Download - Molecular Biology Lecture 8
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BIOL 321 – Regulation of Gene Expression in Eukaryotes
http://micro.magnet.fsu.edu/cells/animalcell.html
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Objectives- Gene “silencing” by histone modifications
- Recruitment of protein complexes to Genes
- Basal machinery
- Transcription factors
- Transcriptional repressors
- Cis-regulatory factors
- Extracellular signals and gene regulation
- Eukaryotic gene regulation at steps after transcription initiation
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1) Control the types and quantities of proteins (gene products) produced
2) Respond to the environment by turning on or off specific genes or groups of genes.
3) Turn genes on or off in the correct sequence during development
Why regulate gene expression?
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1) Cis-Regulatory sequences of DNA (Promoter & enhancer sequences)
2) Transcription factors (= Trans-acting factors) - Nuclear proteins that bind to promoter or enhancer
sequences of genes and stimulate/inhibit transcription
3) Changes to chromatin conformation such as:- DNA methylation- Histone modification – e.g. by acetylation, phosphorylation, etc.
Control of Differential Gene Transcription involves:
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EPIGENETICS
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Gene activity can be modulated in a manner that does not involve changes in the DNA code and these changes can persist through one or more generations. These are called epigenetic effects.
Epigenetics: a change in the expression of a gene that changes the phenotype without permanently changing the gene itself. Typically involving changes in chromatin structure.
EXAMPLES of epigenetic effects: • Chromatin modifications • DNA methylation: can lead to parent-of-origin effects (imprinting)
Epigenetics
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Regulation of Gene Transcription
DNA conformation
Chromatin
Methylation
Transcription
Nuclear Compartment
ON OFF
- DNA supercoiling
- association with thenucleosome
- modification of cytosine
- trans-acting factors
- association with thenuclear matrix
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Nucleosome structure- Positively charged histone proteins bind with each other along with DNA to form a structure called the nucleosome.
- The nucleosome core is composed of two molecules each of the histone proteins: H2A, H2B, H3 and H4.
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Nucleosomes are connected together by linker DNA of variable length and H1 histone.
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Nucleosomes can associate with each other via interactions between histone H1 to form a more compact structure that has been termed, due to its size, the 30 nm fiber.
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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Many different orders of chromatin packing give rise to the highly condensed metaphase chromosome
Two types of chromatin are defined based on chromosome staining properties:
Euchromatin - decondensed and transcriptionally active
Heterochromatin - condensed and transcriptionally inactive
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Modifications include:
- (de) Acetylation - Methylation - Phosphorylation - Ubitquitination
acetylation by HATs
deacetylation byHDACs
Acetylated nucleosome Deacetylated nucleosome
Acetylation and Deacetylation of Histones
HAT = histoneacetyl transferase
HDAC = histonedeacetylase
Histone modifications alter the interaction of the DNA and the histone tails
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N
N
O
N
H H
1
2
34
5
6
N
N
O
N
H H
1
2
34
5
6
H C3
Regulation of gene expression by DNA methylation:
Modification of the DNA base cytosine to 5 methyl-cytosine
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DNA Methylation Is Associated
with Silenced Genes in Mammalian Cells
- Some mammalian genes are
kept silent by methylation
of nearby DNA sequences.
- Methylation of DNA can mark
sites where heterochromatin
subsequently forms.
- DNA methylation lies at the
heart of a phenomenon
called imprinting.
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Switching a gene off through DNA methylation and histone modification
DNA methylation decreases transcription and promotes heterochromatin formation
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Model for Disruption of Normal DNA Methylation in Cancer Cells
NORMAL CELLS-low-level transcription from hypermethylated repetitive DNA results in the generation of RNAi. -Increase in repressive RNAi machinery and, directly or indirectly, DNA methylation (DNMTs and MBPs)-CpG islands are largely hypomethylated and the associated genes (a tumour suppressor gene (TSG) ) are transcribed.
CANCER CELLS-Repetitive DNA tends to lose methylation marks resulting in increased genomic instability. -Repeat hypomethylation near genes or promoters could disrupt transcription of TSGs by several mechanisms, including transcriptional interference
Increase DNA methylation at TSG promoter site decreased TSG expression
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Basal Machinery Maintains Low Levels of Transcription
TBP TFIID
RNA polymerase IITATA BOX
TFIIA TFIIB RNA Transcription
P PPP P
TFIIHTFIIE
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Regulation of gene transcription in eukaryotes
Involves the interaction of the basal transcriptional machinery located at the gene promoter and trans-acting proteins bound to cis-regulatory elements.
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Activation/repressiondomain
Dimerization Domain
DNA Binding Domain
DNA:Protein and Protein:Protein interactions are important for transcription
factor function.
Note the modular structure of transcription factors. Different parts of the protein are responsible for:• DNA binding• Dimer formation – homologous or heterologous• Transcriptional activation (i.e. interaction with basal transcription machinery).
** Dimer formation adds an extra element of complexity and versatility.
Common structural types of transcription factors: 1.Zinc finger2.Helix-turn-helix3.Leucine zipper
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Transcription factor structure
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Some modes of Tf activationInactive condition Active condition
Synthesize Tf protein
Degrade Tf protein
Phosphorylate Tf
Dephosphorylate Tf
Bind an activating ligand
Bind an inhibitory ligand
Bind a partner protein
No protein
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Assembly of the basal transcriptional machinery
Binding of TATA-box binding protein (TBP), TBP associated factors (TAFs),
polymerase II , transcription factors (TFIIs) and an inducible Tf with its coactivator
Inducible Tf
coactiv-
ator
Storey, K.B. 2004. Functional Metabolism: Regulation and Adaptation. Wiley-Liss, Hoboken, NJ
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Example: Hypoxia-inducible transcription factor (HIF-1)
- regulates gene responses to low oxygen
HIF-1α
Inducible
O2-sensitive
HIF-1β (ARNT)
constitutive
Storey, K.B. 2004. Functional Metabolism:
Regulation and Adaptation.
