Title: Gene Regulation in Eukaryotes
1Gene Regulation in Eukaryotes
2Outline of Chapter 17
- How we use genetics to study gene regulation
- Using mutations to identify cis-acting elements
and trans-acting proteins - 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
- RNA splicing
- RNA stability
- mRNA editing
- Translation
- Posttranslational modification
- A comprehensive example of sex determination in
Drosophila
3Regulatory elements that map near a gene are
cis-acting DNA sequences
- cis-acting elements
- Promoter very close to genes initiation site
- Enhancer
- can lie far way from gene
- Can be reversed
- Augment or repress basal levels of transcription
Fig. 17.1 a
4Reporter constructs are a tool for studying gene
regulation
- Sequence of DNA containing genes postulated
regulatory region, but not coding region - Coding region replaced with easily identifiable
product such as ß-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
5Regulatory 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 cis-acting
elements - Known genetically as transcription factors
- Identified by
- Mapping
- Biochemical studies to identify proteins that
bind in vitro to cis-acting elements
Fig. 17.1 b
6In eukaryotes three RNA polymerases transcribe
different sets of genes
- RNA polymerase I transcribes rRNA
- 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 a
7- RNA polymerase III transcribes tRNAs and other
small RNAs (5S rRNA, snRNAs)
Fig. 17.2 b
8- 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
9- 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
10Large 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
11trans-acting proteins control transcription from
class II promoters
- Basal factors bind to the promoter
- TBP TATA box binding protein
- TAF TBP associated factors
- RNA polymerase II binds to basal factors
Fig. 17.4 a
12Activator 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
13- Transcriptional activators bind to specific
enhancers at specific times to increase
transcriptional levels
Fig. 17.5 a
14Examples of common transcription factors
- zinc-finger proteins and helix-loop-helix
proteins bind to the DNA binding domains of
enhancer elements
Fig. 17.5 b
15Some 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
16Localization of activator domains using
recombinant DNA constructs
- 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
Fig. 17.6
17Most 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
18Leucine zipper a common activator protein with
dimerization domains
Fig. 17.7 b
19Repressors diminish transcriptional activity
Fig. 17.8
20Repressors
- 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
21Transcription factors may act as activators or
repressors or have no affect
- Action of transcription factor depends on
- Cell type
- Gene it is regulating
22Specificity 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
23Myc-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
24Myc-Max system is a regulatory mechanism for
switching between activation and repression
- 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
Fig. 17.10
25Gene repression results only when the Max
polypeptide is made in the cell
max gene
Fig. 17.10 b
26Gene activation occurs when both Myc and Max are
made in cell
Fig. 17.10
27The 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
28Proof that cis-acting factor such as LCR is
needed for activation of b-globin gene
Fig. 17.12 b
29One mechanism of activation that brings LCR into
contact with distant globin genes may be DNA
looping
Fig. 17.12 c
30Other mechanisms of gene regulation
- Chromatin structure
- Slows transcription
- Hypercondensation stops transcription
- Genomic imprinting
- 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
31Normal chromatin structure slows transcription
Fig. 17.13
32Remodeling of chromatin mediates the activation
of transcription
Fig. 17.13
33Hypercondensation over chromatin domains causes
transcriptional silencing. This is achieved by
the methylation of cytosine residues
Fig. 17.14
34In 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
35Genomic 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
36- 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
37- H19 promoter is methylated during spermatogenesis
and thus the H19 promoter is not available to the
enhancer and is not expressed
38- 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
39RNA splicing helps regulate gene expression
Fig. 17.16
40Fig. 17.16 b
41RNA 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
42Specialized example of regulation through RNA
stability
Note also the untranslated sequences that help
modulate their translation
Fig. 17.17
43mRNA editing can regulate the function of protein
products e.g., AMPA receptor gene in mammals
Fig. 17.18
44Protein modifications after translation provide a
final level of control over gene function
- Ubiquitination targets proteins for degredation
- Ubiquitin small, highly conserved protein.
- Covalently attaches to other proteins
- Ubiquitinized proteins are marked for degredation
by proteosomes
Fig. 17.19 a
45Sex determination in DrosophilaA comprehensive
example of gene regulation
- Sex specific traits in Drosophila
Fig. 17.20
46(No Transcript)
47(No Transcript)
48The XA 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 XA ratio
through formation of homodimers or heterodimers - Sisterless-A and sisterless-B
49Fig. 17.21
50Hypothesis 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
- Some numerator subunits remain unbound by
denominator elements - Free numerator elements act as transcription
factors at Pe promoter early in development - Males
- 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
51Effects of Sxl mutations
- Recessive Sxl mutations making gene nonfunctional
- Females lethal
- Absence of Sxl allows expression of dosage
compensation genes on X chromosome - Increase transcription of X-linked genes is
lethal - Males
- 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
52Sxl 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
53- 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 - Females - Produces female specific Dsx-F protein
- Males No Tra protein and splicing of Dsx
produces Dsx-M protein
Fig. 17.22
54Dsx-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
55Tra 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