpholino, Embryos

MeSH Terms

Animals
Cell Cycle
DNA Replication*
Retina/cytology
Retina/embryology
Retina/metabolism
RNA, Messenger/genetics
RNA, Messenger/metabolism
Amino Acid Sequence
DNA Damage
Nuclear Pore Complex Proteins/chemistry
Nuclear Pore Complex Proteins/genetics*
Nuclear Pore Complex Proteins/physiology*
Protein Serine-Threonine Kinases/metabolism
Molecular Sequence Data
Zebrafish/genetics*
Zebrafish/growth & development
Zebrafish/physiology
Genes, p53
Apoptosis*
Zebrafish Proteins/chemistry
Zebrafish Proteins/genetics*
Zebrafish Proteins/physiology*
Intestines/cytology
Intestines/embryology
Intestines/metabolism
Mutation*
Transcription Factors/metabolism
Checkpoint Kinase 2

(all 29)

PubMed

18974873 Full text @ PLoS Genet.

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.

Genes / Markers

Figures

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Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
ahctf1	ahctf1^{ti262c/ti262c}	standard conditions	Protruding-mouth	whole organism	RTPCR	Fig. S3
ahctf1	WT	standard conditions	Prim-5	immature eye	ISH	Fig. 3
ahctf1	WT	standard conditions	Long-pec	intestinal bulb	ISH	Fig. 3
ahctf1	WT	standard conditions	Day 5	intestinal bulb	ISH	Fig. 3
ahctf1	WT	standard conditions	Long-pec	liver	ISH	Fig. 3
ahctf1	WT	standard conditions	Prim-5	midbrain	ISH	Fig. 3
ahctf1	WT	standard conditions	Long-pec	optic tectum	ISH	Fig. 3
ahctf1	WT	standard conditions	Long-pec	pancreas	ISH	Fig. 3
ahctf1	WT	standard conditions	Day 5	posterior intestine	ISH	Fig. 3
ahctf1	WT	standard conditions	Long-pec	posterior intestine	ISH	Fig. 3

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
WT	chemical treatment: camptothecin	Day 4	whole organism lethal (sensu genetics), abnormal	text only
WT + MO1-ahctf1 + MO2-ahctf1	standard conditions	Long-pec	optic tectum apoptotic, abnormal	Fig. 2
WT + MO1-ahctf1 + MO2-ahctf1	standard conditions	Long-pec	retina apoptotic, abnormal	Fig. 2
WT + MO1-ahctf1 + MO2-ahctf1	standard conditions	Protruding-mouth	intestine nuclear pore mislocalised, abnormal	Fig. 4
WT + MO1-ahctf1 + MO2-ahctf1	standard conditions	Protruding-mouth	posterior intestine nuclear pore decreased amount, abnormal	Fig. 4
WT + MO1-ahctf1 + MO2-ahctf1	standard conditions	Protruding-mouth	retina nuclear pore decreased amount, abnormal	Fig. 4
WT + MO1-ahctf1 + MO2-ahctf1	standard conditions	Protruding-mouth	retina nuclear pore mislocalised, abnormal	Fig. 4
WT + MO1-ahctf1 + MO2-ahctf1	standard conditions	Day 4	posterior intestine epithelium goblet cell absent, abnormal	Fig. 2
WT + MO1-ahctf1 + MO2-ahctf1	standard conditions	Day 4	retina disorganized, abnormal	Fig. 2
WT + MO1-ahctf1 + MO2-ahctf1	standard conditions	Day 5	exocrine pancreas morphology, abnormal	Fig. 2

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Mutations / Transgenics

Allele	Type	Affected Genomic Region
m74	Point Mutation	polr3b
ti262c	Point Mutation	ahctf1
zdf1	Point Mutation	tp53

Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
ahctf1	MO1-ahctf1	MRPHLNO
ahctf1	MO2-ahctf1	MRPHLNO
ahctf1	MO3-ahctf1	MRPHLNO
tp53	MO3-tp53	MRPHLNO
tp53	MO4-tp53	MRPHLNO

Fish

Fish
ahctf1^{ti262c/ti262c}
ahctf1^{ti262c/ti262c} + MO3-tp53 + MO4-tp53
ahctf1^{ti262c/ti262c}; tp53^zdf1/zdf1
polr3b^m74/m74
WT
WT + MO1-ahctf1 + MO2-ahctf1
WT + MO2-ahctf1

Antibodies

Name	Type	Antigen Genes	Isotypes	Host Organism
Ab1-mcm2	polyclonal	mcm2	IgG1	Rabbit
Ab1-mcm3	polyclonal	mcm3	IgG	Rabbit
Ab1-mcm4	polyclonal	mcm4		Rabbit
Ab1-nucleoporin	monoclonal		IgG1	Mouse
Ab1-rho	monoclonal	rho		Mouse
Ab2-chek1	polyclonal			Rabbit
Ab2-chek2	polyclonal			Rabbit
Ab-2F11	monoclonal	anxa4	IgG1	Mouse
Ab2-h2afx	polyclonal	h2ax		Rabbit
Ab3-chek2	polyclonal	chek2		Rabbit

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Orthology

Engineered Foreign Genes

Mapping

Entity Type	Entity Symbol	Location
Feature	ti262c	Chr: 17 Details
SSLP	z24006	Chr: 17 Details
SSLP	z24082	Chr: 17 Details
SSLP	z54789	Chr: 17 Details

Davuluri et al., 2008 ZDB-PUB-081105-5 PMID:18974873

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

Davuluri et al., 2008

ZDB-PUB-081105-5
PMID:18974873