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Gene Regulation in Eukaryotes
Gene Regulation in
Eukaryotes
Gene Regulation in Eukaryotes Dr. Syahril Abdullah Medical Genetics Laboratory syahril@medic.upm.edu.my

Dr. Syahril Abdullah Medical Genetics Laboratory syahril@medic.upm.edu.my

The Genome

Organism Type Organism Genome Size (bp) Amoeba Amoeba dubia 670 Billion Nematode Caenorhabditis elegans 100
Organism Type
Organism
Genome Size (bp)
Amoeba
Amoeba dubia
670 Billion
Nematode
Caenorhabditis elegans
100 Million
Insect
Apis mellifera (honey bee)
1.7 Billion
Fish
Protopterus aethiopicus
130 Billion

Bacteria e.g. E. coli has genome of 4 mil base pairs - 3000 gene products

Human genome: 3,200,000,000 (3.2 billion) bp (haploid)

- but only 20,000-25,000 gene products

- i.e. 80-90% of human genome do not have direct genetic function !!

- hence redundancy of eukaryotic genome

C-value Enigma there is no correlation between complexity of an organism and its genome size !!

syahril@medic.upm.edu.my

Cellular Differentiation in Higher Eukaryotes

1.

2.

Each mammalian cell contains the same complete set of genome, regardless of which tissues or organs they are from (two copies except haploid cells). Nucleus contains all the necessary information, encoded in DNA, to control the formation of a whole organism Yet different types of mammalian cells express widely different proteins even though each cell has the same complement set of genes

types of mammalian cells express widely different proteins even though each cell has the same complement

Cellular Differentiation in Higher Eukaryotes

3.

In addition, the same type of cells can have different patterns of protein synthesis during different developmental stages, for example the globin genes

developmental stages, for example the globin genes Different members of the globin gene family are transcribed
developmental stages, for example the globin genes Different members of the globin gene family are transcribed

Different members of the globin gene family are transcribed at different stages of human development

the globin genes Different members of the globin gene family are transcribed at different stages of

Lecture Outline

Eukaryotes Transport Control
Eukaryotes
Transport Control

1.

2.

Cellular Differentiation in Higher

The Regulation of Gene Expression

4.1. Genomic Level Control

4.2. Transcriptional Level Control

4.3. mRNA Processing & Nuclear

4.4. Translational Level Control

4.5. Post-Translational Level Control

3.

Review

& Nuclear 4.4. Translational Level Control 4.5. Post-Translational Level Control 3. Review syahril@medic.upm.edu.my

syahril@medic.upm.edu.my

The Regulation of Gene Expression

1.

2.

3.

4.

5.

Genomic Level Control

- involves silencing or expression at chromatin structure or at DNA level.

or expression at chromatin structure or at DNA level. Transcriptional Level Control - involves turning on

Transcriptional Level Control

- involves turning on or off the gene expression

- most important point of control for most genes

mRNA Processing & Nuclear Transport Control

- controlling how the primary RNA transcript is spliced or processed

- some RNAs are selectively transported to the cytoplasm

Translational Level Control

- selecting which mRNAs are translated by ribosomes

- control of mRNA stability

Post-Translational Processing

- at level of protein

- may be modified by various mechanisms like phosphorylation, ligand binding and etc.

- affected by the rates of protein degradation, or its subcellular localization

1. Genomic Level Control

1.

There are transcriptionally active and inactive regions through out the genome.

2.

How are these regions controlled?

3.

A. Methylation of cytosine residues in DNA B. Histone modifications i. Histone Acetylation ii. Histone Methylation C. Chromatin Remodeling These are the types of Epigenetics What is epigenetics? Changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: over; above) -genetics. Changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations.

1. Genomic Level Control : (A) Methylation of Cytosine in DNA

a.

b.

c.

d.

e.

f.

CpG rich region is a short stretch of DNA in which the frequency of CG sequence is higher than other regions in the genome (p=phosphodiester bond). 60-90% of all CpGs are methylated in mammals Unmethylated CpGs are known as CpG island” – located in promoter regions DNA methylation can switch off gene expression

DNA methyltransferase
DNA methyltransferase

i.By impeding the binding of transcriptional proteins (i.e. RNA pol, transcription factors).

ii.Methylated DNA bound by methyl-CpG-binding domain proteins (MBDs) recruits additional proteins .remodel histonesnext slides Active gene (expressed gene) is undermethylated; Inactive (silent) gene is hypermethylated Loss of methyl-CpG-binding protein 2 (MeCP2) = Rett syndrome MBD2 causes transcriptional silencing of hypermethylated genes in cancer

1. Genomic Level Control : (B) Histone Modifications

i. Histone Acetylation

1.

