Download Differential Gene Expression

Biology 4361 - Developmental Biology
Differential Gene Expression
September 25, 2007
Differential Gene Expression
Chromatin structure
Gene anatomy
Gene sequences
Control of gene transcription
enhancers
transcription factors
Epigenetic processes (epigenetic - modification of structure
methylation
imprinting
RNA processing
of DNA rather than sequence)
Chromatin Structure
~140 bp
chromatin
~60 bp
Transcriptional Regulation:
1. “Packing” prevents access
CH3
2. Acetylation ( C O ) state of histones
controls DNA binding
- histone acetyltransferases – allow transcription
- histone deacetylases - repression
3. Histone methylation (CH3) – further repression
Chromatin Configuration
John H. Frenster
heterochromatin
-remains tightly condensed throughout
most of the cell cycle
- replicates later
euchromatin
- “active” chromatin
Anatomy of the Gene
core promoter
region
5’
TATAT
transcription initiation site
transcribed region
+1
upstream
downstream
3’
nRNA Processing
promoter
exon 1
intron 1
exon 2
intron 2
termination region
gene
cleavage signal
transcription, 5’ capping
7-methyl-Gppp
primary transcript
AAUAA
cleavage factor
3’ cleavage,
polyadenylation
cap
poly(A)polymerase
7-methyl-Gppp
AAAAAAA(n)
splicing
7-methyl-Gppp
exon 1
exon 2
AAAAAAA(n)
mature mRNA, ready to be transported to the cytoplasm and translated
Human β-globin Sequence
Production of β-globin
Promoters and Enhancers
RNA polymerase II binds to the promoter region at the TATA box
- however, Pol II cannot initiate transcription alone
- various proteins bind to regulatory sequences upstream and
downstream of transcription initiation site
- promoter region binds Basal Transcription Factors
promoter
region
5’
3’
- basal transcription factors facilitate Pol II binding and activity
Eukaryotic Transcription Initiation Complex
basal transcription factors:
- sequential binding
- binding mediated by
small proteins
- TBP-associated
factors (TAFs)
- mediator complex
1. TFIID complex binds to the TATA box
through its TATA Binding Protein
(TBP) subunit
2. TFIID is stabilized by TFIIA
3. TFIIB and TFIIH join the complex on
the TATA box; TFIIE and TFIIF
associate with RNA polymerase II
Eukaryotic Transcription Initiation Complex
4. RNA polymerase II is positioned
by TFIIB, and its carboxy-terminal
domain (CTD) is bound by TFIID
5. The CTD is phosphorylated by
TFIIH and is released by TFIID;
RNA polymerase II can now
transcribe mRNA
Stabilization of Transcriptional
Initiation Complex by TAFs
TBP – TATA binding protein
TAF(s) – TBP-associated factors
Enhancers
Enhancers are cis-acting regulatory elements
cis – (same or same side); elements that reside on the same DNA strand;
e.g. DNA sequences
trans – (other side); elements that originate from another DNA strand,
e.g. regulatory proteins
Enhancers: DNA sequences that regulate gene expression by affecting the
transcription initiation complex on the promoter.
Function - bind specific regulatory proteins (i.e. specific transcription factors)
Locations - highly variable with respect to the transcribed portion of the gene.
- upstream (5’), downstream (3’), within transcribed region
- close proximity or as many as 106 bp away
Enhancers are modular; e.g. mouse Pax6 gene
- expressed in multiple tissues, each under the influence of a different enhancer
Enhancers
Enhancers differ from promoters:
1) need a promoter to work
2) can work at a distance
3) can work in reverse orientation
Enhancer Generalizations
1. Most gene transcription requires enhancers.
2. Enhancers are the major determinants of differential transcription in
cell types and through developmental stages.
3. There can be multiple signals (e.g. multiple enhancer sites) for a given gene,
and each enhancer can be bound by more than one transcription factor
(not at the same time).
