Transcript Reporter constructs are a tool for studying gene regulation

Gene Regulation in
Eukaryotes
Outline of Chapter 17

How we use genetics to study gene regulation


How genes are regulated at the initiation of transcription






Three polymerases recognize three classes of promoters
Trans-acting proteins control class II promoters
Chromatin structure affects gene expression
Signal transduction systems
DNA methylation regulates gene expression
How genes are regulated after transcription






Using mutations to identify cis-acting elements and trans-acting proteins
RNA splicing
RNA stability
mRNA editing
Translation
Posttranslational modification
A comprehensive example of sex determination in Drosophila
Regulatory elements that map near a
gene are cis-acting DNA sequences

cis-acting elements


Promoter – very close to gene’s initiation site
Enhancer



can lie far way from gene
Can be reversed
Augment or repress basal levels of transcription
Fig. 17.1 a
Reporter constructs are a tool for
studying gene regulation



Sequence of DNA containing gene’s postulated
regulatory region, but not coding region
Coding region replaced with easily identifiable
product such as β-galactosidase (Lac Z) or green
fluorescent protein (GFP)
Reporter constructs can help identify promoters
and enhancers by using in vitro mutagenesis to
systematically alter the presumptive regulatory
region
Regulatory elements that map far from a gene are
trans-acting DNA sequences because they encode
transcription factors

Genes that encode
proteins that interact
directly or indirectly
with target genes cisacting elements


Known genetically as
transcription factors
Identified by:


Fig. 17.1 b
Mapping
Biochemical studies to
identify proteins that
bind in vitro to cisacting elements
In eukaryotes three RNA polymerases
transcribe different sets of genes

RNA polymerase I
transcribes rRNA


Fig. 17.2 a
rRNAs are made of
tandem repeats on
one or more
chromosomes
RNA polymerase I
transcribes one
primary transcript
which is broken
down into 28S, 5.8S,
and 18S by
processing

Fig. 17.2 b
RNA polymerase III transcribes
tRNAs and other small RNAs (5S
rRNA, snRNAs)

RNA polymerase II recognizes cis-acting
regulatory regions composed of one promoter
and one or more enhancers


Promoter contains initiation site and TATA box
Enhancers are distant from target gene

Sometimes called upstream activation sites
Fig. 17.2 c

RNA polymerase II transcribes all protein
coding genes

Primary transcripts are processed by splicing, a
poly A tail is added to the 3’ end, and a 5’ GTP
cap is added
Large enhancer region of Drosophila string gene
• Fourteenth cell
cycle of the fruit
fly embryo
• A variety of
enhancer regions
ensure that string is
turned on at the
right time in each
mitotic domain
and tissue type
Fig. 17.3
trans-acting proteins control transcription
from class II promoters

Basal factors bind to
the promoter
TBP – TATA box
binding protein
 TAF – TBP
associated factors


Fig. 17.4 a
RNA polymerase II
binds to basal
factors
Activator proteins




Also called transcription factors
Bind to enhancer DNA in specific ways
Interact with other proteins to activate and
increase transcription as much as 100-fold
above basal levels
Two structural domains mediate these
functions
DNA-binding domain
 Transcription-activator domain


Transcriptional
activators bind
to specific
enhancers at
specific times to
increase
transcriptional
levels
Fig. 17.5 a
Examples of common transcription factors

zinc-finger
proteins and
helix-loophelix proteins
bind to the
DNA binding
domains of
enhancer
elements
Fig. 17.5 b
Some proteins affect transcription
with out binding to DNA


Coactivator – binds to and affects activator
protein which binds to DNA
Enhancerosome – multimeric complex of
proteins
Activators
 Coactivators
 Repressors
 Corepressors

Localization of activator domains
using recombinant DNA constructs



Fig. 17.6
Fusion constructs
from three parts of
gene encoding an
activator protein
Reporter gene can
only be transcribed
if activator domain
is present in the
fusion construct
Part B contains
activation domain,
but not part A or C
Most eukaryotic activators must
form dimers to function

Eukaryotic transcription factor protein structure


Homomers – multimeric proteins composed of identical
subunits
Heteromers – multimeric proteins composed of
nonidentical subunits
Fig. 17.7 a
Leucine zipper – a common activator protein
with dimerization domains
Fig. 17.7 b
Repressors diminish transcriptional activity
Fig. 17.8
Repressors

Reduction of transcriptional activation but do not
affect basal level of transcription



Activator-repressor competition
Quenching (corepressors)
Some repressors stop basal level of transcription


Binding directly to promoter
Bind to DNA sequences farther from promoter, contact
basal factor complex at promoter by bending DNA
causing a loop where RNA polymerase can not access
the promoter
Transcription factors may act as
activators or repressors or have no affect

Action of transcription factor depends on
Cell type
 Gene it is regulating

Specificity of transcription factor can be
altered by other molecules in cell



yeast a2 repressor –
determines mating
type
Haploid – a2 factor
silences the set of
“a” genes
Diploid – a2 factor
dimerizes with a1
factor and silences
haploid-specific
genes
Fig. 17.9
Myc-Max system is a regulatory mechanism for
switching between activation and repression


Myc polypeptide has an activation domain
Max polypeptide does not have an
activation domain
Fig. 17.10
Myc-Max system is a regulatory mechanism for
switching between activation and repression


Fig. 17.10
As soon as a cell
expresses the myc
gene, the Max-Max
homodimers convert
to Myc-Max
heterodimers that
bind to the
enhancers
Induction of genes
required for cell
proliferation
Gene repression results only when the Max
polypeptide is made in the cell
max gene
Fig. 17.10 b
Gene activation occurs when both
Myc and Max are made in cell
Fig. 17.10
The locus control region is a cis-acting
regulatory sequence that operates sequentially

