Download Matrix Attachment Region

Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Chromatin Boundries
• Observe DNA loops attached to nuclear
scaffold
• DNA loops are 30-90 kb in length
• DNA is attached to Nuclear Matrix
• Attachment region is called Matrix
Attachment Region (or MAR)
Loop (30-60 kb)
MAR
Scaffold
1
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
MARs
Chromatin Boundries
• 200-400 bp; 70% A/T rich; topoisomerase II recognition
site
• Also called Scaffold Attachment Region (SAR)
• No effect in transient transfection
• Confer position independence on integrated genes
Expression
Gene
MAR
Gene
MAR
Episomal
Strong
Integrated
Variable
Strong
Strong
2
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Assays to identify
MARS
Isolate nuclei
extract histones
1. In vivo-
In vivo
In vitro
restriction digest
extract matrix-bound DNA
2. In vitrodegrade all DNA
Add DNA fragments to MAR
Determine which DNA fragments bind
Sequence DNA
3
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Are MARS insulators?
MARs
• Some mammalian MARs will function as insulators in
Drosophila
• Not all MARs have insulator activity
Insulators
• Mammalian MARs can function as insulators in
Drosophila
• Classic insulator is scs and scs’ marking boundries of the
heat shock locus in Drosophila
– Eliminates “position effects”
• gypsy is also an insulator
4
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
LCRs- Review by Higgs
Cell 95:299 (1998)
A. Overview
• 1.Chromosomal environment- differential expression
depending on where located• 2. Position effects observed in
 translocations
 transgenic mice
 cell culture
• 3. Great quote “We hide our ignornace by using terms
“open vs closed”, “permissive vs non-permissive,
“euchromatin vs heterochromatin””
5
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
• 4. Open chromatin
 uncondensed, replicate early, near center of nucleus,
histones hyperacetylated, DNaseI sensitive,
undermethylated
• 5. DNaseI sensitivity
– a. general DNaseI sensitivity- DNA 10-fold more
accessible to DNaseI, covering long regions
– b. DNaseI hypersensitivity- DNA 100-fold more
sensitive to DNaseI
6
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Questions• What defines the origins of replication?
• What directs nuclear sublocalization?
• What sequences define boundries of open
and closed chromatin?
• What organizes higher order chromatin
structures?
7
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Human b-globin gene cluster
• Five erythroid-specific genes
• Arranged in order of expression
• LCR is upstream cluster of 5 (actually 7)
HS sites
• Each HS site binds numerous factors
8
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Human b-globin gene clusterTransgenics
• Each HS site binds numerous factors
9
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Human b-globin gene clusterTransgenics
• Remove LCR, low level expression of all
genes in cluster
• LCR confers copy number dependence
• > 10 additional LCRs identified
• Upstream cluster of 5 HS sites
• Each HS site binds numerous factors
10
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Hispanic deletion
•
•
•
•
Transcriptionally inactive
100kb up and downstream DNAseI resistant
late replicating
Conclude- LCR opens chromatin (is this true??)
11
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Models and observations
12
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Proposed LCR functions
•
•
•
•
•
•
Open chromatin
prevent variegated heterochromatization
Affects timing of replication
Keeps promoters histone-free
Change subcellular localization of b locus
LCR transcription affects rest of locus
expression
• Recruit HATs
13
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Role of intragenic transcription?
• Increase in intragenic transcripts correlates
with globin gene activity
– e.g. e and g intragenic transcripts higher when e
and g transcripts higher
14
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
15
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Human b-globin gene clusterQuestions
• What regulatory properties does the LCR
confer? (gene regulation?)
• Is replication timing directed by gene
activity?
• Is chromatin structure a cause or
consequence of globin gene activation?
16
Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003
Gary A. Bulla, Ph.D.
Model:
Stage-specific activation within subdomains by an LCR holocomplex
“Early”
“Late”
Putative chromatin boundries (small spheres)
17