Gene Regulation in Eukaryotes

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

syahril@medic.upm.edu.my

Lecture Outline

1.  The Genome 2.  Overview of Gene Control 3.  Cellular Differentiation in Higher Eukaryotes 4.  The Regulation of Gene Expression

4.1. Genomic Level Control 4.2. Transcriptional Level Control 4.3. mRNA Processing & Nuclear Transport Control 4.4. Translational Level Control 4.5. Post-Translational Level Control

5.  Review

A darn difficult topic – You better stay awake!

The Genome

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1. Bacteria e.g. E. coli has genome of 4 x 106 base pairs - 3000 gene products

2. Human genome: 3,200,000,000 (3 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 !!

Organism Type Organism Genome Size (bp)

Amoeba Amoeba dubia 670,000,000,000

Nematode Caenorhabditis elegans 100,300,000

Insect Apis mellifera (honey bee) 1,770,000,000

Fish Protopterus aethiopicus 130,000,000,000

Overview of Gene Control

1.  There are many different cell types in a multicellular organism (white blood cells,

neurons, epithelial cells etc)

2.  Each cell type arises from the selective expression of a subset of genes in the

genome.

3.  In many cases, the genetic program that predetermines a cell to be a certain cell

type can be re-programmed to become another type of cell.

4.  In cloning Dolly the sheep, the researcher took the nucleus from a lamb’s udder

and placed it into an egg of which the nucleus has been removed - the

transplanted nucleus regenerated the whole lamb.

Overview of Gene Control

5.  Many biochemical processes are common to all cell types , and thus a majority

of genes are expressed in all cell types (e.g., glycolytic pathway enzymes,

actin, etc.)

6.  Other biochemical processes are specific to certain cells (e.g. hemoglobin in

red blood cells).

7.  In many cases, these tissue-specific genes are highly expressed in one or a

few types of cells and not expressed at all in others.

1.  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

2.  Yet different types of mammalian cells

express widely different proteins even

though each cell has the same complement

of genes

Cellular Differentiation in Higher Eukaryotes

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

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

human development

The Regulation of Gene Expression

1.  Genomic Level Control - involves silencing or expression at chromatin structure

or at DNA level.

2.  Transcriptional Level Control - involves turning on or off the gene expression - most important point of control for most genes

3.  mRNA Processing & Nuclear Transport Control - controlling how the primary RNA transcript is spliced or processed - some RNAs are selectively transported to the cytoplasm

4.  Translational Level Control - selecting which mRNAs are translated by ribosomes - control of mRNA stability

5.  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?

A. Methylation of cytosine residues in DNA

B. Histone modifications

i. Histone Acetylation

ii. Histone Methylation

C. Chromatin Remodeling

3.  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.

a.  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).

b.  60-90% all all CpGs are methylated in mammals

c.  Unmethylated CpGs are known as

“CpG island” – located in promoter regions

d.  DNA methylation can switch off gene expression

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 histones…next slides

e.  Active gene (expressed gene) is undermethylated;

Inactive (silent) gene is hypermethylated

f.  Loss of methyl-CpG-binding protein 2 (MeCP2) = Rett syndrome

MBD2 causes transcriptional silencing of hypermethylated genes in cancer

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

DNA methyltransferase

i. Histone Acetylation 1.  Histone acetyltransferase (HAT) acetylate histone proteins = genes

transcriptionally active

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

Chromatin: DNA + Histones i.  Euchromatin = loosely packed, active genes ii.  Heterochromatin = condensed region, genes

transcriptionally silent. At centromeres

Transcriptionally inactive

Transcriptionally active

1. Genomic Level Control Transcription Factors

RNA Pol

Acetylation Transcription

DNA Methyltransferase 5-methyl-C

Methyl CpG Binding Proteins

Histone Deacetylase

NO Transcription Deacetylation

Transcription factors

Chromatin Compaction Transcriptional Silencing

Association between CpG methylation and histone acetylations

1.  Silencing due to the chromatin compaction. 2.  Interfere with the entry of transcription

factors.

ii. Histone Methylation 1.  Addition of methyl groups to the tail of histone proteins

2.  Activation or repression depending on which amino acids in the tail are

methylated.

3.  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)

1. Genomic Level Control : (B) Histone Modifications

H3K9me

H3K4me

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 trascription

factors to bind to promoters.

1. Genomic Level Control : (C) Chromatin Remodeling

1. Genomic Level Control : DNase I Hypersensitivity

How do we know if the genes are transcriptionally active?

The regions around the genes become highly sensitive to the action of DNase I

Regions known as: DNase I Hypersensitive Sites

Develops about 1kb upstream from the transcription start site

Indicates that these regions adopt a more open configuration.

1. Genomic Level Control

Epigenetic Inheritance?

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

2. Transcriptional Level Control

TATA Box Upstream

Elements

Enhancers/

Silencers

-1 kb -25/-30 bp +1 bp

Promoter 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 Orientation and position independent

Silencers: Inhibits transcription Also orientation and position independent

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

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 receptor (a kind of

specific TF) protein

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

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."

Primary mRNA transcript of fibronectin gene

Fibroblast mRNA

Liver mRNA

Exon EIIIB

Exon EIIIA

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

5’ 3’

Fibronectin Gene

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

ribonucleases.

•  Poly A tail stabilizes mRNA transcripts.

•  Hormones can stabilize certain mRNA transcripts

Milk

Gene for Casein DNA

mRNA Casein

Gene for Casein DNA

mRNA Casein Ribonuclease

Digest

Milk

Gene for Casein DNA

mRNA Casein Ribonuclease

Prolactin Prevents Digestion

4. Translational Level Control

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.

ii. Kozak sequence – RccAUGG, where R is a purine (A or G) 3 bases upstream of the start codon, follow by another G. Translation more efficient with Kozak sequence.

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

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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

RNAi

4. Translational Level Control : RNAi Mechanisms

1. RNA Cleavage 2. Inhibition of Translation 3. Transcriptional Silencing

RISC: RNA-induced silencing complex

RITS: RNA-induced transcriptional silencing

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.

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?

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

Review

The End

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