Eukaryotic Gene Regulation

PART II
How Genes Are Regulated
Gene Regulation in Eukaryotes
OUTLINE
2.1 Overview of Eukaryotic Gene Regulation 2.2 Control of Transcription Initiation 2.3 Epigenetic Effects 2.4 Regulation After Transcription (siRNA and miRNA)
The focus of this course is on human genetics
Genetics has powerful tools for understanding human biology

Paradigm shift from studying one gene or protein at a time to studying interacting networks of many genes and proteins Molecular studies can lead to predictive and preventive medicine DNA diagnostics can be used to generate a genetic profile of an individual Design of therapeutic drugs to prevent or minimize symptoms of gene-based diseases
2
Modern genetic techniques

Genetic dissection of model organisms Inactivate a gene and observe the consequences
Genome sequencing Human Genome Project Model organisms and other organisms Understanding higher-order processes that arise from interacting biological networks
Genomics can rapidly analyze thousands of genes High-throughput DNA sequencing and genotyping Large-scale DNA arrays (chips)
The Human Genome Project

First formally discussed in 1985
Officially began in 1990 estimated 15 years, $3 billion
Systems approach to biology and medicine Study of the interplay of the elements in a biological system as it undergoes genetic perturbation or biological activation Development of technologies to analyze the genome and the complete biochemical machinery of cells 3-5% of budget committed to studying the ethical, legal, and social implications (ELSI) of human genome mapping Social and personal repercussions are generating new areas of biological concern
4
Global analysis of genes and their mRNAs

Genomics
Studies whole genomes: global analysis of chromosomal

features and gene products
the development and application of more effective mapping,

sequencing, and computational tools
Predict and verify the existence and functions of previously

undefined genes using molecular biology tools
High throughput instruments - DNA sequencers and DNA arrays Platforms all components needed for automated acquisition of data
5
Important implications of genetics to social issues

Entire genetic profiles of individuals will become available
This genetic information can be used to help people

Make predictions about future possibilities and risks Or, genetic information could also be used to restrict people's lives Genetic Information Nondiscrimination Act was passed by the US federal government in 2008
Prohibits discrimination on the basis of genetic tests by insurance companies and employers
Important implications of genetics to social issues (continued)

Proper interpretation of genetic information and understanding of statistical concepts is essential
Regulation and control of new technology

Transgenic technology (genetic engineering) is routine in many animals Should genetic engineering of human embryos be allowed? Guidelines must be established to prevent misuse of new knowledge in human genetics
Overview of eukaryotic gene regulation

Eukaryotes use complex sets of interactions
Regulated interactions of large networks of genes

Each gene has multiple points of regulation
transcription takes place in the nucleus and translation takes place in the cytoplasm
Genes are turned on or off in the right place and time Differentiation and precise positioning of tissues and organs during embryonic development
8
Key regulatory events in eukaryotes
Multiple steps where production of the final gene product can be regulated in eukaryotes
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16
10
Control of transcription initiation
Three types of RNA polymerases in eukaryotes RNA pol I transcribes rRNA genes RNA pol II transcribes all protein-coding genes (mRNAs) and micro-RNAs RNA pol III transcribes tRNA genes and some small regulatory RNAs
11
RNA polymerase II transcription

RNA pol II catalyzes synthesis of the primary transcript, which is complementary to the template strand of the gene Most RNA pol II transcripts undergo further processing to generate mature mRNA
RNA splicing removes introns

Addition of 5' GTP cap protects RNA from degradation Cleavage of 3' end and addition of 3' polyA tail
12
cis-acting elements: promoters and enhancers

Promoters usually directly adjacent to the gene
Include transcription initiation site
A A A T T Allow basal level of transcription

Often have TATA box: TATA Enhancers can be far away from gene Augment or repress the basal level of transcription
13
trans-acting factors interact with cis-acting elements to control transcription initiation

Direct effects of transcription factors:
Through binding to DNA

Indirect effect of transcription factors: Through proteinprotein interactions
14
Use of reporter genes to identify trans-acting factors in transcriptional regulation
15
Basal transcription factors

Basal transcription factors assist the binding of RNA pol II to promoters Key components of basal factor complex: TATA box-binding protein (TBP)
Bind to TATA box First of several proteins to assemble at promoter
TBP-associated factors (TAFs)

Bind to TBP assembled at TATA box
RNA pol II associates with basal complex and initiates basal level of transcription
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16 16
Basal factors bind to promoters of all protein-encoding genes

Ordered pathway of assembly at promoter: 1. TBP binds to TATA box
2. TAFs bind to TBP

3. RNA pol II binds to TAFs
17
Activators are transcription factors that bind to enhancers

Activators are responsible for much of the variation in levels of transcription of different genes Increase levels of transcription by interacting directly or indirectly with basal factors at the promoter 3-dimensional complex of proteins and DNA Mechanisms of activator effects on transcription Stimulate recruitment of basal factors and RNA pol II to promoters Stimulate activity of basal factors already assembled on promoters Facilitate changes in chromatin structure
Binding of activators to enhancers increases transcriptional levels

