position effect variegation wakimoto cell 93:321, 1998 “the mosaic phenotype caused by a...
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Position Effect Variegation
Wakimoto Cell 93:321, 1998
“The mosaic phenotype caused by a chromosomal position effect in which a rearrangement breakpoint displaced the white gene from its normal euchromatic location and placed it in the vicinity of heterochromatin”
1930- first described
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Position effect variegation- pp448, Alberts
Definition- Translocation of a gene from a euchromatic region to a heterochromatic region resulting in inactivation of nearby heterochromatic genes.
– Called “heterochromatic spreading”, but is an incomplete definition
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Position Effect Variegation
Wild-type
Variegated
The white gene produces red eyes
Dorer and Henikoff Cell 77:993, 1994
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PEV- effect of transgene repeats
Single copy tandem repeat
three repeats
four repeats
Heterochromatin
• Single copy of “mini-white” locus inserted near centromere in null-white fly strain
Thus, repeat number and orientation affect PEV
Inverted repeat
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PEV can be suppressed by modifiers
Six-copy mini-white gene
at 50C in Su(var)295 flys
controls
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Models of PEVA. Cis-spreading
1. Cis-spreading, block factor binding
2. Cis-spreading, form repressor complex with factors
Thus, more spreading = more variegation
Advanced Molecular and Cellular Biology Bio4751 Spring 2003Gary A. Bulla, PhD
Note: Histone acetylation effects PEV
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Models of PEVA. Cis-spreading
Problems with cis-spreading model
• Some hetero-euchromatin rearrangements induce PEV several megabases away
• PEV is sensitive to interchromosomal interactions
• Thus, trans-interactions are suggested
Advanced Molecular and Cellular Biology Bio4751 Spring 2003Gary A. Bulla, PhD
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ModelsB. Nuclear compartment model
A trans-effect modelEvidence in support-• Centromeres and most heterochromatin is located at one
end of nucleus, telomeres at opposite end
• Displaced heterochromatic regions interact with other heterochromatic regions– prevented by modifiers of PEV
• However- have not yet correlated measured transcriptional activity and nuclear localization
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How does PEV Occur? Lets look at Telomere Position Effect
RAP1-
• Telomere Position Effect- Rap1 in complex with SIR proteins (SIR2/SIR3/SIR4) and histones H4 + H3– Functions- heterochromatin assembly; recruitment of SIR proteins
Folding-back mechanism
Advanced Molecular and Cellular Biology Bio4751 Spring 2003Gary A. Bulla, PhD
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Models of PEV
• Over 120 modifiers (enhancers and suppressors) of PEV identified
• Only some are directly involved• HP-1, Su(var)3-7 both co-localize
to heterochromatin, interact in yeast two hybrid assay– Neither binds DNA
Advanced Molecular and Cellular Biology Bio4751 Spring 2003Gary A. Bulla, PhD
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What about genes normally active in heterochromatin?
• Flys have over 20 expressed genes located in heterochromatin
• >7 of these genes require placement in heterochromatin for normal expression
• If place into euchromatic region- PEV results!
• 1/2 of mutations that suppress PEV of euchrom. genes also enhance PEV of heterochrom. genes
Advanced Molecular and Cellular Biology Bio4751 Spring 2003Gary A. Bulla, PhD
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What about genes normally active in heterochromatin?
Heterochromatin binding proteins interact with transcription factors to activate transcritpion
or mediate long-range enhancer-promoter communication
Thus, Rap1p may may have repressor role in euchromatin, activator role in heterochromatin
Advanced Molecular and Cellular Biology Bio4751 Spring 2003Gary A. Bulla, PhD
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How is PEV maintained?
• No current model is satisfactory• Not DNA methylation -(Flys don’t do this)• GAGA protein binds to heterochromatin,
remains throughout cell cycle• DNA must be “tagged” to maintain a given
level of PEV during subsequent cell divisions • Competition for factors at each cell cycle?
Advanced Molecular and Cellular Biology Bio4751 Spring 2003Gary A. Bulla, PhD
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Recent result • What happens if have two genes (GFP and mini-
white) near centromere?
Ahmad and Henikoff, Cell 104:839, 2001.
Gal4-responsive Green Fluor. Protein Mini-white gene
Centromere
Advanced Molecular and Cellular Biology Bio4751 Spring 2003Gary A. Bulla, PhD
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• Observe GFP expression is variegated next to heterochromatin
GFP in euchromatin
GFP in near heterochromatin
•And as increase Gal4, suppress variegation
High GAL4
Low GAL4
Euchrom. Heterochrom.
Ahmad and Henikoff, Cell 104:839, 2001.
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What happens to a nearby gene (the mini-white gene)?
Miniwhite in euchromatin
GAL4? YesNo
Miniwhite near heterochromatin
No Yes
Thus, GAL4 binding counteracts silencing at nearby mini-white locus
Ahmad and Henikoff, Cell 104:839, 2001.
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Can GFP and mini-white variegation be uncoupled?
•Thus, GFP and mini-white silencing can be uncoupled.!•Heterochromatic boundary may be within 2 kb of DNA. •Heterochomatic spreading in not continuous
Ahmad and Henikoff, Cell 104:839, 2001.
Miniwhite near heterochromatin
Note- GFP on, mini-white off!!