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

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

Position Effect Variegation

Wild-type

Variegated

The white gene produces red eyes

Dorer and Henikoff Cell 77:993, 1994

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

PEV can be suppressed by modifiers

Six-copy mini-white gene

at 50C in Su(var)295 flys

controls

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

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

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

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

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

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

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

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

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

• 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.

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.

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!!