Wiley-Liss, Hoboken, NJ
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HIF-1 regulationHigh Oxygen -- HIF-1α is hydroxylated & quickly degraded by the proteosome
Low/no Oxygen -- HIF-1α stabilized, moves to the nucleus & dimerizes with HIF-1β to activate multiple genes that enhance oxygen delivery to tissues and/or energy supply via glycolysis
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• Adaptive to changes in tissue oxygenation
• Increases glycolysis, Apoptosis resistance and Invasion/Metastasis
HIF-1 regulation in tumors
cells
Metabolic adaptation:
Apoptosis resistance:
Invasion/metastasis:
ALDA, GADPH, GLUT1, GLUT3, HK1, HK2, LDHA, PFK,
ADM, EPO, ET1, IGF2, NOS2, TGFA
AMF, CATHD, CMET, FN1, KRT14, KRT18, KRT19, MMP2, UPAR, VIM
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HIF-1 and Tumour Growth
- Frequently found to be
overexpressed in tumour cells as a
result of genetic alteration- Pre-clinical studies has
shown that HIF-1
inhibition has marked
effects on tumour
growth
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Cis-regulatory sequences
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Folding of DNA can cause interactions between enhancer or repressor proteins bound to distant cis-elements and the basal transcriptional machinery.
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Different genes possess the same cis-regulatory sequences!
This provides spatial and temporal coordination of gene regulation.
1) Stage specific expression during embryonic development.
2) Tissue specific expression
3) Response to external stimuli: hypoxia, heat, other stresses, hormones, etc.
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Gene CGene CGene BX X XGene AEnhancer Enhancer Enhancer
Transcription A Transcription B Transcription C
Steroid Hormone
Steroid hormone + Steroid receptor
Nucleus
Cytoplasm
Coordinate regulation of gene expression- Often several different genes must be activated at the same time- Coordinate gene activation is easily accomplished if all genes share a
common promoter or enhancer sequence
E.g.SteroidHormoneaction
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Enhancer that confers hormone responsiveness
Glucocorticoid-binding sequenceHormonal non-responsive viral gene
(thymidine kinase)
Ligation and selection
Thymidine kinase induced
Stimulate with glucocorticoidSome cells incorporate the
Recombinant gene into theirchromosome
Restriction enzymesRestriction enzymes
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( Mouse fibroblast)
An enhancer that is required for cell type-specific gene transcription
Myeloma cell line producingIgG
VDJ Intron C
Remove portionsof introns
Mouse myelomas lacking heavy chain genes
Transfect withaltered genes
Isolate and clone immunoglobulin heavy chain
Extract mRNA, separate on a gel, blot to paper, and hybridize with radioactive CDNA fragment
IgG mRNA IgG mRNAPosition of normal C mRNA
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Transcriptional level control– Differential gene transcription – the major
mechanism of selective protein synthesis– Governed by a large number of proteins known
as transcription factors– Two functional classes of transcription factors
• General transcription factors• Specific transcription factors
– Main topic of discussion
Summary: Control of Gene Expression
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Specific transcription factors– A single gene controlled by many regulatory
sites – bind different regulatory proteins– A single regulatory protein may become attached
to numerous sites on the genome– Cells respond to environmental stimuli by
synthesizing different transcription factors• Bind to different sites on DNA
– Extent of transcription depends on the particular combination of transcription factors activated
Summary: Control of Gene Expression
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Activation of Transcription– Hormones that affect transcription of genes include insulin,
thyroid hormone, glucagon and glucocorticoids• All affect transcription factors which bind to DNA• DNA sites bound by transcription factors are termed – response elements
– e.g. Glucocorticoids stimulate gene expression by binding to a specific DNA sequence termed – a glucocorticoid response element (GRE)
– Same GRE is located upstream from different genes on different chromosomes
• Thus, a single stimulus – ie. elevated glucocorticoid levels – can simultaneously activate a range of genes needed in a comprehensive response to stress
Summary: Control of Gene Expression
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Activation of Transcription– Expression of genes also regulated by more distant DNA
elements termed enhancers– Can be experimentally moved without affecting their
ability to enhance gene expression– Might be 1000’s or 10,000’s of base pairs upstream or
downstream from the gene• Happens because enhancer can be brought into close proximity
to the gene when DNA forms loops
– Promoters and enhancers cordoned off from other genes by sequences called insulators
Summary: Control of Gene Expression
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Activation of TranscriptionA transcription factor bound to an enhancer may act
via the following mechanisms:• Recruit general transcription factors and DNA
polymerase II to the core promoter• Stabilize the transcription machinery located in the
core promoter• Interact with an intermediary termed a coactivator
– Co-activators are large complexes with 15 to 20 subunits– Do not directly bind DNA– Interact with a range of transcription factors
Summary: Control of Gene Expression
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Structure of Transcription Factors– Contain different domains which mediate the different
functions – at least two domains are key• DNA-binding domain• Activation domain
– Many also form dimers & have a dimerization domain– Some common transcription factor motifs are
• Zinc finger Helix-loop-helix• Leucine zipper
– Shared feature• Structurally stable framework so that specific DNA recognizing
sequences are correctly positioned
Summary: Control of Gene Expression