molecular biology lecture 8

BIOL 321 – Regulation of Gene Expression in Eukaryotes

http://micro.magnet.fsu.edu/cells/animalcell.html

Objectives- Gene “silencing” by histone modifications

- Recruitment of protein complexes to Genes

- Basal machinery

- Transcription factors

- Transcriptional repressors

- Cis-regulatory factors

- Extracellular signals and gene regulation

- Eukaryotic gene regulation at steps after transcription initiation

1) Control the types and quantities of proteins (gene products) produced

2) Respond to the environment by turning on or off specific genes or groups of genes.

3) Turn genes on or off in the correct sequence during development

Why regulate gene expression?

1) Cis-Regulatory sequences of DNA (Promoter & enhancer sequences)

2) Transcription factors (= Trans-acting factors) - Nuclear proteins that bind to promoter or enhancer

sequences of genes and stimulate/inhibit transcription

3) Changes to chromatin conformation such as:- DNA methylation- Histone modification – e.g. by acetylation, phosphorylation, etc.

Control of Differential Gene Transcription involves:

EPIGENETICS

Gene activity can be modulated in a manner that does not involve changes in the DNA code and these changes can persist through one or more generations. These are called epigenetic effects.

Epigenetics: a change in the expression of a gene that changes the phenotype without permanently changing the gene itself. Typically involving changes in chromatin structure.

EXAMPLES of epigenetic effects: • Chromatin modifications • DNA methylation: can lead to parent-of-origin effects (imprinting)

Epigenetics

Regulation of Gene Transcription

DNA conformation

Chromatin

Methylation

Transcription

Nuclear Compartment

ON OFF

- DNA supercoiling

- association with thenucleosome

- modification of cytosine

- trans-acting factors

- association with thenuclear matrix

Nucleosome structure- Positively charged histone proteins bind with each other along with DNA to form a structure called the nucleosome.

- The nucleosome core is composed of two molecules each of the histone proteins: H2A, H2B, H3 and H4.

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Nucleosomes are connected together by linker DNA of variable length and H1 histone.

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Nucleosomes can associate with each other via interactions between histone H1 to form a more compact structure that has been termed, due to its size, the 30 nm fiber.

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Many different orders of chromatin packing give rise to the highly condensed metaphase chromosome

Two types of chromatin are defined based on chromosome staining properties:

Euchromatin - decondensed and transcriptionally active

Heterochromatin - condensed and transcriptionally inactive

Modifications include:

- (de) Acetylation - Methylation - Phosphorylation - Ubitquitination

acetylation by HATs

deacetylation byHDACs

Acetylated nucleosome Deacetylated nucleosome

Acetylation and Deacetylation of Histones

HAT = histoneacetyl transferase

HDAC = histonedeacetylase

Histone modifications alter the interaction of the DNA and the histone tails

N

N

O

N

H H

1

2

34

5

6

N

N

O

N

H H

1

2

34

5

6

H C3

Regulation of gene expression by DNA methylation:

Modification of the DNA base cytosine to 5 methyl-cytosine

DNA Methylation Is Associated

with Silenced Genes in Mammalian Cells

- Some mammalian genes are

kept silent by methylation

of nearby DNA sequences.

- Methylation of DNA can mark

sites where heterochromatin

subsequently forms.

- DNA methylation lies at the

heart of a phenomenon

called imprinting.

Switching a gene off through DNA methylation and histone modification

DNA methylation decreases transcription and promotes heterochromatin formation

Model for Disruption of Normal DNA Methylation in Cancer Cells

NORMAL CELLS-low-level transcription from hypermethylated repetitive DNA results in the generation of RNAi. -Increase in repressive RNAi machinery and, directly or indirectly, DNA methylation (DNMTs and MBPs)-CpG islands are largely hypomethylated and the associated genes (a tumour suppressor gene (TSG) ) are transcribed.

CANCER CELLS-Repetitive DNA tends to lose methylation marks resulting in increased genomic instability. -Repeat hypomethylation near genes or promoters could disrupt transcription of TSGs by several mechanisms, including transcriptional interference

Increase DNA methylation at TSG promoter site decreased TSG expression

Basal Machinery Maintains Low Levels of Transcription

TBP TFIID

RNA polymerase IITATA BOX

TFIIA TFIIB RNA Transcription

P PPP P

TFIIHTFIIE

Regulation of gene transcription in eukaryotes

Involves the interaction of the basal transcriptional machinery located at the gene promoter and trans-acting proteins bound to cis-regulatory elements.

Activation/repressiondomain

Dimerization Domain

DNA Binding Domain

DNA:Protein and Protein:Protein interactions are important for transcription

factor function.

Note the modular structure of transcription factors. Different parts of the protein are responsible for:• DNA binding• Dimer formation – homologous or heterologous• Transcriptional activation (i.e. interaction with basal transcription machinery).

** Dimer formation adds an extra element of complexity and versatility.

Common structural types of transcription factors: 1.Zinc finger2.Helix-turn-helix3.Leucine zipper

Transcription factor structure

Some modes of Tf activationInactive condition Active condition

Synthesize Tf protein

Degrade Tf protein

Phosphorylate Tf

Dephosphorylate Tf

Bind an activating ligand

Bind an inhibitory ligand

Bind a partner protein

No protein

Assembly of the basal transcriptional machinery

Binding of TATA-box binding protein (TBP), TBP associated factors (TAFs),

polymerase II , transcription factors (TFIIs) and an inducible Tf with its coactivator

Inducible Tf

coactiv-

ator

Storey, K.B. 2004. Functional Metabolism: Regulation and Adaptation. Wiley-Liss, Hoboken, NJ

Example: Hypoxia-inducible transcription factor (HIF-1)

- regulates gene responses to low oxygen

HIF-1α

Inducible

O2-sensitive

HIF-1β (ARNT)

constitutive

Storey, K.B. 2004. Functional Metabolism:

Regulation and Adaptation.

