Regulation of Gene Expression in Eukaryotes

Eukaryotic aspects that affect gene expression

Eukaryotic chromosomes have DNA wound around histone proteins condensation/decondensation is a level of regulation Eukaryotes have a nucleus transport of mRNA into cytoplasm can be regulated

Eukaryotes process their RNA alternative splicing is a level of regulation

Eukaryotic mRNA has a longer half-life allows for more translational control of regulation Eukaryotes are multicellular differential gene expression allows for specialization of cell types

Levels of Gene Expression Regulation in Eukaryotes

Regulation of Transcription in Eukaryotes

Activator Regulatory Transcription Factors bind to Enhancers Repressor Regulatory Transcription Factors bind to Negative Regulatory Elements Chromatin Remodelers

Basal vs. Regulatory Transcription Factors in Eukaryotes

Basal Transcription Factors
Recruit and Stabilize RNA Polymerase Bind to the basal promoter region (-30 to -90 bp) ( TATA box, CAAT box, or GC box) ARE NOT directly responsive to signals DO NOT regulate transcription

Regulatory Transcription Factors

Can Affect Basal Transcription Factor Binding Bind to Enhancers and Negative Regulatory Elements (which can be far from the basal promoter region) ARE responsive to signals DO Regulate transcription

Both are types of proteins

Properties of Enhancers or Negative Regulatory Elements

DNA sequences Can act at long distances (kilobases) in either orientation May be upstream, downstream, or within genes regulated Recruit activator or repressor Regulatory Transcription Factors Can act on heterologous (unrelated) promoters Enhancers are bound by activator regulatory transcription factors Negative Regulatory Elements are bound by repressor regulatory transcription factors

Design an experiment to ectopically express Gene N in muscle cells.

(Gene N is usually only expressed in neurons)

Ectopic eye formation due to ectopic eyeless expression

Dpp Enhancer

Heterologous promoter

Eyeless gene

Jang, C.-C. et al. Development 2003;130:2939-2951

Mechanism of Activator Regulatory Transcription Factors

Basal vs. Regulatory Transcription Factors in Eukaryotes

Basal Transcription Factors
Recruit and Stabilize RNA Polymerase Bind to the basal promoter region (-30 to -90 bp) ( TATA box, CAAT box, or GC box) ARE NOT directly responsive to signals DO NOT regulate transcription

Regulatory Transcription Factors

Can Affect Basal Transcription Factor Binding Bind to Enhancers and Negative Regulatory Elements (which can be far from the basal promoter region) ARE responsive to signals DO Regulate transcription

Both are types of proteins

Figure 11.25: Transcriptional activation by recruitment

Mechanism of Activator Regulatory Transcription Factors

Regulatory Transcription Factors Contain Domains and Motifs

Domains Regions of the protein that have a particular function-DNA binding, protein-protein interaction, interaction with basal transcription factors
Motifs Conserved three-dimensional structural patterns that allow for a specific function-in this case DNA binding

Activator Transcription Factor Domains

True Activators have: 1) A DNA Binding Domain that binds to a specific Enhancer DNA sequence 2) A Transactivating Domain that positively interacts with basal transcription factors or chromatin remodeling complexes

Transactivation domain binds to basal transcription factors

Transactivation Domain

DNA Binding Motifs

Helix-turn-Helix Zinc finger Leucine zipper basic Helix-Loop-Helix

Homeobox Helix-turn-Helix Activators

Zinc Finger

Leucine Zipper

Basic Helix-Loop-Helix
Dimerizes Protein-protein interaction domain in helix 1 Basic region binds DNA

Regulation of Transcription in Eukaryotes

Regulatory Transcription Factors binding to Enhancers or Negative Regulatory Elements Chromatin Remodelers: Anti-repressor Activators Repressors

Chromatin Remodeling

Figure 11.27: Function of chromatin-remodeling complexes

Chromatin Remodeler Systems

SWI/SNF, NURF complex ATP hydrolysis dependent Histone acetyltransferases (HATs)

Models for SWI/SNF Mediated chromatin remodeling

Chromatin Remodeling

Chromatin Remodeler Systems

SWI/SNF, NURF complex ATP hydrolysis dependent Histone acetyltransferases (HATs)

Chromatin Remodeling by Histone Acetylases and Histone Deacetylases

Levels of Gene Expression Regulation in Eukaryotes

Post-transcriptional regulation RNAi Alternative splicing selective mRNA export to cytoplasm mRNA stability

Post-translational modifications Proteolysis Phosphorylation Methylation Acetylation

RNA interference (RNAi)

Small RNA molecules specifically block translation of genes Two types of RNAs
siRNA miRNA

RNAi
siRNA
small/short interfering RNAs or silencing RNAs Double stranded (ds)RNAs 20-25 bases Endogenous siRNAs identified/occur in plants Synthetic siRNAs now made to knockout gene products

miRNA
microRNAs 22ish nts- single stranded RNA with stem-loop

Figure 11.35: Mechanisms of gene silencing by the siRNA and miRNA pathways

Levels of Gene Expression Regulation in Eukaryotes

When are genes regulated?

In response to environmental factors (nutrients, heat, toxic compounds etc.) During development as cells become specialized In response to inter-cellular signals involved in MANY cellular processes

Levels of Gene Expression Regulation

Transcription
Selective nuclear RNA processing Selective mRNA translation Post-translational protein modification

Techniques to study transcription

Individual Genes Levels of expression Northern Blots Reporter Assays Real-Time RT-PCR Regulatory Transcription Factors binidng DNA Gel shifts and ChIP assays
Genome-wide Microarrays Expression profiling

Northern Blots

Reporter Genes as Tools to Study Transcriptional Regulation

Luciferase (tissue culture) lacZ: b-galactosidase (animals- in vivo) Green Fluorescent Protein (animals-in vivo) GUS-b-glucoronidase (plants- in vivo)

The Genetic Elements Regulating Tissue-Specific Transcription Can Be Identified by Fusing Reporter Genes to Suspected Enhancer Regions of the Genes Expressed in Particular Cell Types

Real Time RT PCR

Reverse transcriptase (RT)
mRNA DNA

Real Time PCR

Techniques to study transcription

Individual Genes Levels of expression Northern Blots Reporter Assays Real-Time RT-PCR Regulatory Transcription Factors binidng DNA Gel shifts and ChIP assays
Genome-wide Microarrays Expression profiling

Techniques to study DNA-protein interactions

Gel Shifts (Electromobility shift assays)

Chromatin immunoprecipitation assays

Gel Shifts

Microarrays

Techniques to study protein levels

Western Blots Immunohisto(cyto)chemistry