Low level transcription occurs when only basal factors are bound to promoter
When basal factors and activators are bound to DNA, rate of transcription increases
19
Domains within activators

Activator proteins have two functional domains
Sequence-specific DNA binding domain

Binds to enhancer
Transcription-activator domain
Interacts with other transcriptional regulatory proteins
Some activators have a third domain Responds to environmental signals

Example - steroid hormone receptors
DNA-binding domains of activator proteins

Interacts with major groove of DNA Specific amino acids have high-affinity binding to specific nucleotide sequence
The three best-characterized motifs:

Helix-loop-helix (HLH)
Helix-turn-helix (HTH)
Zinc finger
21
Repressor proteins suppress transcription initiation through different mechanisms

Some repressors have no effect on basal transcription but suppress the action of activators Compete with activator for the same enhancer OR Block access of activator to an enhancer Some repressors eliminate virtually all basal transcription from a promoter Block RNA pol II access to promoter OR Bind to sequences close to promoter or distant from promoter
Repressor proteins that act through competition with an activator protein

Repressor binds to the same enhancer sequence as the activator Has no effect on the basal transcription level
23
Repressor proteins that act through quenching an activator protein

Quenchers bind to the activator but do not bind to DNA
Type I: Repressor blocks the DNA-binding domain
Type II: Repressor blocks the activation domain
24
Complex regulatory regions enable fine-tuning of gene expression

Each gene can have many regulatory proteins
In humans, ~2000 genes encode transcriptional regulatory proteins

Each regulatory protein can act on many genes
Each regulatory region can have dozens of enhancers

Enhanceosome multimeric complex of proteins and other small molecules that associate with an enhancer
Enhancers can be bound by activators and repressors with varying affinities

Different sets of cofactors and corepressors compete for binding to activators and repressors
Chromatin structure and epigenetic effects

Chromatin structure can affect transcription
Nucleosomes can sequester promoters and make them inaccessible to RNA polymerase and transcription factors
Histone modification and DNA methylation Chromatin remodeling and hypercondensation Epigenetic changes changes in chromatin structure that are inherited from one generation to the next DNA sequence is not altered
26
Chromatin reduces transcription
27
Genomic imprinting results from transcriptional silencing

Genomic imprinting expression of a gene depends on whether it was inherited from the mother or father Occurs with some genes of mammals Epigenetic effect (no change in DNA sequence)
Paternally imprinted gene is transcriptionally silenced if it was transmitted from the father
Maternal allele is expressed
Maternally imprinted gene is transcriptionally silenced if it was transmitted from the mother
Paternal allele is expressed
Methylation of complementary strands of DNA in genomic imprinting

Epigenetic state can be maintained across cell generations through the action of DNA methylases
29
The resetting of genomic imprints during meiosis

Epigenetic imprints remain throughout the lifespan of the mammal In germ cells, epigenetic imprints are reset at each generation During meiosis, imprints are erased and new ones are set
30
Genomic imprinting and human disease

Examples: two syndromes associated with small deletions in chromosome 15 At least two genes within this region are differently imprinted Praeder-Willi syndrome occurs when the deletion is inherited from the father Angelman syndrome occurs when the deletion is inherited from the mother
Affected individuals have mental retardation and development disorders
31
Inheritance patterns of disorders resulting from mutations in imprinted genes

These pedigrees may appear to be instances of incomplete penetrance, but are distinctly different
32
Regulation after transcription

Posttranscriptional regulation can occur at any step
At the level of RNA

Splicing, stability, and localization Example alternative splicing of mRNA
Generates more diversity of proteins Common feature in eukaryotes
At the level of protein

Synthesis, stability, and localization
33
Some small RNAs are responsible for RNA interference (RNAi)

Specialized RNAs that prevent expression of specific genes through complementary base pairing Small (21 30 nt) RNAs Micro-RNAs (miRNAs) and small interfering RNAs (siRNAs)
First miRNAs (lin-4 and let-7) identified in C. elegans Nobel prize to A. Fire and C. Mello in 2006
Posttranscriptional mechanisms for gene regulation mRNA stability and translation May also affect chromatin structure
Primary transcripts containing miRNA

Most miRNAs are transcribed by RNA polymerase II from noncoding DNA regions that generate short dsRNA hairpins
35
miRNA processing
Drosha excises stem-loop from primary miRNA (pri-miRNA) to generate pre-miRNA of ~ 70 nt Dicer processes pre-miRNA to a mature duplex miRNA One strand is incorporated into miRNA-induced silencing complex (RISC)
36
Two ways that miRNAs can down-regulate expression of target genes

