Introduction to
Genetic AnalysisTENTH EDITION
Introduction to
Genetic AnalysisTENTH EDITION
Griffiths • Wessler • Carroll • Doebley
© 2012 W. H. Freeman and Company
CHAPTER 12Regulation of Gene Expression
in Eukaryotes
CHAPTER OUTLINE12.1 Transcriptional regulation in eukaryotes: an overview
12.2 Lessons from yeast: the GAL system
12.3 Dynamic chromatin
12.4 Short-term activation of genes in a chromatin environment
12.5 Long-term inactivation of genes in a chromatin environment
12.6 Gender-specific silencing of genes and whole chromosomes
12.7 Post-transcriptional gene repression by miRNAs
The first cloned mammal
Dolly, the Finn Dorset lamb in 1996 and her surrogate Scottish Blackface mother
Dolly and Bonnie
globinmyosin
erythrocytemuscle cell
All genes
housekeeping
All cells have the same genome, but each cell expresses only a subset of all genes
Cells differ in gene expression
Gene regulation at multiple levels
TransportLocalizationModificationComplex formationDegradation
Many regulatory proteins have to import into nucleus
Promoter-proximal elements are necessary for efficient transcription
Point mutations throughout the promoter region were analyzed for their effects on transcription rates. The height of each line represents the transcription level relative to a wild-type promoter or promoter-proximal element (1.0).
Transcription factors need multiple functional domains
1. A domain that recognizes a DNA regulatory sequence (the protein’s DNA-binding site)
2. A domain that interacts with one or more proteins of the transcriptional apparatus (RNA polymerase or a protein associated with RNA polymerase)
3. A domain that interacts with proteins bound to nearby regulatory sequences on DNA such that they can act cooperatively to regulate transcription
4. A domain that influences chromatin condensation either directly or indirectly
5. A domain that acts as a sensor of physiological conditions within the cell
Direct or indirect (by interacting with other proteins)
Transcriptional activator proteins bind to UAS elements in yeast
UAS: Upstream Activation Sequences
Binding site for Gal4
Can be far from promoter
Transcriptional activator proteins recruit the transcriptional machinery
Co-activator:Does not directly bind DNA
Specific recognition of target sequenceEnhancer can function
far away from promoter
Enhancer can function upstream or downstream, even far away
Combinations of regulatory proteins control cell types
Mating type
Combinations of binding partners => different binding specificities
A nucleosome is composed of DNA wrapped around eight histones
Histone octamer (H2A2H2B2H32H42)
DNA exposed on the outside
The structure of chromatin
Euchromatin(loose)
Heterochromatin
•Condensed
•Repetitive sequences
•Late replicating
•Genes silenced
Chromatin remodeling exposes regulatory sequences
Shifting of nucleosome position
Exposes regulatory sequences
Linker DNA: sensitive to nuclease
Nucleosomal DNA: protected from nuclease digestion
Use nuclease sensitivity to determine chromatin state (open/closed) or nucleosome position
+ SWI-SNF + ATP
The SWI-SNF complex for chromatin remodeling
Yeast mutant screen
sugar nonfermenting (snf)
Mating type switch (swi)
swi2=snf2
swi2/snf2 (“switch-sniff”) locus
SWI-SNF complex
Modifications of histone tails results in chromatin remodeling
Histone tails are exposed, can be modified
Modifies Lysine (K) and Arginine (R) (basic aa)
Acetylation: negative charges => repulsion
Inheritance of chromatin states
Epigenetic memory: heritable traits (over rounds of cell division and sometimes transgenerationally) that do not involve changes to the underlying DNA sequence. (e.g. chromatin state)
A model for the inheritance of DNA methylation
In mammals, 70-80% of CG are methylated genome-wide.CpG island: clusters around gene promoter
hemi-methylated
maintenance
Enhanceosomes recruit chromatin remodelers
Enhancers contain binding sites for many transcription factors, which bind and interact cooperatively.
Mating-type switching is controlled by recombination of DNA cassettes
ds break in MAT made by HO endonuclease
=> gene conversion
silent information regulators (SIR)Sir2 (HDAC)
Gene silencing is caused by the spread of heterochromatin
w+ is expressed in some cells => not a mutation in w gene
Clonal => epigenetic memory
Position-effect variegation (PEV)
Heterochromatin in Drosophila chromosomes
~30% of genome
H3K4me2, enriched in euchromatinH3K9me2, enriched in heterochromatin
Some genes enhance or suppress the spread of heterochromatin
Enhancer
Suppresor
Su(var)2-5 = HP1 (heterochromatic protein 1)Su(var)3-9 = histone methylatransferase
Steps required for imprinting
Igf2: maternal imprinting (inactive)H19: paternal imprinting (inactive)
H19Igf2
Barr body and Lyon Hypothesis of X inactivation
Murray Barr: discoverer
Mary Lyon
Epigenetic memory
Xi: H3K9me, histone hypoacetylation, DNA hypermethylation~ heterochromatin
Xist RNA covers one of the two copies of the X chromosome
RNA fluorescent in situ hybridization (FISH)
metaphase chromosomes
female fibroblast cell line
Xist expression => cis-inactivation