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

Dolly in Royal Museum of Scotland

Ian Wilmut

Dolly

Dolly Parton

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

Overview of transcriptional regulation

nucleus(membrane)

chromatin

Gene regulation at multiple levels

TransportLocalizationModificationComplex formationDegradation

Many regulatory proteins have to import into nucleus

Promoter-proximal elements precede the promoter of a eukaryotic gene

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)

Model Organism Yeast

Brewer’s yeast Baker’s yeast

Saccharomyces cerevisiaeBudding yeast

The Gal pathway

Expressed at low level

Induced by galactose

Regulated by Gal4

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 are modular

Reporter gene

domain swap

Transcriptional activator proteins may be activated by an inducer

Galactose: inducing signal

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

Transcriptional complexes

Combinations of regulatory proteins control cell types

Mating type

Combinations of binding partners => different binding specificities

The structure of chromatin

~150 bp linker DNA

A nucleosome is composed of DNA wrapped around eight histones

Histone octamer (H2A2H2B2H32H42)

DNA exposed on the outside

The structure of chromatin

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

Histone modifications

Histone modifications

Acetylation of histones

Histone acetyltransferase (HAT)

Histone de-acetylase (HDAC)

Histone modifications

Alternative modifications on the same residue

Histone code

Regulation of gene expression by histone acetylation

Histone deacetylation can turn off gene transcription

HDAC(corepressor)

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)

Methylation of DNA

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 help recruit the transcriptional machinery

Enhanceosomes recruit chromatin remodelers

Enhancers contain binding sites for many transcription factors, which bind and interact cooperatively.

Enhancer-blocking insulators prevent enhancer activation

Model for how enhancer-blocking insulators might work

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

Multiple states of Lysine methylation

Heterochromatin may spread farther in some cells than in others

Barrier insulators stop the spread of heterochromatin

Genomic imprinting

Genomic imprinting

Phenotype depends on the parental origin of the genes

Inactivation of genes and chrosomomes

Genomic imprinting

No changes in DNA sequence

mouse/human ~100 imprinted genes

Genomic imprinting requires insulators

DNA methylation

imprinting control region

Unusual inheritance of imprinted genes

Steps required for imprinting

Igf2: maternal imprinting (inactive)H19: paternal imprinting (inactive)

H19Igf2

X inactivation

Dosage compensation for X chromosome female: XX male: XY

Barr body and Lyon Hypothesis of X inactivation

Murray Barr: discoverer

Mary Lyon

Epigenetic memory

Xi: H3K9me, histone hypoacetylation, DNA hypermethylation~ heterochromatin

Xist

non-coding

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

A model for X-chromosome inactivation

Possible models for the repression of translation by miRNA