Transcript Gene_expression

Unit 2.2: Introduction to Control of Gene Expression
Objectives:
-learn why gene expression is important
-learn the basic concepts of gene expression
-become familiar with promoters, cis-regulatory modules,
transcription factors
-see examples of experimental methods used to assay how gene
expression is regulated
Reading:
Maston GA, Evans SK, Green MR. (2006). Transcriptional
regulatory elements in the human genome. Annu Rev Genomics
Hum Genet, 7:29-59.
Every cell has the same DNA and therefore
the same genes. But different genes need to be
“on” and “off” in different types of cells.
Therefore, gene expression must be regulated.
liver
embryo
muscle
bone
sperm
(The first statement on this slide is not completely true. Which of these cells
does not have exactly the same DNA as the other? Can you think of any other
examples of cells in your body that have different DNA than most of the
others?)
Gene expression must be regulated in
several different dimensions—
In time:
10 wks
6 mos
14 wks
1 day
12 mos
18 mos
At different stages of the life cycle, different genes need to be on and off.
© M. Halfon, 2007
In space:
Paddock S.W. (2001). BioTechniques 30: 756 - 761.
Each colored stripe in this fly embryo shows the expression
of a different gene or set of genes. The spatial regulation of
these genes allows the embryo to be divided up into different
regions that will give rise to the head, the internal organs, the
abdomen, etc.
and in abundance:
Clyde et al. (2003). Nature 426:849-853
Note how the gene whose expression is indicated in blue varies
in abundance from strong expression (bold arrow) to weak
(thin arrow) within its expression domain. These differences in
strength of gene expression have important functional
consequences.
Some of the many areas in which regulated
gene expression plays a critical role are
illustrated on the following slide. Gene
regulation is important not only during
development but also in mediating common
variation between individuals, diseases and
birth defects, and evolution.
Importance of gene regulation
common variation
behavior
pattern
evolution
chromosome
inactivation
metabolism
pathology (mutation)
© M. Halfon, 2006
Gene Regulation and Nutrition:
Development (organs, cell types)
muscle
liver (diseased)
fat
embryo
embryo
brain
intestines
With respect to nutrition, gene regulation is important to guide
the development of organs, tissues, and cell types required to
ingest, digest, and metabolize nutrients.
Gene Regulation and Nutrition:
Metabolism (enzymes)
high carb/low fat
(sustained) 
insulin 
increased transcription
fatty acid synthase
acetyl-CoA carboxylase
Your long-term diet can lead to permanent changes in your body’s
gene expression profile
Genes can be regulated at many levels
DNA
RNA
TRANSCRIPTION
PROTEIN
TRANSLATION
The “Central Dogma”
click here and then select “Control of Gene Expression in
Eukaryotes (959.0K)” for an animation on gene expression
Control of Gene Expression—Transcription Factors
Transcription factors (TFs) are proteins that bind to
the DNA and help to control gene expression. We
call the sequences to which they bind transcription
factor binding sites (TFBSs), which are a type of
cis-regulatory sequence.
Determining Transcription Factor Binding Sites
One way to determine where a TF binds is
to use DNAseI footprinting, which takes
advantage of the ability of the enzyme
DNAseI to non-specifically cleave DNA. A
bound TF “protects” the DNA from
cleavage, leaving a visible “footprint” when
the digested DNA is visualized by gel
electrophoresis.
Figure 8-54. The DNA footprinting technique. (A) This
technique requires a DNA molecule that has been labeled
at one end (see Figure 8-24B). The protein shown binds
tightly to a specific DNA sequence that is seven
nucleotides long, thereby protecting these seven
nucleotides from the cleaving agent. If the same reaction
were performed without the DNA-binding protein, a
complete ladder of bands would be seen on the gel (not
shown). (B) An actual footprint used to determine the
binding site for a human protein that stimulates the
transcription of specific eucaryotic genes. These results
locate the binding site about 60 nucleotides upstream from
the start site for RNA synthesis. The cleaving agent was a
small, iron-containing organic molecule that normally cuts
at every phosphodiester bond with nearly equal frequency.
