Introduction to Genetic Analysis TENTH EDITION Introduction to Genetic Analysis TENTH EDITION Griffiths Wessler Carroll Doebley © 2012 W. H. Freeman and Company CHAPTER 12 Regulation of Gene Expression in Eukaryotes

CHAPTER OUTLINE 12.1Transcriptional regulation in eukaryotes: an overview 12.2Lessons from yeast: the GAL system 12.3Dynamic chromatin 12.4Short-term activation of genes in a chromatin environment 12.5Long-term inactivation of genes in a chromatin environment 12.6Gender-specific silencing of genes and whole chromosomes 12.7Post-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

globin myosin erythrocyte muscle 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 Transport Localization Modification Complex formation Degradation 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 yeastBaker’s yeast Saccharomyces cerevisiae Budding 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 sequence Enhancer 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 bplinker DNA

A nucleosome is composed of DNA wrapped around eight histones Histone octamer (H2A 2 H2B 2 H3 2 H4 2 ) 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

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 euchromatin H3K9me2, 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

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