Human b-globin gene cluster contains five
genes that can all be regulated by a distant
LCR (locus control region)
Fig. 17.12 a
Proof that cis-acting factor such as LCR is
needed for activation of b-globin gene
Fig. 17.12 b
One mechanism of activation that brings LCR into contact
with distant globin genes may be DNA looping
Fig. 17.12 c
Other mechanisms of gene regulation

Chromatin structure



Genomic imprinting


Slows transcription
Hypercondensation stops transcription
Silences transcription selectively if inherited from one
parent
Some genes are regulated after transcription





RNA splicing can regulate expression
RNA stability controls amount of gene product
mRNA editing can affect biological properties of protein
Noncoding sequences in mRNA can modulate translation
Protein modification after translation can control gene
function
Normal chromatin structure slows
transcription
Fig. 17.13
Remodeling of chromatin mediates
the activation of transcription
Fig. 17.13
Hypercondensation over chromatin domains
causes transcriptional silencing. This is
achieved by the methylation of cytosine
residues
Fig. 17.14
In mammals hypercondensation is
often associated with methylation

It is possible to determine the methylation state of
DNA using restriction enzymes that recognize the
same sequence, but are differentially sensitive to
methylation
Fig. 17.14
Genomic imprinting results from chromosomal events that
selectively silence genes inherited from one parent

1980s, in vitro fertilization experiments in mice
demonstrated pronuclei from two females could
not produce a viable embryos

Experiments with transmission of Ig f 2
deletion showed mice inheriting deletion from
male were small. Mice inheriting deletion from
female were normal.
Figure 15.15 a

H19 promoter is methylated during
spermatogenesis and thus the H19 promoter
is not available to the enhancer and is not
expressed


Epigenetic effect – whatever silences the maternal or
paternal gene is not encoded in the DNA. The factor is
outside the gene, but is heritable
Methylation can be maintained across generations by
methylases that recognize methyl groups on one strand and
respond by methylating the opposite strand
Fig. 15.15 c
RNA splicing helps regulate gene
expression
Fig. 17.16
Fig. 17.16 b
RNA stability provides a mechanism for
controlling the amount of gene product

Cellular enzymes slowly shorten the poly-A
tail. mRNA then degrades.
Length of poly-A tails of mRNAs affects the
speed at which mRNAs are degraded after they
leave the nucleus.
 Histone transcripts receive no poly-A tail


mRNA quickly degrades after S phase of cell cycle
Specialized
example of
regulation
through
RNA
stability
Note also the untranslated sequences that help modulate their translation
Fig. 17.17
mRNA editing can regulate the function of protein
products – e.g., AMPA receptor gene in mammals
Fig. 17.18
Protein modifications after translation provide a
final level of control over gene function

Ubiquitination targets
proteins for
degredation

Ubiquitin – small,
highly conserved
protein.


Fig. 17.19 a
Covalently attaches to
other proteins
Ubiquitinized proteins
are marked for
degredation by
proteosomes
Sex determination in Drosophila
A comprehensive example of gene regulation
Sex specific traits in Drosophila
Fig. 17.20
The X:A ratio regulates expression
of the Sex lethal (sxl) gene

Key factors of sex determination

Helix-loop-helix proteins encoded by genes on
the autosomes


Denominator elements
Helix-loop-helix proteins encoded by genes on
the X chromosome
Numerator elements – monitor the X:A ratio
through formation of homodimers or heterodimers
 Sisterless-A and sisterless-B

Fig. 17.21
Hypothesis to explain why flies with more numerator
homodimers transcribe Sxl early in development

Numerator subunit homodimers may function as transcription
factors that turn on Sxl

Females



Males




Some numerator subunits remain unbound by denominator elements
Free numerator elements act as transcription factors at Pe promoter early in
development
Carry half as many X-encoded numerator subunits
All numerator proteins are bound by abundant denominator elements
Pe promoter is not turned on
The Sxl protein expressed early in development in females
regulates its own later expression through RNA splicing

Females


Sxl protein produced early in development catalyzes the synthesis of more of
itself through RNA splicing of the PL transcript
Males

No Sxl transcript in early development results in a unproductive transcript in
later development from the PL promoter with a stop codon near the beginning of
the transcript
Effects of Sxl mutations

Recessive Sxl mutations making gene nonfunctional

Females – lethal



Males



Absence of Sxl allows expression of dosage compensation genes on X
chromosome
Increase transcription of X-linked genes is lethal
No Sxl expression
No affect on phenotype
Dominant Sxl mutations that allow expression even in XY
embryos

Females


No affect because they normally produce the protein
Males


Repression of genes used in dosage compensation
No hypertranscription of X-linked genes and do not have enough X-linked gene
product to survive
Sxl triggers a cascade of splicing

Sxl influences splicing of RNAs in other genes

e.g., transformer (tra)


Presence of Sxl produces functional protein
Absence of Sxl results in nonfunctional protein
Fig. 17.22

Cascade of splicing continues

e.g., doublesex (dsx)

Tra protein synthesized in females along with Tra2 protein
(produced in males and females) influences splicing of dsx


Fig. 17.22
Females - Produces female specific Dsx-F protein
Males – No Tra protein and splicing of Dsx produces Dsx-M
protein
Dsx-F and Dsx-M are transcription factors
that determine somatic sexual characteristics

Alternative forms of Dsx bind to YP1 enhancer, but have
opposite effects of expression on YP1 gene


Dsx-F is a transcriptional activator
Dsx-M is a transcriptional repressor
Fig. 17.23
Tra and Tra-2 proteins also help
regulate the expression of Fruitless

Primary fru mRNA transcript made in both sexes


Presence of tra protein in females causes alternative splicing
encoding fru-F
Absence of tra protein in males produces fru-M
Fig. 17.24