Histone

acetyltransferase

(HAT)

acetylate

histone

proteins

transcriptionally active

(HAT) acetylate histone proteins transcriptionally active = genes 2.   From previous slide: MBDs bound to

=

genes

2.From previous slide: MBDs bound to methylated CpG, recruits histone deacytelases (HDAC) – takes away the acetyl group = genes transcriptionally inactive.

1. Genomic Level Control : (B) Histone Modifications

1. Genomic Level Control : (B) Histone Modifications Transcriptionally inactive Transcriptionally active Chromatin: DNA +

Transcriptionally inactive

Transcriptionally active

Transcriptionally inactive Transcriptionally active Chromatin: DNA + Histones i.   Euchromatin = loosely

Chromatin: DNA + Histones

i.

Euchromatin = loosely packed, active genes

ii.

Heterochromatin = condensed region, genes transcriptionally silent. At centromeres

packed, active genes ii.   Heterochromatin = condensed region, genes transcriptionally silent. At centromeres

1. Genomic Level Control

Transcription Factors RNA Pol Transcription Acetylation DNA 5-methyl-C Methyltransferase Histone Deacetylase NO
Transcription Factors
RNA Pol
Transcription
Acetylation
DNA
5-methyl-C
Methyltransferase
Histone
Deacetylase
NO Transcription
Deacetylation
Transcription factors
Chromatin Compaction
Transcriptional Silencing

Methyl CpG Binding Proteins

Association between CpG methylation and histone acetylations

1.

Silencing due to the chromatin compaction.

2.

Interfere

with

the

entry

of

transcription

factors.

1. Genomic Level Control : (B) Histone Modifications

ii. Histone Methylation

1.

2.

Addition of methyl groups to the tail of histone proteins Activation or repression depending on which amino acids in the tail are

3.

methylated.

For activation of transcription:

- Addition of methyl at lysine 4 in the tail of H3 histone protein (H3K4me3)

- Frequently found in promoters of transcriptionally active genes. (NURF) = Nucleosome Remodeling Factor

4. For repression of transcription

- Addition of methyl at lysine 9 in the tail of H3 histone protein (H3K9me3)

H3K4me H3K9me
H3K4me
H3K9me

1. Genomic Level Control : (C) Chromatin Remodeling

1.

Some transcription factors & regulatory proteins alter chromatin structure without altering the chemical structure of the histones directly.

2.

Known as:

Chromatin Remodeling Complex.

3.

They bind directly to particular sites on DNA and reposition nucleosomes, allowing transcription factors to bind to promoters.

bind directly to particular sites on DNA and reposition nucleosomes, allowing transcription factors to bind to

1. Genomic Level Control

Epigenetic Inheritance?

How histone modifications, nucleosome positioning & other types of epigenetic marks might be maintained is still unclear

histone modifications, nucleosome positioning & other types of epigenetic marks might be maintained is still unclear
5’ UTR 3’ UTR
5’ UTR
3’ UTR

2. Transcriptional Level Control

Promoter

Enhancers/ Upstream TATA Box Silencers Elements -1 kb -25/-30 bp +1 bp
Enhancers/
Upstream
TATA Box
Silencers
Elements
-1 kb
-25/-30 bp
+1 bp

Start of translation: AUG

Promoters: A DNA sequence to which RNA Pol binds prior to initiation of transcription. Contains a sequence called TATA box (7 bp consensus sequence 5’ -TATA[A/

T]A[A/T]-3’).

Enhancers: To stimulate/increase the activity of the promoters

Silencers: Inhibits transcription

Transcription Factors (TFs): Bind to regulatory DNA sequences (promoters, enhancers) to regulate transcription Two types: (i) Basal TFs (eg. TFIIA, TFIIB)- bind at promoters, assisting RNA pol (ii) Specific TFs (eg. Sp1, C-Jun) – bind at specific enhancers

2. Transcriptional Level Control

2. Transcriptional Level Control

2. Transcriptional Level Control

Hormonal Effects on Enhancer

Human metallothionein protein – 1.Regulation of zinc (Zn) & copper (Cu) in blood, detoxification of heavy metals, function of immune system, neuronal development. Synthesized in kidney and liver. 2.Usually expressed at very low level 3.Gene expression can be activated by cadmium(Cd), copper(Cu) ions or by glucocorticoid hormone.

When glucocorticoid hormone is released, it binds to the glucocorticoid protein receptor (a kind of specific TF).

