Download HGD- Gene Regulation in Eukaryotes.pptx

Gene Regulation in
Eukaryotes
Dr. Syahril Abdullah
Medical Genetics Laboratory
[email protected]
Lecture Outline
1. 
2. 
3. 
4. 
The Genome
Overview of Gene Control
Cellular Differentiation in Higher Eukaryotes
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!
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The Genome
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
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
C-value Enigma
there is no correlation between complexity of an organism and its genome size !!
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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.
Cellular Differentiation in Higher Eukaryotes
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
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.
1. Genomic Level Control : (A) Methylation of Cytosine in DNA
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).
DNA methyltransferase
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 : (B) Histone Modifications
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
Transcriptionally inactive
Transcriptionally active
Chromatin: DNA + Histones
i.  Euchromatin = loosely packed, active genes
ii.  Heterochromatin = condensed region, genes
transcriptionally silent. At centromeres
1. Genomic Level Control
Transcription Factors
RNA Pol
Transcription
DNA
Methyltransferase
Methyl CpG Binding
Proteins
NO Transcription
Acetylation
5-methyl-C
Histone
Deacetylase
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.
1. Genomic Level Control : (B) Histone Modifications
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)
H3K4me
- 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)
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 trascription
factors to bind to promoters.
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
Promoter
Enhancers/
Upstream
Silencers
Elements
-1 kb
Start of
translation: AUG
TATA Box
-25/-30 bp
+1 bp
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 positiondependent 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."
Fibronectin Gene
Primary mRNA transcript
of fibronectin gene
Exon
EIIIB
Exon
EIIIA
5’
3’
Fibroblast
mRNA
Liver mRNA
- 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.
Gene for Casein
• 
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.
Prolactin
Prevents
Digestion
• 
Poly A tail stabilizes mRNA transcripts.
• 
Hormones can stabilize certain mRNA transcripts
mRNA Casein
Milk
DNA
Ribonuclease
Ribonuclease
Digest
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
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
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4. Translational Level Control : RNAi Mechanisms
1. RNA Cleavage
RISC: RNA-induced
silencing complex
2. Inhibition of Translation
3. Transcriptional Silencing
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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