4. Transcription is regulated by the interaction of transcription factors bound to
enhancers and the transcription initiation complex assembled at the promoter.
5. Enhancers are combinatorial. Various DNA sequences regulate temporal and
spatial gene expression; these can be mixed and matched.
6. Enhancers are modular. A gene can have several enhancer elements, each of
which may turn it on in different sets of cells.
7. Enhancers generally activate transcription by remodeling chromatin to expose
the promoter, or by facilitating the binding of RNA polymerase to the promoter
by stabilizing TAFs.
8. Enhancers can also inhibit transcription (aka Silencers).
Method – Reporter Genes
Reporter gene construct: enhancer elements for Pax6 attached to
structural genes for reporter molecules.
exons for Pax6
mouse – Pax6
LacZ – β-galactosidase
Drosophila
add a gene for reporter protein, e.g.
β-galactosidase (blue)
luciferase (produces light)
green fluorescent protein (GFP)
Transcription Factors
Proteins that bind to enhancer or promoter regions
- activate or repress transcription
Most bind to specific DNA sequences (e.g. enhancers)
Transcription factors are grouped together in families, based on structural similarities
- families share common framework in DNA binding sites
- slight differences in binding sites cause differences in recognition
estrogen receptor zinc finger domain
Transcription Factor Domains
Three major domains:
1. DNA-binding
- recognizes particular DNA sequence
transcription factor - engrailed
Transcription Factor Domains
Three major domains:
1. DNA-binding
- recognizes particular DNA sequence
2. trans-activation – activates or represses transcription
- often involved with proteins involved in binding RNA polymerase II;
e.g. TFIIB, TFIIE
- often involved with enzymes that modify histones
3. protein-protein interaction domain
- promotes dimerization
- allows it to be modulated by TAFs or other transcription factors
Transcription Factor Domains
Transcription factor MITF
- basic helix-loop-helix
- homodimer is the
functional protein
The trans-activating domain is contained in the center of the protein.
- when bound to a promoter or enhancer, the protein is able to bind
a TAF (p300/CBP)
- TAF p300/CBP is a histone acetyltranferase
Enhancer Modules
Enhancers are modular: e.g. Pax6 gene expressed in the eye, pancreas,
nervous system.
- also expressed in different genes within these tissues
Within these modules, transcription factors work in a combinatorial fashion.
Transcription factors operate in cascades: one stimulates the production
of several others.
DNA Methylation
- found in vertebrates; not Drosophila, nematodes, inverts
- methylation stabilizes nucleosome;
stable nucleosome = transcriptional repression
- degree of methylation is proportional to degree of transcription
DNA Methylation
- absence of methylation correlates with tissue-specific expression
- methylation patterns maintained throughout cell division by
DNA (cytosine-5)-methyltransferase
Genomic Imprinting
Special case of DNA methylation
Alleles from maternal and paternal genome are differentially methylated.
Methylation patterns can be distinguished based on resulting phenotypes.
Dosage Compensation
Mammals, Drosophila, nematodes – XX = female, XY = male;
diploid XXs have double the X gene products as diploid XYs
Drosophila – transcription rate of male X is doubled
C. elegans – both Xs partially repressed (♀ = hermaphrodite)
Mammals - Inactivation of a single X chromosome in mammalian XX cells
XX cell
Barr bodies
early embryo – both active
later embryo –
only one active
X Chromosome inactivation
Lyon hypothesis:
1. Both X chromosomes active in very early female development.
2. One X is inactivated in each cell
3. Inactivation is random
4. The process is irreversible. All progeny cells will retain the same
inactivation pattern
♀ calico cat:
early
late
- heterozygous for coat color
- genes contained on
X chromosome
X chromosome inactivation
a-Tropomyosin alternative splicing
striated muscle
striated muscle’
myoblast
smooth muscle
neuroblast/muscle
hepatoma
brain