Interactions between Activators and RNA Polymerase in Prokaryotic Gene Regulation

What are the common and different problems that prokaryotic and eukaryotic transcription machineries have to face?

Interactions between Activators and RNA Pol II Holoenzyme in Eukaryotic Gene Regulation

Model for Cooperative Assembly of an Activated Transcription-Initiation Complex

Identification of Tissue- and Gene-Specific Subunits of the General Transcription Apparatus

ovarian-specific TAF105

TBP-related factors (TRFs)

Nucleosome is the Basic Unit of Chromatin Structure

Histone Modification: an Important step in Gene Regulation

In Gel Assay for Detecting Histone Acetyltransferase (HAT) Activity

Histone Acetylation and Gene Expression

– Correlation between histone acetylation and gene expression.

– Genetic identification of GCN5 and related genes.

– Biochemical purification of HAT from Tetrahymena.

– Discovery of the Gcn5-containing complexes.

– Recruiting of HAT to specific promoters by activators (chromatin IP).

– An ever-growing list of eukaryotic HATs.

Histone Deacetylation and Gene Repression

• Unacetylated histones are positively charged, have higher affinity for DNA, thus may prevent access of general transcription factors to promoters.

• Genetic identification of yeast proteins that repress transcription of multiple genes. (RPD3, etc)

• Biochemical purification of histone deacetylase from human (HDAC).

• DNA-binding repressors target histone deacetylases to specific promoters.

ARTKQTARKSTGGKAPRKQLATKAARKSAP H3

ARTKQTARKSTGGKAPRKQLATKAARKSAP H3

Ac

histone deacetylase (HDAC)+ histone methyltransferase (HMT)

*histone demethylase +HAT

Me

Concerted Actions of Multiple Histone Modifying Enzymes in Gene Regulation

transcriptionally active

transcriptionally inactive

The “histone code” theory: modifications may serve as docking sites for other chromatin regulatory proteins

Chromatin-Remodeling factors Participate in Activation at Some Promoters

• Genetic identification of the Swi/Snf complex in yeast.

• Swi/Snf complex induces changes in chromatin structure in an ATP dependent manner.– Nuclease sensitivity assay.

– Nucleosome array assay.

• Chromatin-remodeling complexes in higher eukaryotes.

Biochemical Activities of ATP-Dependent Chromatin Remodeling Complexes

Models Depicting Different Orders of Action by Regulators and Chromatin Remodeling Complexes

Fundamental Elements of Eukaryotic Transcriptional Control

Chromatin Immunoprecipitation (ChIP) for Detecting In Vivo Protein-DNA Interaction

Common Approaches for Identifying Protein-Protein Interactions

(A) Classic Genetic Screen

A B a B a b a B

healthy sick dead(synthetic lethal screen)

healthy(suppressor hunt)

high-copy suppressor ormutational suppressor

Strategies for a Suppressor Hunt: (1) Mutational Approach; (2) High-Copy Suppression

Common Approaches for Identifying Protein-Protein Interactions

(B) Yeast Two-Hybrid Screen

Common Approaches for Identifying Protein-Protein Interactions(C) Biochemical Purification

co-immunoprecipitation glutathione-S-transferase (GST) pulldown

Potential Pitfalls of co-IP

Identification of Associated Proteins by mass spectrometry

To succeed as a theorist, you have to be good.To succeed as an experimentalist, you only have to be lucky.As an experimentalist, you can go through life kicking over a lot of stones, and,if you’re lucky, you’ll find something. ------John B. Fenn, 2002 Nobel Laureate

Multiple Levels of Transcriptional Control in Eukaryotic Cells

• Changes of chromatin structure– Histone modification

– Chromatin remodeling

• Enhancer-binding proteins and diverse core promoter transcription complexes

generate tissue-specific and gene-specific transcription.

• Regulation of the regulators– Regulating protein activity

– Regulating protein levels

– Generating protein diversity

– Regulating protein location

Life Cycle of Saccharomyces cerevisiae (budding yeast)

Mating-Type Loci on Chromosome III of S. cerevisiae

Mating-type switching is determined by three genetic loci.MAT: actively transcribedHML silencedHMRa: silenced

A normal haploid cells switches its mating type each generation.

Mating-type information is transferred from HML or HMR to MAT.

Repression of the silent loci is critical to haploid cells.

Functions of Sir Proteins at the Three Silent Loci in Yeast

SIR2 is a Conserved NAD-Dependent Histone Deacetylase

The link between metabolic rate and aging. Calorie restriction increases longevity.

NAD is required by glycolytic enzymes to oxidize glucose. NAD is also required by SIR2 to deacetylate histones.

In budding yeast, Sir2 is a limiting component of longevity.

Is SIR2 a Link between Metabolic Rate and Aging?

P53: Guardian of the Genome

• A site-specific DNA-binding transcription factor.

• A critical component of the cellular mechanisms that respond to genotoxic stress, e.g. DNA damage. hypoxia, etc.

• p53 is activated upon exposure to stress stimuli.

• p53 regulates expression of downstream target genes to arrest cell cycle progression or induce apoptosis.

• Mutations in p53 are the most frequent seen genetic alterations in human cancer.

Transcriptional Activation by p53

Stabilization of p53 by ARF-Dependent Sequestration of MDM2

Formation of a p53-MDM2 complex in the nucleus stimulates export to the cytoplasm and subsequent degradation.Stress-dependent phsophorylation of p53 masks the nuclear export sequences in p53 and also weakens p53-MDM2 interaction.ARF also binds MDM2 and sequesters it in the nucleolus.

Inactive State Active State

Post-Translational Modification of p53

Lipid-Soluble Hormones Control the Activities of Nuclear Receptors

Hormone-Dependent Nuclear Translocation of Glucocorticoid Receptor (GR)

Model of Hormone-Dependent Gene Activation by GR

Disassembly of Transcriptional Regulatory Complexes by Molecular Chaperones

receptor

hormone

chaperones

active regulatory complex

A”suicide” model for concerted transcriptional activation and destruction

Model of IFN- Mediated Gene Activation by Stat1

Microarray Assay Serial Analysis of Gene Expression (SAGE)

Whole Genome Approaches for Gene Expression Study

Gene Expression Profiling Predicts Clinical Outcome of Breast CancerT

um

ors

70 prognostic marker genes

Good prognosis:no distant metastasesover 5 years

Poor prognosis:distant metastaseswithin 5 years

Future Perspectives

– Discovery of new co-activators and co-repressors.

– Higher-order chromatin structure.

– Mechanism of integrating multiple signals.

– Cross talk with other nuclear processes.

– High throughput methods for studying gene expression

– Connections with human diseases.