Gene Regulation in Eukaryotes

1
Gene Regulation in Eukaryotes
2
Outline 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

3
Regulatory 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
4
Reporter 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

5
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 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
6
In 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

10
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
11
trans-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
12
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

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• Transcriptional activators bind to specific
  enhancers at specific times to increase
  transcriptional levels

Fig. 17.5 a
14
Examples 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
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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

16
Localization 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
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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
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Leucine zipper a common activator protein with
dimerization domains
Fig. 17.7 b
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Repressors diminish transcriptional activity
Fig. 17.8
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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

21
Transcription factors may act as activators or
repressors or have no affect

• Action of transcription factor depends on
• Cell type
• Gene it is regulating

22
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
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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
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Myc-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
25
Gene repression results only when the Max
polypeptide is made in the cell
max gene
Fig. 17.10 b
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Gene activation occurs when both Myc and Max are
made in cell
Fig. 17.10
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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
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Proof that cis-acting factor such as LCR is
needed for activation of b-globin gene
Fig. 17.12 b
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One mechanism of activation that brings LCR into
contact with distant globin genes may be DNA
looping
Fig. 17.12 c
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Other 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

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Normal chromatin structure slows transcription
Fig. 17.13
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Remodeling of chromatin mediates the activation
of transcription
Fig. 17.13
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Hypercondensation over chromatin domains causes
transcriptional silencing. This is achieved by
the methylation of cytosine residues
Fig. 17.14
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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
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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

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

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• 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
39
RNA splicing helps regulate gene expression
Fig. 17.16
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Fig. 17.16 b
41
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

42
Specialized example of regulation through RNA
stability
Note also the untranslated sequences that help
modulate their translation
Fig. 17.17
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mRNA editing can regulate the function of protein
products e.g., AMPA receptor gene in mammals
Fig. 17.18
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Protein 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
45
Sex determination in DrosophilaA comprehensive
example of gene regulation

• Sex specific traits in Drosophila

Fig. 17.20
46
(No Transcript)
47
(No Transcript)
48
The 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

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Fig. 17.21
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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
• 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

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Effects 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

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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
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
54
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
55
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