1. Position Effect Variegation
1930- first described
“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”
Wakimoto Cell 93:321, 1998

2. 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

3. Position Effect Variegation
The white gene produces
red eyes
Variegated
Wild-type
Dorer and Henikoff Cell 77:993, 1994

4. PEV- effect of transgene repeats
Single copy of “mini-white” locus inserted near centromere
in null-white fly strain
Heterochromatin
tandem repeat
Inverted repeat
Single copy
three repeats
four repeats
Thus, repeat number and orientation affect PEV

5. PEV can be suppressed by modifiers
Six-copy
mini-white gene
at 50C in
Su(var)295 flys
controls

6. Models of PEV A. Cis-spreading
Advanced Molecular and Cellular Biology Bio4751 Spring 2003
Gary A. Bulla, PhD
Models of PEV A. Cis-spreading
Note: Histone acetylation effects PEV
1. Cis-spreading, block factor binding
2. Cis-spreading, form repressor complex with factors
Thus, more spreading = more variegation

7. Models of PEV A. Cis-spreading
Advanced Molecular and Cellular Biology Bio4751 Spring 2003
Gary A. Bulla, PhD
Models of PEV A. 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

8. Models B. Nuclear compartment model A trans-effect model
Evidence 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

9. How does PEV Occur? Lets look at Telomere Position Effect
Advanced Molecular and Cellular Biology Bio4751 Spring 2003
Gary A. Bulla, PhD
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

10. Advanced Molecular and Cellular Biology Bio4751 Spring 2003
Gary 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

11. What about genes normally active in heterochromatin?
Advanced Molecular and Cellular Biology Bio4751 Spring 2003
Gary 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

12. What about genes normally active in heterochromatin?
Advanced Molecular and Cellular Biology Bio4751 Spring 2003
Gary 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

13. How is PEV maintained? No current model is satisfactory
Advanced Molecular and Cellular Biology Bio4751 Spring 2003
Gary 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?

14. Recent result
Advanced Molecular and Cellular Biology Bio4751 Spring 2003
Gary A. Bulla, PhD
What happens if have two genes (GFP and mini-white) near centromere?
Mini-white gene
Gal4-responsive Green Fluor. Protein
Centromere
Ahmad and Henikoff, Cell 104:839, 2001.

15. GFP in near heterochromatin
Ahmad and Henikoff, Cell 104:839, 2001.
GFP in
euchromatin
GFP in near heterochromatin
Observe GFP expression is variegated next to heterochromatin
High GAL4
Low GAL4
Euchrom.
Heterochrom.
And as increase Gal4, suppress variegation

16. What happens to a nearby gene (the mini-white gene)?
Ahmad and Henikoff, Cell 104:839, 2001.
Miniwhite near heterochromatin No
Yes
Miniwhite in
euchromatin
GAL4?
Yes No
Thus, GAL4 binding counteracts silencing at
nearby mini-white locus

17. Can GFP and mini-white variegation be uncoupled?
Ahmad and Henikoff, Cell 104:839, 2001.
Can GFP and mini-white variegation be uncoupled?
Miniwhite near heterochromatin
Note- GFP on, mini-white off!!
Thus, GFP and mini-white silencing can be uncoupled.!
Heterochromatic boundary may be within 2 kb of DNA.
Heterochomatic spreading in not continuous