Transcript Position Effect Variegation

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
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
The white gene produces
red eyes
Variegated
Wild-type
Dorer and Henikoff Cell 77:993, 1994
PEV- effect of transgene repeats
• Single copy of “mini-white” locus inserted near centromere
in null-white fly strain
Heterochromatin
Inverted repeat
Single copy
tandem repeat
three repeats
four repeats
Thus, repeat number and orientation affect PEV
PEV can be suppressed by
modifiers
controls
Six-copy
mini-white gene
at 50C in
Su(var)295 flys
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
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
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
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
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
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
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 longrange enhancerpromoter
communication
Thus, Rap1p may may
have repressor role in
euchromatin, activator
role in heterochromatin
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?
Advanced Molecular and Cellular Biology Bio4751 Spring 2003
Gary A. Bulla, PhD
Recent result
• What happens if have two genes (GFP and miniwhite) near centromere?
Gal4-responsive Green Fluor. Protein
Mini-white gene
Centromere
Ahmad and Henikoff, Cell 104:839, 2001.
Ahmad and Henikoff, Cell 104:839, 2001.
GFP in
GFP in near
euchromatin heterochromatin
• Observe GFP
expression is
variegated next to
heterochromatin
•And as increase High GAL4
Gal4, suppress
variegation
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?
No
Yes
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?
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