(B, courtesy of Michele Sawadogo and Robert Roeder.)
Source: Alberts et al., Molecular Biology of the Cell
Determining Transcription Factor Binding Sites
Other methods include
- EMSA (gel shift)
- SELEX (Systematic Evolution of Ligands
by EXponential enrichment)
-protein-binding microarrays
-ChIP-chip/ChIP-seq
Control of Gene Expression—Transcription Factors
Most transcription factors can bind to a range of similar
sequences. We call this a binding “motif.”
Wasserman, W. W. and A. Sandelin (2004). Nat Rev Genet 5(4): 276-287.
(We can represent these motifs in various ways, which
we will see in Unit 2.5)
Control of Gene Expression
Transcription factor binding sites are found within larger
functional units of the DNA called cis-regulatory elements.
There are two main type of cis-regulatory elements: promoters,
and cis-regulatory modules (sometimes called “enhancers”).
cis-regulatory module (CRM)
TFBS
transcription factor binding site (TFBS)
TFBS
Image adapted from Wolpert, Principles of Development
Control of Gene Expression: Promoters
Every gene has a promoter, the DNA sequence immediately
surrounding the transcription start site. The promoter is the site
where RNA polymerase and the so-called general transcription
factors bind.
Control of Gene Expression: CRMs
Additional gene regulation takes place via the cis-regulatory
modules (CRMs), which can be located 5’ to, 3’ to, or within
introns of a gene. CRMs can be very far away from the gene
they regulate—over 50 kb—and other genes might even lie in
between!
cis-regulatory
module (CRM)
TFBS
TFBS
transcription factor binding site (TFBS)
click here and then select “Transcription Complex and Enhancers
(586.0K)” for an animation on gene expression
cis-Regulatory Modules (enhancers)
Genes are often regulated in a modular fashion—discrete cis-regulatory elements
(CRMs, “enhancers”) dictate a specific spatio-temporal expression pattern, shown
here by purple stain. A gene might have many CRMs, each responsible for a
different part of its overall expression pattern.
eve
Map of 3’ regulatory region of Drosophila even skipped (Fujioka et al. 1999)
Looking at cis-regulatory modules:
Reporter Genes
How can we identify and study CRMs? To do this we use a
reporter gene assay. In such an assay, we use recombinant
DNA methods to test if a DNA sequence can regulate the
expression of a gene whose expression we can easily identify
(a “reporter gene”). The jellyfish green fluorscent protein
(GFP) gene is often used, as the encoded protein emits green
light when exposed to light of the proper wavelength. We can
test for CRM activity in transfected cells in culture, or even
better, in a transgenic animal:
Looking at cis-regulatory modules:
Reporter Genes
cis-regulatory module (CRM)
TFBS
TFBS
CRM
e.g., from Myosin Heavy
Chain gene
muscle
transcription factor binding site (TFBS)
minimal promoter
green fluorescent protein
Looking at cis-regulatory modules:
Reporter Genes
CRM
minimal promoter
green fluorescent protein
reporter construct
transfect cells
make transgenic animal
A nutritional example: the lactase gene
© M. Halfon, 2007
A nutritional example: the lactase gene
Many adult humans cannot metabolise lactose (milk sugar). A single nucleotide
polymorphism (SNP), i.e., a one basepair difference in DNA sequence,
correlates with activation of the lactase promoter and with lactose
tolerance/intolerance.
Moreover, this simple change can be seen to affect the binding activity of a
transcription factor, Oct-1, to the relevant CRM.
There are likely to be many such instances of how changes in gene regulation
affect nutrition, health, and disease, although most remain to be discovered.
agataatgtagTccctggcctca
agataatgtagCccctggcctca
ability to
activate
Oct-1
binding
phenotype
++
++
tolerant
+
+
intolerant
Olds, L. C. and E. Sibley (2003). Hum. Mol. Genet. 12(18): 2333-2340.