Glucocorticoid receptor protein (+glucocorticoid) recognizes a specific enhancer called Glucocorticoid Response Element (GRE) in the metallothionein gene and binds to it -- this activates expression of the metallothionein gene.

Response elements function in response to transient increase in the level of a substance or a regulatory hormone

2. Transcriptional Level Control

Insulator

1.

Also known as boundary element

2.

What it is? DNA sequences that block or insulate the effect of enhancers in position- dependent manner

2.   What it is? DNA sequences that block or insulate the effect of enhancers in

3. mRNA Processing and Nuclear Transport Control

1.

Splicing: The process of cutting the pre-mRNA to remove the introns and joining

together the exons.

2.Alternative splicing: is a process that occurs in which the splicing process of a

pre-mRNA transcribed from one gene can lead to different mature mRNA

molecules and therefore to different protein."

Fibronectin Gene

Exon

Exon

EIIIB

EIIIA

Primary mRNA transcript of fibronectin gene

5’ 3’
5’
3’
Fibroblast mRNA
Fibroblast
mRNA

Liver mRNA

of fibronectin gene 5’ 3’ Fibroblast mRNA Liver mRNA - exons EIIIA and EIIIB are spliced

- exons EIIIA and EIIIB are spliced out in liver mRNA transcript

A single gene can code for two or more related proteins, depending on how the exons/ introns are spliced

3. mRNA Processing and Nuclear Transport Control

1.

Speed of Transport of mRNA Through the Nuclear Pores Evidence suggests that this time may vary.

2.

Longevity of mRNA mRNA can last a long time. For example, mammalian red blood cells eject their

nucleus but continue to synthesize hemoglobin for several months. This

indicates that mRNA is available to produce the protein even though the DNA is

gone.

Ribonucleases are enzymes that destroy mRNA. • mRNA has noncoding nucleotides at either end of the

molecule – contain info about the number of times

mRNA is transcribed before being destroyed by

Prolactin

Prevents

Digestion

ribonucleases. Poly A tail stabilizes mRNA transcripts.

Hormones can stabilize certain mRNA transcripts

Gene for Casein

Gene for Casein

Gene for Casein

DNA DNA DNA
DNA
DNA
DNA
mRNA Casein mRNA Casein mRNA Casein Milk Milk
mRNA Casein
mRNA Casein
mRNA
Casein
Milk
Milk

Ribonuclease Ribonuclease Digest

4. Translational Level Control

4. Translational Level Control 5’ Untranslated Region (5’ UTR) Starts from transcription start site to just

5’ Untranslated Region (5’ UTR) Starts from transcription start site to just before the initiation codon (ATG) Contains sequence that regulate translation efficiency i.Binding site for proteins that may effect the translation e.g. Iron responsive elements (also in 3’UTR) – regulate gene expression in response to iron.

3’ Untranslated Region (3’ UTR) Starts from stop codon, end before poly A tail. Contains regulatory sequence for efficient translation i.For cystoplasmic localization of mRNA ii.Binding site for :

SECIS elements – direct ribosome to translate codon UGA as selenocysteines. MicroRNA (a type of RNAi)

4. Translational Level Control

A bit about RNA interference (RNAi)

1.

From DNA, transcribed but not translated

2.

About 30% of human genes regulated by RNA interference

3.

In eukaryotes, fungi, plants, animals

genes regulated by RNA interference 3.   In eukaryotes, fungi, plants, animals RNAi syahril@medic.upm.edu.my

RNAi

syahril@medic.upm.edu.my

5. Post-Translational Processing

These mechanisms act after the protein has been produced

1.

Protein cleavage and/or splicing. The initial polypeptide can be cut into different functional pieces, with different patterns of cleavage occurring in different tissues. In some cases, different pieces may be spliced together.

In some cases, different pieces may be spliced together. e.g. Bovine proinsulin is a precursor to

e.g. Bovine proinsulin is a precursor to the hormone insulin. It must be cleaved into 2 polypeptide chains and about 30 amino acids must be removed to form insulin.

5. Post-Translational Processing

2.

Chemical modification. Protein function can be modified by addition of methyl, acetyl, alkyl, phosphoryl, or glycosyl groups. E.g. How can phosphorylation control enzyme activity?

E.g. How can phosphorylation control enzyme activity? Addition of phosphate causes conformational changes to the

Addition of phosphate causes conformational changes to the protein. Opens up the active site for catalytic process.

Review

Review
The End
The End

Let’s have a 5 min break before my next class!

Dr. Syahril Abdullah Medical Genetics Laboratory syahril@medic.upm.edu.my