When complementarity is perfect: When complementarity is imperfect:
Target mRNA is degraded
Translation of mRNA target is

repressed
37
siRNAs detect and destroy foreign dsRNAs

Two biological sources of dsRNAs that are precursors of siRNAs (pri-RNAs) Transcription of both strands of an endogenous genomic sequence Arise from exogenous virus pri-RNAs are processed by Dicer siRNA pathway targets dsRNAs for degradation siRNAs are very useful experimental tools to selectively knock down expression of target genes To study function of a gene, dsRNAs for that gene can be introduced into cells
siRNA video
http://www.youtube.com/watch?v=Fa4skYBJHoI
39
The cellular components of gene expression

Mutations in genes encoding gene products for transcription, RNA processing, translation, and protein processing are often lethal Some mutations in tRNA genes can suppress mutations in proteincoding genes
40
Health consequences of mutations in genes encoding DNA repair enzymes
Xeroderma pigmentosum: Mutations in one of seven genes encoding enzymes involved in nucleotide excision repair
Hereditary forms of colorectal cancer (not shown): Mutations in human homologs of bacterial genes (MutS and MutL) involved in mismatch repair
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7 41
Impact of unrepaired mutations

Germ line mutations occur in gametes or in gamete precursor cells Transmitted to next generation Provide raw material for natural selection
Somatic mutations occur in non-germ cells

Not transmitted to next generation of individuals Can affect survival of an individual Can lead to cancer
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7 42
Alkaptonuria: An inborn error of metabolism
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7
43
The molecular basis of sickle-cell anemia

GluVal substitution at sixth amino acid affects the three-dimensional structure of the hemoglobin b chain
Abnormal protein aggregates cause sickle shape of red blood cells
44
Sickle-cell anemia is pleiotropic
45
A comprehensive example: Mutations that affect vision

The cellular basis of vision The molecular basis of vision
46
How mutations modulate light and color perception
Autosomal dominant disorder
47
Levels of polypeptide structure
Interactions that determine the three-dimensional conformation of a polypeptide
48
Levels of polypeptide structure (cont)

1o structure is the amino acid sequence
2o structure is the characteristic geometry of localized regions
49
Levels of polypeptide structure (cont)
3o structure is the complete three-dimensional arrangement of a polypeptide
50
Multimeric proteins are complexes of polypeptide subunits

Identical subunits Non-identical subunits
51
Multimeric proteins are complexes of polypeptide subunits (cont)

One polypeptide in different proteins
52

Eukaryotic Gene Regulation

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PART II

How Genes Are Regulated

Gene Regulation in Eukaryotes

The focus of this course is on human genetics

Genetics has powerful tools for understanding human biology

Modern genetic techniques

The Human Genome Project

Officially began in 1990 estimated 15 years, $3 billion

Global analysis of genes and their mRNAs

Studies whole genomes: global analysis of chromosomal

the development and application of more effective mapping,

Predict and verify the existence and functions of previously

Important implications of genetics to social issues

This genetic information can be used to help people

Important implications of genetics to social issues (continued)

Regulation and control of new technology

Overview of eukaryotic gene regulation

Regulated interactions of large networks of genes

Key regulatory events in eukaryotes

Control of transcription initiation

RNA polymerase II transcription

RNA splicing removes introns

cis-acting elements: promoters and enhancers

Include transcription initiation site

A A A T T Allow basal level of transcription

trans-acting factors interact with cis-acting elements to control transcription initiation

Through binding to DNA

Use of reporter genes to identify trans-acting factors in transcriptional regulation

Basal transcription factors

TBP-associated factors (TAFs)

Basal factors bind to promoters of all protein-encoding genes

2. TAFs bind to TBP

Activators are transcription factors that bind to enhancers

Binding of activators to enhancers increases transcriptional levels

Domains within activators

Sequence-specific DNA binding domain

Some activators have a third domain Responds to environmental signals

DNA-binding domains of activator proteins

The three best-characterized motifs:

Repressor proteins suppress transcription initiation through different mechanisms

Repressor proteins that act through competition with an activator protein

Repressor proteins that act through quenching an activator protein

Type II: Repressor blocks the activation domain

Complex regulatory regions enable fine-tuning of gene expression

In humans, ~2000 genes encode transcriptional regulatory proteins

Each regulatory region can have dozens of enhancers

Enhancers can be bound by activators and repressors with varying affinities

Chromatin structure and epigenetic effects

Chromatin reduces transcription

Genomic imprinting results from transcriptional silencing

Methylation of complementary strands of DNA in genomic imprinting

The resetting of genomic imprints during meiosis

Genomic imprinting and human disease

Affected individuals have mental retardation and development disorders

Inheritance patterns of disorders resulting from mutations in imprinted genes

Regulation after transcription

At the level of RNA

At the level of protein

Some small RNAs are responsible for RNA interference (RNAi)

Primary transcripts containing miRNA

Two ways that miRNAs can down-regulate expression of target genes

Target mRNA is degraded

Translation of mRNA target is