Wiley-Liss, Hoboken, NJ

HIF-1 regulationHigh Oxygen -- HIF-1α is hydroxylated & quickly degraded by the proteosome

Low/no Oxygen -- HIF-1α stabilized, moves to the nucleus & dimerizes with HIF-1β to activate multiple genes that enhance oxygen delivery to tissues and/or energy supply via glycolysis

• Adaptive to changes in tissue oxygenation

• Increases glycolysis, Apoptosis resistance and Invasion/Metastasis

HIF-1 regulation in tumors

cells

Metabolic adaptation:

Apoptosis resistance:

Invasion/metastasis:

ALDA, GADPH, GLUT1, GLUT3, HK1, HK2, LDHA, PFK,

ADM, EPO, ET1, IGF2, NOS2, TGFA

AMF, CATHD, CMET, FN1, KRT14, KRT18, KRT19, MMP2, UPAR, VIM

HIF-1 and Tumour Growth

- Frequently found to be

overexpressed in tumour cells as a

result of genetic alteration- Pre-clinical studies has

shown that HIF-1

inhibition has marked

effects on tumour

growth

Cis-regulatory sequences

Folding of DNA can cause interactions between enhancer or repressor proteins bound to distant cis-elements and the basal transcriptional machinery.

Different genes possess the same cis-regulatory sequences!

This provides spatial and temporal coordination of gene regulation.

1) Stage specific expression during embryonic development.

2) Tissue specific expression

3) Response to external stimuli: hypoxia, heat, other stresses, hormones, etc.

Gene CGene CGene BX X XGene AEnhancer Enhancer Enhancer

Transcription A Transcription B Transcription C

Steroid Hormone

Steroid hormone + Steroid receptor

Nucleus

Cytoplasm

Coordinate regulation of gene expression- Often several different genes must be activated at the same time- Coordinate gene activation is easily accomplished if all genes share a

common promoter or enhancer sequence

E.g.SteroidHormoneaction

Enhancer that confers hormone responsiveness

Glucocorticoid-binding sequenceHormonal non-responsive viral gene

(thymidine kinase)

Ligation and selection

Thymidine kinase induced

Stimulate with glucocorticoidSome cells incorporate the

Recombinant gene into theirchromosome

Restriction enzymesRestriction enzymes

( Mouse fibroblast)

An enhancer that is required for cell type-specific gene transcription

Myeloma cell line producingIgG

VDJ Intron C

Remove portionsof introns

Mouse myelomas lacking heavy chain genes

Transfect withaltered genes

Isolate and clone immunoglobulin heavy chain

Extract mRNA, separate on a gel, blot to paper, and hybridize with radioactive CDNA fragment

IgG mRNA IgG mRNAPosition of normal C mRNA

Transcriptional level control– Differential gene transcription – the major

mechanism of selective protein synthesis– Governed by a large number of proteins known

as transcription factors– Two functional classes of transcription factors

• General transcription factors• Specific transcription factors

– Main topic of discussion

Summary: Control of Gene Expression

Specific transcription factors– A single gene controlled by many regulatory

sites – bind different regulatory proteins– A single regulatory protein may become attached

to numerous sites on the genome– Cells respond to environmental stimuli by

synthesizing different transcription factors• Bind to different sites on DNA

– Extent of transcription depends on the particular combination of transcription factors activated

Summary: Control of Gene Expression

Activation of Transcription– Hormones that affect transcription of genes include insulin,

thyroid hormone, glucagon and glucocorticoids• All affect transcription factors which bind to DNA• DNA sites bound by transcription factors are termed – response elements

– e.g. Glucocorticoids stimulate gene expression by binding to a specific DNA sequence termed – a glucocorticoid response element (GRE)

– Same GRE is located upstream from different genes on different chromosomes

• Thus, a single stimulus – ie. elevated glucocorticoid levels – can simultaneously activate a range of genes needed in a comprehensive response to stress

Summary: Control of Gene Expression

Activation of Transcription– Expression of genes also regulated by more distant DNA

elements termed enhancers– Can be experimentally moved without affecting their

ability to enhance gene expression– Might be 1000’s or 10,000’s of base pairs upstream or

downstream from the gene• Happens because enhancer can be brought into close proximity

to the gene when DNA forms loops

– Promoters and enhancers cordoned off from other genes by sequences called insulators

Summary: Control of Gene Expression

Activation of TranscriptionA transcription factor bound to an enhancer may act

via the following mechanisms:• Recruit general transcription factors and DNA

polymerase II to the core promoter• Stabilize the transcription machinery located in the

core promoter• Interact with an intermediary termed a coactivator

– Co-activators are large complexes with 15 to 20 subunits– Do not directly bind DNA– Interact with a range of transcription factors

Summary: Control of Gene Expression

Structure of Transcription Factors– Contain different domains which mediate the different

functions – at least two domains are key• DNA-binding domain• Activation domain

– Many also form dimers & have a dimerization domain– Some common transcription factor motifs are

• Zinc finger Helix-loop-helix• Leucine zipper

– Shared feature• Structurally stable framework so that specific DNA recognizing

sequences are correctly positioned

Summary: Control of Gene Expression